Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones

SOX2, a universal marker of pluripotent stem cells, is a transcription factor that helps control embryonic development in vertebrates; its expression persists in neural stem/progenitor cells into adulthood. Considering the critical role of the SOX2 transcription factor in the regulation of genes required for self-renewal and pluripotency of stem cells, we developed and characterized SOX2-overexpressing NT2/D1 cell clones. Using Southern blot and semi-quantitative RT-PCR, we confirmed integration and expression of exogenous SOX2 in three NT2/D1 cell clones. Overexpression of the SOX2 gene was detected in two of these clones. SOX2 overexpression in NT2/D1 cell clones resulted in altered expression of key pluripotency genes OCT4 and NANOG. Furthermore, SOX2-overexpressing NT2/D1 cell clones entered into retinoic acid-dependent neural differentiation, even when there was elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of neural markers (neuroD1 and synaptophysin) was higher in induced cell clones than in induced parental cells. The cell clone with SOX2 overexpression had an approximately 1.3-fold higher growth rate compared to parental cells. SOX2 overexpression did not increase the population of cells undergoing apoptosis. Taken together, we developed two SOX2-overexpressing cell clones, with constitutive SOX2 expression after three weeks of retinoic acid treatment. SOX2 overexpression resulted in altered expression of pluripotency-related genes, increased proliferation, and altered expression of neural markers after three weeks of retinoic acid treatment

Hemijski aspekt uticaja jona kobalta na aktivnost ATP-aza

The influence of Co2+ ions on the activities of Na+/K+-ATPase and Mg2+-ATPase, enzymes from rat brain synaptic plasma membrane, was studied. The aim of this study was to investigate the inhibition of both ATPases activities by exposure to cobalt ions as a function of experimentally added CoSO4. The "free" Co2+ concentrations in the reaction mixture were also calculated and discussed. CoSO4 induced a dose-dependent inhibition of both enzymes. The IC50 values of Co2+, as calculated from the experimental curves, were 168 mu M for Na+/K+-ATPase and 262 mu M for Mg2+-ATPase, and for the recalculated free Co2+ concentration 75.4 mu M for Na+/K+-ATPase and 136 mu M for Mg2+-ATPase. The obtained linear Dixon's plot for Na+/K+-ATPase implies equilibrium binding of cobalt with inhibitory sites on the enzyme. The kinetic parameters for both enzymes in presence and absence of CoSO4 were calculated from the experimental data. The results of the kinetic analysis show that inhibition of Na+/K+-ATPase induced by CoSO4 is non-competitive, and for Mg2+-ATPAse that there are two sites of different sensitivities or two different enzymes.Ispitan je uticaj Co2+ jona na aktivnost Na+/K+ -ATPaze i Mg2+ -ATP-aze, enzima sinaptozomalne membrane mozga pacova. Cilj rada je bio da se ispita inhibicija aktivnosti oba enzima izazvana izlaganjem jonima kobalta kao funkcija eksperimentalno dodatog CoSO4. Takođe je izračunata i diskutovana "slobodna" koncentracija Co2+ u reakcionoj smeši. Utvrđeno je da kobalt inhibira enzime u koncentraciono zavisnom smislu. Vrednosti IC50 izračunate iz eksperimentalnih krivih su: 168 μM za Na+/K+ -ATP-azu i 136 μM za Mg2+ -ATP-azu. Linearan Dixon-ov plot za Na+/K+ -ATP-azu ukazuje na ravnotežno vezivanje kobalta. Izračunati su kinetički parametri oba enzima u prisustvu i odsustvu CoSO4. Inhibicija Na+/K+-ATP-aze izazvana CoSO4 je nekompetitivna, dok Mg2+ -ATP-aza ima dva mesta vezivanja različitog afiniteta ili pak dva različita enzima

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

Vasić, V. et al. Na+,K+-ATPase as the Target Enzyme for Organic and Inorganic Compounds. Sensors 2008, 8, 8321-8360

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Hyperbaric oxygenation as an adjuvant therapy for traumatic brain injury: a review of literature

In recent years significant amount of data have been published in the filed of hyperbaric oxygenation (HBO) and traumatic brain injury (TBI). The main rational for the research in this field is that in TBI patients with the existence of dormant neural tissues that maintain cellular homeostasis but are unable to participate in neurotransmission, the addition of HBO provides a favourable environment by which neuronal reactivation can be achieved. As hyperbaric oxygen therapy is not all-or-nothing phenomena and the consequences of TBI can vary from mild to moderate and severe, it is important to evaluate each TBI patient before referring him/her to hyperbaric oxygen therapy (HBOT). Reports from the clinical trial that were investigating the effects of HBO on severe TBI show promising results. For example, significant reduction in mortality rates and improvement in favourable neurological outcomes were reported. However, conflicting results have been reported from trials that investigated the effects of HBO on mild and moderate TBI. The results from the experimental studies indicate that HBO can preserve mitochondrial function, reduce apoptosis and neuroinflammation and promote neuronal plasticity. Therefore, conducting of methodologically-based multicentric clinical trials is necessary to determine proper guidelines for inclusion of TBI patients in HBOT. As many reports have stated that even a few exposures to HBO can contribute to the recovery process, future research must be aimed at establishing most effective HBO protocol for TBI patients

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

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

Temperature and Al3+ influence on electrophoretic mobility of porcine pepsin

The influence of temperature and different concentrations of Al3+ on pepsin electrophoretic mobility was investigated. The increase of Al3+ concentrations causes the decrease the electrophoretic mobility of enzyme. Also the increase of temperature induced the same effect. The influence of both temperature and Al3+ ion concentrations is additive.Physical chemistry 2006 : 8th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 26-29 September 200

Kućne posete - podrška deci sa smetnjama u razvoju i njihovim porodicama

Kućne posete porodici od strane profesionalaca različitih profila su prepoznata domaća i međunarodna praksa u radu sa porodicama dece sa smetnjama u razvoju. Međutim, u prethodnom periodu uočavamo da se ne praktikuju u okviru Predškolske ustanove Čukarica. Razloge tome možemo tražiti, između ostalog, i u za nas osetljivom pitanju postavljanja formalnog odnosa profesionalaca i roditelja, koji je posetama porodičnoj sredini naročito doveden u pitanje. Kućne posete su kao oblik rada profesionalaca u Predškolskoj ustanovi Čukarica razvijane u sklopu projekta „Vrtići bez granica 2“, nakon čega su horizontalnom razmenom došle i do naše razvojne grupe. Prepoznali smo značaj ovog oblika rada, koji nam otvara put ka autentičnoj slici snaga i izazova porodice, u njenom realnom kontekstu, i omogućava direktniji put intervencije i podrške. Preispitali smo dileme oko profesionalne granice u ovom načinu rada. Istraživali smo prilike u kojima porodici možemo pristupiti u njenom realnom kontekstu, pre svega timskim definisanjem ciljeva, zadataka, trajanja poseta, prepoznavanjem profesionalaca koji ih realizuju, kao i dobiti za porodicu. Naročito kada se radi o roditeljima dece sa smetnjama u razvoju imamo situaciju da profesionalci uglavnom nisu značajnije posvetili pažnju razgovoru sa njima, približavanju stanja njihovog deteta, razumevanju kako da mu pristupe, već se zadržavaju na medicinskoj terminologiji, izveštajima, uz kratkotrajne preglede ili kontrole. S obzirom na to da predškolska ustanova uključuje decu uzrasta do 6 godina, kada se teme vezane za razvojne teškoće deteta tek otvaraju u porodici, smatramo da je sem rane stimulacije dece, krucijalan upravo naš rad sa roditeljima, razrešenje njihovih izazova i podrška koju im u datom momentu pružamo. Ukoliko kao opšti cilj postavimo kvalitetniju brigu o deci i partnerski odnos sa roditeljima, ovaj oblik rada nudi nam optimalnu mogućnost približavanja tim ciljevima. U narednom periodu, značajan je dijalog svih profesionalaca iz predškolskih ustanova o mogućnostima realizovanja kućnih poseta porodicama