Acetylcholinesterase and ATPases: targets of biologically active compounds

Acetylcholinesterase is a serine hydrolase whose key biological role is the termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The reversible inhibition of brain acetylcholinesterase is the major therapeutic target in the treatment of Alzheimer’s disease associated with loss of cholinergic neurons in the brain and the decreased level of acetylcholine, whereas toxic effects are related to irreversible modulators of the enzyme activity. Na+/K+-ATPase is a transmembrane protein regulating many cellular functions, involving those associated with tumor cell growth. In addition, particular Na+/K+-ATPase subunits are expressed in some cancer cells and changes in Na+/K+-ATPase activity and relative subunit abundance were detected in various carcinoma cell lines. Accordingly, design and synthesis of novel compounds have been directed towards new modulators of Na+/K+-ATPase, which selectively target these cellular abnormalities. Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are plasma membrane bound enzymes representing the major part of purinergic signaling. Increased E-NTPDases levels were observed in cancer cells due to their abnormal cellular growth and proliferation. Accordingly, the decrease of E-NTPDase activity could be regarded as a new approach in the development of antitumor drugs. This presentation will be focused on metal-based compounds such as polyoxometalates which are discrete, negatively charged metal-oxo clusters of early d-block metal ions in high oxidation states, surrounded by oxygen atoms. These compounds were approved to exhibit a variety of biological actions such as anticancer, antimicrobial and antidiabetic properties. However, the mechanism of their bioactivities has not been completely understood yet. It has been assumed that polyoxometalates interact with different enzyme families extracellularly located on the plasma membrane such as phosphatases and ecto-nucleotidases. This presentation will primarily be directed to acetylcholinesterase, Na+/K+-ATPase and E-NTPDases as potential targets of polyoxometalate pharmacological and toxicological activities.PDSS 2022 : Virtual event : September 8-9, 2022, Paris, France

Time – dependent inhibition of electric eel ache induced by chlorpyrifos

The aim of the work was to investigate the influence of contact time between acetylcholinesterase (AChE) and chlorpyrifos, on the sensitivity of earlier developed AChE based bioanalytical method for detection and determination of organophosphates in water samples. The IC50 values were obtained from the concentration- dependent responses of AChE activity to chlorpyrifos and they decreased with the increasing the contact time. The results indicated that the sensitivity of AChE based bioassay can be improved by increasing the time of incubation, but this comes at the expense of additional analysis time. In addition, the inhibition parameters of chlorpyrifos induced inhibition of AChE were determined.Physical chemistry 2008 : 9th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 200

Aggressive intraabdominal fibromatosis

Background. Intraabdominal or mesenteric fibromatosis is a rare benign nonmetastatic neoplasm that appears as a sporadic lesion or in patients with familiar polyposis. Its evolution is unpredictable. About 10% of the cases show a very aggressive growth. Case report. We presented a 22-year-old woman in whom an aggressive intraabdominal fibromatosis had appeared during the first pregnancy as a well circumscribed ovoid tumor, involving the terminal ileum, the caecum, the ascending colon, the right kidney, the ureter, and the right common iliac artery. The tumor was excised with right colectomy, nephroureterectomy and resection of the involved artery using arterial reconstruction with graft interposition. Two years after the surgery the patient developed an inoperable tumor recurrence with a fatal outcome. Conclusion. In spite of a successful surgical excision during the original surgery intraabdominal or mesenteric fibromatosis might have an aggressive evolution leading to an inoperable tumor recurrency and a fatal outcome

Interaction of some polyoxotunstates with acetylcholinesterase

Polyoxometalates (POMs) are polyanionic oligomeric aggregates of transition metal ions, such as tungsten, molybdenum, vanadium, etc. held together by oxygen bridges, with a high density of negative charge. They are relatively stable, some even highly stable in aqueous solutions at biological pH values. In addition to applications in catalysis, separations, analysis, and as electrondense imaging agents, some of these complexes have been shown to exhibit biological activity in vitro as well as in vivo ranging from anti-cancer, antibiotic, and antiviral to antidiabetic effects. Recent investigations reported some polyoxotungstates as reversible inhibitors of acetylcholinesterase (AChE), making them potential anti-Alzheimer’s drugs. AChE is a serine hydrolase mainly found at neuromuscular junctions and cholinergic brain synapses. Its principal biological role is the termination of impulse transmission at cholinergic synapses. Reversible inhibitors of AChE mostly have therapeutic applications, while toxic effects are associated with irreversible AChE activity modulators. Reversible inhibitors play an important role in the pharmacological manipulation of the enzyme activity, and have been applied in the diagnostic and/or treatment of various diseases such as: myasthenia gravis, AD, postoperative ileus, bladder distention, glaucoma, as well as antidote to anticholinergic overdose. The effect of four new synthesized polyoxotungstates soluble in water on AChE activity was studied. AChE is purified from electric eel and commercially available. The enzyme was treated in vitro with polyoxotungstates in the concentration range from 1 × 10-7 to 1 × 10-3 mol/L at 37ºC for 15 minutes, and the incubation time was 12 min. The obtained dependence remaining enzyme activity vs. the inhibitor concentration fitted the sigmoidal function. IC50values, indicating the enzyme sensitivity toward the inhibitor and the inhibitory capacity of the analyzed compounds, were determined from the inhibition sigmoidal curves. Na10[H2W12O42] × 27H2O did not markedly reduce AChE activity at the highest investigated concentration (1 mmol/L). K7[SiV3W9O40] × 10H2O exhibited a weak inhibitory potential, causing 50% decrease in the enzyme activity at 5 × 10-4 mol/L. However, AChE sensitivity in the presence of K7[Ti2PW10O40] was several hundred times higher, reaching IC50 at 1.15 × 10-6 mol/L. Furthermore, (NH4)14[NaP5W30O110] × 31H2O demonstrated the strongest capacity to inhibit AChE. In the presence of its low concentration of 2 × 10-8 mol/L, the enzyme activity was noticeably reduced related to the control value (obtained without inhibitor), while 50% decrease in AChE activity was achieved at 3.8 × 10-7 mol/L.Fourth International Conferenceon Radiation and Applications in Various Fields of Research, RAD 2016, May 23-27, 2016, Niš, Serbi

Stanje oralnog zdravlja dece obolele od nasledne distrofičke bulozne epidermolize

Background/Aim. Epidermolysis bullosa is a group of rare, genetic connective tissue diseases that cause blisters in the skin and mucosal membranes. The aim of this study was to evaluate the oral health status of patients with epidermolysis bullosa dystrophica and level of knowledge and opinion of parents about the implementation of preventive measures and quality of dental care of affected children. Methods. This study included a group of 17 patients from Serbia suffering from dystrophic epidermolysis bullosa and matched control group. Dental caries status was assessed using the Klein-Palmer index. Oral hygiene status was verified with oral hygiene indices, simplified plaque index, and calculus index as described by Green and Vermillion. The gingiva was assessed as healthy or inflamed (gingivitis) on the basis of any changes in color, shape, size and surface texture. The condition of oral mucosa has been registered on the basis of inspection of the lips, tongue, a floor of the mouth, mouth vestibule and palate. The level of knowledge and the impressions of parents about the application of preventive measures were investigated through two questionnaires specifically designed for this study. Results. In both dentitions, there was the highest percentage of caries teeth. In primary dentition average value of the modified plaque index was 1.4 ± 1.14 and modified calculus was 0.7 ± 1. On permanent teeth average plaque index was 2 ± 0.4, and average calculus 1.6 ± 0.6. Statistically, significant higher values were found in permanent dentition in percentage distribution of decayed, missing, filled teeth and also for plaque and calculus indices between affected children and the control group. Most common findings on mucosa were microstomia (76.5%) and ankyloglossia (88.2%). Conclusion. The absence of protocol between the treating physician and the dentist and not sufficiently informed parents are leading to inadequate dental care. The implementation of preventive measures is of most importance to decrease the risk of severe complications that are difficult to be managed.Uvod/Cilj. Bulozna epidermoliza je grupa retkih, genetski predisponiranih bolesti vezivnog tkiva koja se karakteriše formiranjem bula (mehurova) u koži i mukoznim membranama. Cilj rada bio je da se ispita stanje oralnog zdravlja dece obolele od distrofične bulozne epidermolize, kao i nivo znanja i mišljenje roditelja o primeni preventivnih mera i kvalitetu stomatološke zaštite obolele dece. Metode. U studiju je bilo uključeno 17 bolesnika sa distrofičnom buloznom epidermolizom iz Srbije i odgovarajuća kontrolna grupa. Stanje zuba ustanovljeno je Klein Palmerovim indeksom karijesnih, ekstrakovanih plombiranih zuba - KEP indeksom. Stanje oralne higijene analizirano je indeksima oralne higijene (pojednostavljeni plak indeks i indeks zubnog kamenca po Green-Vermillionu). Stanje gingive je na osnovu inspekcije ocenjivano kao zdrava gingiva ili gingivitis, u zavisnosti od toga da li su joj bili promenjeni boja, oblik, veličina ili struktura. Stanje oralne sluzokože evidentirano je na osnovu inspekcije usana, jezika, poda usne duplje, vestibuluma, plika i frenuluma, kao i mekog i tvrdog nepca. Nivo znanja i utisci roditelja o primeni preventivnih mera i kvalitetu stomatološke zaštite dece obolele od bolozne epidermolize, ispitivani su kroz dva upitnika specijalno dizajnirana za potrebe ove studije. Rezultati. U obe denticije bilo je najviše karijesno obolelih zuba. Za mlečnu denticiju prosečan modifikovani plak indeks iznosio je 1,4 ± 1,14, a modifikovani kalkulus indeks 0,7 ± 1. U stalnoj denticiji vrednosti prosečnog plak indesa i kalkulus indeksa redom su bile 2 ± 0,4 i 1,6 ± 0,6. Utvrđena je statistički značajna razlika u procentualnoj zastupljenosti karijesnih, ekstrahovanih i plombiranih zuba stalne denticije između dece obolele od bulozne epidermolize i kontrolne grupe, kao i za vrednosti plak i kalkulus indeksa. Najčešće promene oralne sluzokože bile su mikrostomija (76,5%) i ankiloglosija (88,2%). Zaključak. Odsustvo protokolarne saradnje između ordinirajućeg lekara i stomatologa, kao i nedovoljna informisanost roditelja, glavni su razlozi lošeg stanja oralnog zdravlja. Primena preventivnih mera je od najvećeg značaja kako bi se sprečio nastanak komplikacija koje je teško sanirati kod ove grupe bolesnika

Enzymes as a platform for drug development

Polyoxometalates are negatively charged polyanions containing early transition metal ions in their high oxidation state surrounded by bridged oxygen. Firstly, these metal-based clusters were used as promising agents in electron-dense imaging, separations, catalysis, and analysis. In recent years, numerous studies in vitro and in vivo found that these nanocomplexes possess a variety of biological effects including antidiabetic, anticancer, and antibiotic actions. Despite these observed properties, the mechanism of their biological activities has not been completely elucidated so far. On the other hand, the results of enzymatic studies revealed their inhibiting influence on physiologically important extracellular enzymes such as phosphatases, esterases, and ecto-nucleotidases, which are considered target enzymes for the approved biological actions. Accordingly, the overview of the in vitro influence of selected polyoxo-vanadates, -tungstates, and – palladates on cholinesterase, ATPase, and phosphatase activities will be given in this presentation. Cholinesterases, enzymes located on the postsynaptic plasma membrane, have a key role in nerve impulse transmission and were confirmed as the targets of drugs for neurological diseases, which are regularly used in clinical practice. Moreover, ATPases and phosphatases were found to be included in the proliferation and migration of tumor cells, thus the inhibition of these enzymes was found as the mechanism of some anticancer drug actionSimpozijum „Stremljenja i novine u medicini“ Medicinskog fakulteta u Beogradu, Beograd, 04-08. decembra, 2023

The influence of diazinon and its metabolites on acetylcholinesterase, NA+/K+-ATPase and antioxidant enzymes in rat brain synaptosomes

The aim of this study was to investigate neurotoxic potential and oxidative stress responses of diazinon and its metabolites, diazoxon and 2-isopropyl-6-methyl-4-pyrimidinol using synaptosomes as a model system. Synaptosomes were isolated from the brain of Wistar albino rats and incubated at 37oC for 1 hour in the presence of selected concentrations of the investigated compounds. Acetylcholinesterase, Na+/K+-ATPase and antioxidant enzymes activities were determined by standard spectrophotometric methods. Diazinon induced concentration-dependent acetylcholinesterase and Na+/K+-ATPase inhibition, while the activity of catalase, superoxide dismutase and glutathione peroxidase was not significantly affected. Increasing concentrations of diazoxon, oxo analog of diazinon, caused almost complete acetylcholinesterase and Na+/K+-ATPase inhibition, and activated antioxidant enzymes: catalase (up to 25%), superoxide dismutase (up to 55%) and glutathione peroxidase (up to 30%). Unlike diazoxon, diazinon hydrolysis product, 2-isopropyl-6-methyl-4-pyrimidinol did not remarkably change the activities of the investigated enzymes, except superoxide dismutase that was stimulated up to 25%. The obtained results suggest that neurotoxic and prooxidative potential of diazinon, thioorganophoshate used as a commercial insecticide preparation, significantly reinforces mostly due to its transformation to diazoxon in the metabolic pathways.Third International Conferenceon Radiation and Applications in Various Fields of Research, RAD 2015, June8-12, 2015, Budva, Montenegr

Toksikološka ispitivanja tokom razvijanja novih lekova i medicinskih sredstava

Toksikološka ispitivanja predstavljaju osnovu prekliničkih ispitivanja novih lekova i medicinskih sredstava. Ovaj tip studija može predstavljati presudni korak u procesu procene daljeg razvoja lekova i medicinskih sredstava. Svedoci smo sve većeg interesovanja industrije i šire akademske zajednice za ovaj tip istraživanja, pa je upoznavanje sa vodičima za sprovođenje ovih studija, potpomognuto primerima iz prakse od ključnog značaja za njihovu edukaciju. Uzimajući sve ovo u obzir neophodno je da šira akademska zajednica bude bolje upoznata sa značajem ovih studija i pravilnim načinom njihovog sprovođenja. Od velikog je značaja upoznati zainteresovane strane o načinu sprovođenja toksikoloških studija, pre svega sa zakonskim okvirima koji se moraju poštovati u toku njihovog sprovođenja. Neophodno je zainteresovanim stranama iz šire akademske zajednice i industrije ukazati na etičke izazove na koje se nailazi tokom sprovođenja jedne toksikološke studije, pogotovu ako se u obzir uzme koliko su ovakva ispitivanja sa etičkog aspekta osetljiva. Postoji i potreba da se razjasni kako ispitivanje različitih toksikoloških aspekata zahteva i različite pristupe u sprovođenju ovih studija. Tako se npr. međusobno se razlikuju studije koje ispituju akutnu ili hroničnu toksičnost, studije u kojima se ispituju različiti načini primene (per os, intravenski,...) lekova i medicinskih sredstva i sl. Sve ove informacije bi trebalo da razjasne zainteresovanim stranama kako pravilno sprovesti toksikološke studije i kako iz njih dobiti relevantne rezultate, a da se pri tome ne krše zakonske odredbe niti etičke norme.Simpozijum „Stremljenja i novine u medicini“ Medicinskog fakulteta u Beogradu, Beograd, 04-08. decembra, 2023

Application of ultra performance liquid chromatography (uplc) for determination of Na+ /K+ atpase activity

An ion-pair ultra performance liquid chromatography (IP-UPLC) method was developed to obtain a sensitive and efficient means for quantification of ADP in order to follow the decrease of Na+ /K+ ATPase activity after its exposure to different inhibitors. The concentrations of ADP obtained after hydrolysis of ATP in the presence of enzyme depends on enzyme activity. Simultaneously with the chromatographic determination of ADP, the spectrophotometric determination of phosphates liberated after the hydrolysis of ATP was done.Physical chemistry 2008 : 9th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 200

Methionine versus homocysteine: future directions in cardiovascular research

L-methionine, the principal sulfur-containing amino acid in proteins, has important roles in cell physiology as an antioxidant, and in the breakdown of fats and heavy metals. Methionine is the precursor of homocysteine, and participates in the methylation and transsulfuration pathways. Elevated total plasma homocysteine (hyperhomocysteinemia) is associated with atherosclerosis, thromboembolic disease and cancer. Whether homocysteine per se or a coincident metabolic abnormality (homocysteine-related compounds, thiolactone metabolites) causes vascular disease is still an open question. Animals with genetic hyperhomocysteinemia have so far not displayed atheromatous lesions. However, when methionine-rich diets are used to induce hyperhomocysteinemia, vascular pathology is often observed. Such studies have not distinguished the effects of excess dietary methionine from those of hyperhomocysteinemia. It is known that high methionine diet, not only red meat for example lamb, beef, pork but also chicken meat can induce cardiovascular dysfunction but the mechanisms are unclear. It has been hypothesized that a diet rich in methionine can malfunction the cardiovascular system in three ways: (1) by augmenting oxidative stress, (2) by inflammatory manifestations, and (3) by matrix/vascular remodeling. However, some evidence indicates that an excess of methionine can be harmful for other systems, and can increase the risk of developing type-2 diabetes, certain types of cancer, brain alterations such as schizophrenia, and memory impairment. However this is still controversial because previous studies suggesting the use of L-methionine as a treatment for depression and other diseases indicate that it might also improve memory (role in brain function). Thus, the direction of our research is to further elucidate mechanisms of cardiovascular and neural effects of homocysteine vs. methionine overload.HISPA 2017 : HISPA TRILATERAL "Cardiovascular challenges on Balkan roads crossing" : Abstracts : September 15-17, 2017, Zlatibor