Papers by Ivanka Markovic
Applied Sciences
An efficient transfection is a crucial step for the introduction of epigenetic modification in ho... more An efficient transfection is a crucial step for the introduction of epigenetic modification in host cells, and there is a need for an optimized transfection process for individual model systems separately. Mouse pancreatic αTC1-6 cells, which act as an attractive model system for epigenetic cell reprogramming and diabetes treatment, were transiently transfected with two different transfection methods: the chemical method with polyethyleneimine (PEI) and nucleofection as a physical transfection method. Flow cytometry and fluorescent microscopy examination of GFP expression showed that transfection efficiency was affected by the size of plasmids using both transfection methods. Subsequently, the Cas9 mRNA expression confirmed successful transfection with EpiCRISPR plasmid, whereas the cell physiology remained unchanged. The adjusted nucleofection protocol for αTC1-6 cells transfected with an EpiCRISPR mix of plasmids reached 71.1% of GFP-positive transfected cells on the fifth post-tr...
Medicinski podmladak, 2019
Parkinson's disease (PD) is defined as a chronic neurodegenerative disorder with a gradual demise... more Parkinson's disease (PD) is defined as a chronic neurodegenerative disorder with a gradual demise of dopaminergic (DA) neurons in substantia nigra pars compacta. Although molecular mechanisms of DA neurons' degeneration are still insufficiently understood, different studies based on animal, toxic and genetic models, as well as post-mortem brain tissue analyses revealed several common features of neuronal death in SN: mitochondrial dysfunction, increased ROS production and impaired proteostasis. Each of these mechanisms might be, at least in part, related to adenosine monophosphate-activated protein kinase (AMPK). The AMPK is a major intracellular energy sensor. It can be activated by different types of metabolic stress, mediated by at least three different kinases LKB1, CAMKKβ, and TAK1, in conditions of decreased cellular ATP and/or increased Ca 2+ level. Once activated, AMPK promotes catabolic pathways, inhibits mTORC1, and stimulates autophagy, mitochondrial biogenesis, and turnover. Different studies revealed a growing body of evidence suggesting an important role of AMPK in PD. Many of them have documented the protective effect of AMPK activation in different PD models by facilitating mitochondrial quality control, enhancing autophagic clearance of defective mitochondria and protein aggregates. However, some studies have shown the detrimental effect of AMPK activation in DA neurons in advanced stages of neuronal damage, where prolonged activation could inhibit protein synthesis and impair synaptic integrity and plasticity. In this review, we will try to summarize the literature data regarding the role of AMPK in PD pathogenesis.
SIDERITIS SCARDICA Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, ha... more SIDERITIS SCARDICA Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and N-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and N-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.
Psychiatry Research, 2021
Transforming growth factor beta (TGF-β), which has a role as a regulatory cytokine, has not been ... more Transforming growth factor beta (TGF-β), which has a role as a regulatory cytokine, has not been widely investigated in patients with major depressive disorder (MDD) who experienced childhood trauma. The aim of our study was to investigate the differences in circulating TGF-β levels between the patients with major depressive disorder (MDD) with and without child maltreatment (CM) history, and to compare them to the corresponding control subjects' groups (with or without CM). Blood samples were obtained from 55 patients, fulfilling DSM-IV-R criteria for a current MDD episode without psychotic symptoms, and 45 healthy controls, matched for age and gender. Participants were administered the Childhood Trauma Questionnaire (CTQ). Serum TGF-β concentration was determined by enzyme-linked immunosorbent assay. The concentration of TGF-β was significantly higher in patients with MDD with CM history, compared to MDD patients with no CM, as well as both control groups. Furthermore, we have shown that the combined effect of CM history and MDD affected TGF-β levels in adulthood, which was not observed in the control group with CM. These results indicate that MDD patients with the experience of CM have altered immune-regulatory response, and they may constitute a specific subtype within this heterogenic disorder (ecophenotype).
Psychiatry Research, 2018
Little is known about the correlation between IL-6 and childhood abuse and neglect which may be r... more Little is known about the correlation between IL-6 and childhood abuse and neglect which may be risk factors for the development of affective disorders in adulthood. The aim of this study was to analyze differences in serum concentrations of IL-6 between patients with major depressive disorder and healthy controls, and to investigate possible correlations with adverse childhood experiences. Peripheral venous blood samples were obtained from 64 patients who fulfilled DSM-IV-R criteria for a current major depressive episode without psychotic symptoms (MDD) and 53 healthy controls, matched for age and gender. Participants were assessed by the Beck Depression Inventory (BDI), Childhood Trauma Questionnaire (CTQ), Hamilton Depression Rating Scale (HDRS) and Hamilton Anxiety Rating Scale (HARS). The concentration of IL-6 was significantly higher in patients with major depressive disorder compared to healthy controls. The total score of childhood trauma questionnaire highly statistically significantly correlated with IL-6 levels in patient group. Persons who were physically abused, physically neglected and emotionally abused had higher levels of IL-6. Interleukin 6 as a pro-inflammatory immune marker could be an important developmental mediator linking physical and emotional abuse in early life with the development of depressive disorder in adulthood.
Melanoma research, Feb 1, 2018
Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortal... more Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O,O-diethyl-(S,S)-ethylenediamine-N,N'di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for invest...
Chemical biology & drug design, 2017
This study presents the synthesis, characterization, and antitumor action of five new Pt(II) halo... more This study presents the synthesis, characterization, and antitumor action of five new Pt(II) halogenido, chlorido, and iodido complexes with edda type of ligands. (S,S)-Ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoic acid dihydrochloride and its methyl, ethyl, and n-propyl esters were prepared according to the previously reported procedure. All investigated complexes were characterized by IR, ESI-MS ((1) H, (13) C, and HMBC) NMR spectroscopy, and elemental analysis. Their cytotoxic action was investigated in four human tumor cell lines: promyelocytic (HL-60) and lymphocytic (REH) leukemia, glioma (U251), and lung carcinoma (H460). Cell viability was assessed by acid phosphatase and LDH assay, while oxidative stress and cell death parameters were analyzed by flow cytometry. The results showed that novel Pt(II) complexes exhibited antitumor action superior to precursor ligands, with iodido complexes being more efficient than corresponding chlorido complexes. Human promyelocytic ...
Experimental Cell Research, 2017
suppression sensitizes glioma cells to IMP dehydrogenase inhibition-induced apoptotic death, Expe... more suppression sensitizes glioma cells to IMP dehydrogenase inhibition-induced apoptotic death, Experimental Cell Research,
Journal of neurology, 2016
Both homo- (causing autosomal-recessive Gaucher's disease; GD) and heterozygous mutations in ... more Both homo- (causing autosomal-recessive Gaucher's disease; GD) and heterozygous mutations in the glucocerebrosidase gene (GBA) are associated with Parkinson's disease (PD), and represent the most robust known genetic susceptibility factors identified in PD. Since the accumulation of α-synuclein has been considered critical to the pathogenesis of PD among several possible pathways through which glucocerebrosidase (GCase) deficiency may promote the pathogenesis of PD, particular attention was given to the reciprocity with α-synuclein levels, lysosomal dysfunction, endoplasmatic reticulum-Golgi trafficking of GCase, dysregulation of calcium homeostasis and mitochondrial abnormalities. The proportion of PD patients that carry GBA mutations is estimated to be approximately between 5 and 10 %. Individual PD patients with or without GBA mutations cannot be discriminated on clinical or pathological grounds. However, GBA mutation carriers may have slightly earlier age at PD onset, mo...
Advances in Behavioral Biology, 1996
Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2016
Phencyclidine (PCP) acts as a non-competitive antagonist of glutamatergic N-methyl-D-aspartate re... more Phencyclidine (PCP) acts as a non-competitive antagonist of glutamatergic N-methyl-D-aspartate receptor. Its perinatal administration to rats causes pathophysiological changes that mimick some pathological features of schizophrenia (SCH). Numerous data indicate that abnormalities in mitochondrial structure and function could be associated with the development of SCH. Mitochondrial dysfunction could result in the activation of apoptosis and/or autophagy. The aim of this study was to assess immediate and long-term effects of perinatal PCP administration and acute restraint stress on the activity of respiratory chain enzymes, expression of apoptosis and autophagy markers and ultrastructural changes in the cortex and hippocampus of the rat brain. Six groups of rats were subcutaneously treated on 2nd, 6th, 9th and 12th postnatal day (P), with either PCP (10mg/kg) or saline (0,9% NaCl). One NaCl and one PCP group were sacrificed on P13, while other two NaCl and PCP groups were sacrificed on P70. The remaining two NaCl and PCP groups were subjected to 1h restraint stress prior sacrifice on P70. Activities of respiratory chain enzymes were assessed spectrophotometrically. Expression of caspase 3 and AIF as markers of apoptosis and Beclin 1, p62 and LC3, as autophagy markers, was assessed by Western blot. Morphological changes of cortical and hippocampal ultrastructure were determined by transmission electron microscopy. Immediate effects of perinatal PCP administration at P13 were increased activities of complex I in the hippocampus and cytochrome c oxidase (COX) in the cortex and hippocampus implying mitochondrial dysfunction. These changes were followed by increased expression of apoptotic markers. However the measurement of autophagy markers at this time point has revealed decrease of this process in cortex and the absence of changes in hippocampus. At P70 the activity of complex I was unchanged while COX activity was significantly decreased in cortex and increased in the hippocampus. Expressions of apoptotic markers were still significantly higher in PCP perinatally treated rats in all investigated structures, but the changes of autophagy markers have indicated increased level of autophagy also in both structures. Restraint stress on P70 has caused increase of COX activity both in NaCl and PCP perinatally treated rats, but this increase was lower in PCP group. Also, restraint stress resulted in decrease of apoptotic and increase of autophagy processes especially in the hippocampus of PCP perinatally treated group. The presence of apoptosis and autophagy in the brain was confirmed by transmission electron microscopy. In this study we have demonstrated for the first time the presence of autophagy in PCP model of SCH. Also, we have shown increased sensitivity of PCP perinatally treated rats to restraint stress, manifested in alterations of apoptotic and autophagy markers. The future studies are necessary to elucidate the role of mitochondria in the pathophysiology of SCH and putative significance for development of novel therapeutic strategies.
European Neuropsychopharmacology, 2014
Background: Phencyclidine (PCP) is non-competitive antagonist of N-methyl-D-aspartate (NMDA) rece... more Background: Phencyclidine (PCP) is non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors [1]. Perinatal PCP administration to rodents represents one of the more compelling animal models of schizophrenia. There is evidence that oxidative stress mediated through free radicals in the central nervous system is involved in the pathophysiology of this disease [2]. Still, immediate functional changes of astroglial cells under PCP influence remain unknown. Aim: Establishing the astrocytes cell culture from brains of 7 days old Wistar rats treated with saline (control group) or PCP (PCP group). Determine the presence of oxidative stress, changes in the acidic cytoplasmic content, caspase activation and astrocytes' cell cycle. Materials and Methods: Wistar rats were treated on the 2 nd and 6 th postnatal day (PD) with either phencyclidine (10 mg/kg) or saline and sacrificed on 7PD. Astrocytes cultures were established from the obtained brains (8/group) according to the method developed by McCarthy and de Vellis [3]. At the same time astrocyte culture was established from brains of newborn rats in order to obtain conditioned medium. After approximately 3 weeks during which both culture groups were maintained by changing medium with fresh composed of new standard growth medium plus conditioned medium (2/3:1/3), astrocytes were detached by trypsinization and analyzed by flow cytometry after staining with appropriate fluorochromes. Flow cytometric analysis of cells stained with appropriate fluorochromes was employed for measurement of superoxide production (DHE), mitochondrial membrane potential (JC-1), caspase activation (Apostat), DNA fragmentation (propidium iodide), and acidic cytoplasmic content (Acridine-orange). Results: No significant increase in superoxide production, determined by measuring the DHE-FL2 fluorescence intensity, was observed in astrocytes cultivated from PCP group, compared to astrocytes from control group. However, astrocytes obtained from PCP group showed mitochondrial membrane depolarization since FL1/FL2 ratio was 1.8 in PCP group in comparison with 1 in control. Furthermore, G1-Scell cycle arrest was detected in PCP group astrocytes. Nevertheless, postnatal treatment with PCP failed to induce caspase activation since no increase in Apostat-FL1 fluorescence intensity was detected in PCP group compared to control. This result suggests the absence of perinataly administrated PCP induced oxidative stress mediated apoptosis. Autophagy can play a pro-survival role in cells under the stress conditions, but if extensive, activated inappropriately or in cells which are unable to die by apoptosis, it can be the type of cell death. Our experimental results show the absence of significant difference in red fluorescence intensity originating from lysosomes between PCP-treated and control group implicating that autophagy does not play an important role in PCP-induced effects on astrocytes. Conclusion: Since the signaling between astrocytes and neurons at the tripartite synapse plays a significant role during formation of neural circuits, the changes observed in our investigation could be involved in the development of glioneurotransmission imbalance suggested in SCH.
Advances in Behavioral Biology, 1996
Transport of tiazofurin (2-β-D-ribofuranosyl thiazole-4-carboxamide) across the blood-brain barri... more Transport of tiazofurin (2-β-D-ribofuranosyl thiazole-4-carboxamide) across the blood-brain barrier (BBB) was studied using brain vascular perfusion method in the guinea pig. The obtained results demonstrate that brain clearance of 3H tiazofurin significantly differs from zero, suggesting that this molecule penetrates from blood into the brain. The values of tiazofurin brain clearance are very close to the values obtained for neuropeptides and other so called “slow penetrating molecules” (regarding the blood brain barrier). Addition of increasing concentrations of unlabelled tiazofurin to the perfusing medium caused a significant decrease in the uptake of [3H] labelled tiazofurin. Therefore, penetration of tiazofurin from blood into brain seems to be a saturable process. Presence of increasing concentrations of unlabelled adenosine has similar effect as the presence of unlabelled tiazofurin in the perfusing medium. However, it did not cause complete inhibition of tiazofurin brain uptake.
Mnoge ćelije u CNS-u preuzimaju nukleozide iz vanćelijske tečnosti i koriste ih za sintezu nukleo... more Mnoge ćelije u CNS-u preuzimaju nukleozide iz vanćelijske tečnosti i koriste ih za sintezu nukleotida u putevima uštede. Nukleozidi se kroz membranu astrocita transportuju putem dve genske familije nukleozidnih transportera: ekvilibrativnih (ENT1 i ENT2) koji omogućavaju jednosmernu olakšanu difuziju i koncentrativnih (CNT2) koji omogućavaju jednosmerni sekundarno aktivni transport kroz ćelijsku membranu. Cilj ovog istraživanja je bio ispitivanje ekspresije različitih klasa nukleozidnih transportera na primarno kultivisanim astrocitima pacova, kao i uticaj hronično povećane koncentracije adenozina u vanćelijskom medijumu na stepen njihove ekspresije. Eksperimenti su rađeni u primarnoj kulturi astrocita pacova soja Wistar. Ćelije su u toku 21 dana kontinuirano tretirane adenozinom u koncentracijama od 25µM i 100µM. Kvantifikacija genske ekspresije je vršena RT-qPCR analizom na svakih 7 dana. i izražena je kao ∆ΔCt vrednost u odnosu na vrednost koamplifikovanog β-aktina. Rezultati su ...
European Journal of Inorganic Chemistry, 2015
ABSTRACT Copper(II) complexes with the condensation derivative ofmethyl hydrazinoacetate and 2-ac... more ABSTRACT Copper(II) complexes with the condensation derivative ofmethyl hydrazinoacetate and 2-acetylpyridine were synthe-sized. The X-ray crystal structures for both complexes re-vealed that they are polymerized isomers. A common featureof both complexes is the bidentate coordination of the ligandby one hydrazone and one pyridine nitrogen atom. In themonomeric complex, the copper(II) center is tetracoordinate,whereas dimerization through chlorido bridges results in apentacoordinate arrangement about the metal ions in the di-mer. The electronic and magnetic properties of both com-plexes are discussed on the basis of their X-ray structures,electron paramagnetic resonance (EPR) spectroscopy studies,and superconducting quantum interference device (SQUID)magnetization measurements combined with DFT calcula-tions. Magnetostructural comparisons with structurally sim-ilar copper(II) complexes are also provided, and a possiblecorrelation has been established. The antitumor activities ofthe CuIIcomplexes were investigated against six differentcancer cell lines, and the results suggest that the antigliomaaction of the dimeric species is based on oxidative-stress-me-diated phosphatidylserine externalization and caspase acti-vation, which indicate apoptosis. http://onlinelibrary.wiley.com/doi/10.1002/ejic.201500349/pdf
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Papers by Ivanka Markovic