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Hyssopus officinalisL. is a well-known aromatic plant used in traditional medicine and the foodand cosmetics industry. The aim of this study is to assess the antioxidant, genotoxic, antigenotoxic andcytotoxic properties of characterized hyssop essential oils and methanol extracts. Chemical compositionwas analyzed by gas chromatography - mass spectrometry (GC-MS) and liquid chromatography with diodearray detection and mass spectrometry (LC-DAD-MS), respectively. Antioxidant activity was examinedby 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) tests; genotoxicand antigenotoxic activity were examined by the comet assay, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) test against tumor cell lines (SW480,MDA-MB 231, HeLa) and non-transformed human lung fibroblast cell lines (MRC-5). The essential oilswere rich in monoterpene hydrocarbons (e.g., limonene; 7.99–23.81%), oxygenated monoterpenes (1,8-cineole; 38.19–67.1%) and phenylpropanoids (methyl eugenol; 0.00–28.33%). In methanol extracts, the mostabundant phenolics were chlorogenic and rosmarinic acid (23.35–33.46 and 3.53–17.98 mg/g, respectively).Methanol extracts expressed moderate to weak antioxidant activity (DPPH IC50= 56.04–199.89μg/mL,FRAP = 0.667–0.959 mmol Fe2+/g). Hyssop preparations significantly reduced DNA damage in humanwhole blood cells, induced by pretreatment with hydrogen peroxide. Methanol extracts exhibited selectiveand potent dose- and time-dependent activity against the HeLa cell line. Results of the current studydemonstrated notableH.officinalismedicinal potential, which calls for further investigation.

Aim: Newly synthesized platinum(IV) complexes with ethylenediamine-N,N’-diacetate ligands (EDDA-type) (butyl-Pt and pentyl-Pt) were investigated against two cancer (A549 lung, and HTB 140 melanoma) and one non-cancerous (MRC-5 embryonic lung fibroblast) human cell lines. Materials and Methods: The effects of these agents were compared with those of cisplatin after 6-, 24- and 48-h treatment. Sulforhodamine-B (SRB) assay was performed to estimate the cytotoxic effect, while the inhibitory effect on cell proliferation was measured using 5-bromo-2,-deoxyuridine (BrdU) incorporation assay. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by these agents was determined by electrophoretic analysis of DNA, flow cytometry and by western blot analysis of proteins involved in induction of apoptosis. The effects of gamma irradiation, alone and in combination with platinum-based compounds, were examined by clonogenic and SRB assays. Results: All examined platinum-based compounds had inhibitory and antiproliferative effects on A549 cells, but not on HTB140 and MRC-5 cells. Butyl-Pt, pentyl-Pt and cisplatin arrested the cell cycle in the S-phase and induced apoptotic cell death via regulation of expression of B-cell lymphoma 2 (BCL2) and BCL2-associated X (BAX) proteins. Platinum-based compounds increased the sensitivity of A549 cells to gamma irradiation. Butyl-Pt and pentyl-Pt showed better antitumour effects against A549 cells than did cisplatin, by interfering in cell proliferation and the cell cycle, and by triggering apoptosis. Conclusion: The effects of gamma irradiation on tumour cells may be amplified by pre-treatment of cells with platinum-based compounds.

Aim: Newly synthesized platinum(IV) complexes with ethylenediamine-N,N’-diacetate ligands (EDDA-type) (butyl-Pt and pentyl-Pt) were investigated against two cancer (A549 lung, and HTB 140 melanoma) and one non-cancerous (MRC-5 embryonic lung fibroblast) human cell lines. Materials and Methods: The effects of these agents were compared with those of cisplatin after 6-, 24- and 48-h treatment. Sulforhodamine-B (SRB) assay was performed to estimate the cytotoxic effect, while the inhibitory effect on cell proliferation was measured using 5-bromo-2,-deoxyuridine (BrdU) incorporation assay. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by these agents was determined by electrophoretic analysis of DNA, flow cytometry and by western blot analysis of proteins involved in induction of apoptosis. The effects of gamma irradiation, alone and in combination with platinum-based compounds, were examined by clonogenic and SRB assays. Results: All examined platinum-based compounds had inhibitory and antiproliferative effects on A549 cells, but not on HTB140 and MRC-5 cells. Butyl-Pt, pentyl-Pt and cisplatin arrested the cell cycle in the S-phase and induced apoptotic cell death via regulation of expression of B-cell lymphoma 2 (BCL2) and BCL2-associated X (BAX) proteins. Platinum-based compounds increased the sensitivity of A549 cells to gamma irradiation. Butyl-Pt and pentyl-Pt showed better antitumour effects against A549 cells than did cisplatin, by interfering in cell proliferation and the cell cycle, and by triggering apoptosis. Conclusion: The effects of gamma irradiation on tumour cells may be amplified by pre-treatment of cells with platinum-based compounds.

Uvod/cilj U poslednjoj deceniji zabeleženo je povećanje rezistencije Grampozitivnih koka na antibiotike iz grupe makrolida, linkozamida i streptogramina A/B (MLSa/b). Složeni mehanizmi rezistencije na MLS antibiotike uključuju: modifikaciju ribozoma, aktivno izbacivanje antibiotika i modifikaciju antibiotika. Porodica gena odgovornih za kodiranje enzima koji vrše metilaciju ribozoma je označena skraćenicom erm (od engl. erythromycin resistance methylase) i dovode do pojave unakrsne rezistencije na makrolide, linkozamide i streptogramine grupe B (MLSb). Profil rezistencije poznat kao MLSb fenotip može biti: inducibilan (iMLSb) i konstitutivan (cMLSb). Sojevi sa erm posredovanom eritromicin rezistencijom mogu se pokazati osetljivim na klindamicin u rutinskom disk difuzionom testiranju osetljivosti. Ciljevi Utvrditi učestalost fenotipova i gena rezistencije na MLS antibiotike kod Gram-pozitivnih koka na području jugoistočne Srbije. Metode MLS fenotipovi rezistencije: konstitutivni (cMLSb), inducibilni (iMLSb), M/MSb i LSa/b, bili su utvrđeni dupli-disk difuzionom metodom (D-testom). Ispitano je 2366 kliničkih izolata Gram-pozitivnih koka, sakupljenih u toku jednogodišnjeg perioda u Centru za mikrobiologiju Zavoda za javno zdravlje Vranje. Geni koji kodiraju MLSb rezistenciju (ermA, ermB, ermC, msrA, msrB, mefA/E, lnuA, lnuB, lsaA i lsaC) su identifikovani korišćenjem multipleks PCR-a. Rezultati Najveću učestalost rezistencije na eritromicin imali su izolati meticilin-rezistentnih koagulaza-negativnih stafilokoka (MRCNS, 91,5%), zatim meticilin-rezistentnih S. aureus (MRSA, 86,4%) i enterokoka (80,8%). Niža, ali još uvek visoka stopa rezistencije na eritromicin je bila prisutna i kod drugih izolata Gram-pozitivnih koka. Nasuprot makrolidima, izuzev za izolate Enterococcus spp. (96,7%), Streptococcus pneumoniae (35,9%) i MRSA (28,4%), drugi izolati Grampozitivnih koka su pokazali značajno nižu rezistenciju na klindamicin. Otkrili smo da je iMLSb fenotip najučestaliji kod, Introduction The increase of resistance to macrolide, lincosamide, and streptogramin a/b antibiotics (MLSa/b) among Gram-positive cocci has been reported in the last decade. The complex mechanisms of resistance to MLS antibiotics include: modification of ribosome, active efflux of antibiotics and antibiotic inactivation. The gene family called erm (erythromycin resistance methylase) that encoding enzymes responsible for ribosome methylation leads to cross- resistance to macrolides, lincosamides and streptogramin b (MLSb resistance phenotype). The antimicrobial resistance profile known as MLSb resistance phenotype can be either inducible (iMLSb) and constitutive (cMLSb). Macrolide resistant strains carrying erm gene can show false sensitivity to clindamycin, using routine antimicrobial susceptibility test. Objective This study investigated the prevalence of MLS resistance phenotypes and genes in Gram-positive cocci in region of the Southeast Serbia. Methods MLS resistance phenotypes: constitutive (cMLSb), inducible (iMLSb), M/MSb, and LSa/b, were determined by the double-disk diffusion method. We have analyzed 2366 clinical isolates of Gram-positive cocci, collected during 1-year period at the Center for Microbiology of the Public Health Institute in Vranje, Serbia. The MLS resistance genes (ermA, ermB, ermC, msrA, msrB, mefA/E, lnuA, lnuB, lsaA, and lsaC) were identified by multiplex PCR. Results The highest frequencies of isolates resistant to erythromycin were found among methicillin-resistant coagulase-negative staphylococci (MRCNS, 91.5%), methicillin-resistant S. aureus (MRSA, 86.4%) and Enterococcus strains (80.8%). Lower, but still high resistance rate to erythromycin was found among other Gram-positive cocci. Contrary to macrolides, except for Enterococcus spp. (96.7%), S. pneumoniae (35.9%), and MRSA (28.4%), other Gram-positive cocci showed significantly lower resistance to clindamycin. We have found that iMLSb is the most prevalent phenotype in staphyloco

In order to make a progress in discovering a new agents for chemotherapy with improved properties and bearing in mind the fact that substituted 3-hydroxy-3-pyrrolin-2-ones belong to a class of biologically active compounds, series of novel 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones were synthesized and characterized by spectral (UV–Vis, IR, NMR, ESI-MS), X-ray and elemental analysis. All compounds were examined for their cytotoxic effect on human cancer cell lines HeLa and MDA-MB 231 and normal fibroblasts (MRC-5). Four compounds, 3-hydroxy-1-(p-tolyl)-4-(2-thienylcarbonyl)-5-(4-chlorophenyl)-2,5-dihydro-1H-pyrrol-2-one (D10), 3-hydroxy-1-(3-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D13), 3-hydroxy-1-(4-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D14), and 3-hydroxy-1-(4-chlorophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D15), that showed the highest cytotoxicity against malignant cells and the best selectivity towards normal cells were selected for further experiments. Results obtained by investigating mechanisms of cytotoxic activity suggest that selected 3-hydroxy-3-pyrrolin-2-one derivatives in HeLa cells induce apoptosis that is associated with S phase arrest (D13, D15, and D10) or unrelated to cell cycle distribution (D14). Additionally, to better understand their suitability for potential use as anticancer medicaments we studied the interactions between biomacromolecules (DNA or BSA) and D13 and D15. The results indicated that D13 and D15 have great affinity to displace EB from the EB-DNA complex through intercalation [K sv = (3.7 ± 0.1) and (3.4 ± 0.1) × 10 3 M −1 , respectively], an intercalative mode also confirmed through viscosity measurements. K a values, obtained as result of fluorescence titration of BSA with D13 and D15 [K a = (4.2 ± 0.2) and (2.6 ± 0.2) × 10 5 M, respectively], support the fact that a sign

In order to make a progress in discovering a new agents for chemotherapy with improved properties and bearing in mind the fact that substituted 3-hydroxy-3-pyrrolin-2-ones belong to a class of biologically active compounds, series of novel 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones were synthesized and characterized by spectral (UV–Vis, IR, NMR, ESI-MS), X-ray and elemental analysis. All compounds were examined for their cytotoxic effect on human cancer cell lines HeLa and MDA-MB 231 and normal fibroblasts (MRC-5). Four compounds, 3-hydroxy-1-(p-tolyl)-4-(2-thienylcarbonyl)-5-(4-chlorophenyl)-2,5-dihydro-1H-pyrrol-2-one (D10), 3-hydroxy-1-(3-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D13), 3-hydroxy-1-(4-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D14), and 3-hydroxy-1-(4-chlorophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D15), that showed the highest cytotoxicity against malignant cells and the best selectivity towards normal cells were selected for further experiments. Results obtained by investigating mechanisms of cytotoxic activity suggest that selected 3-hydroxy-3-pyrrolin-2-one derivatives in HeLa cells induce apoptosis that is associated with S phase arrest (D13, D15, and D10) or unrelated to cell cycle distribution (D14). Additionally, to better understand their suitability for potential use as anticancer medicaments we studied the interactions between biomacromolecules (DNA or BSA) and D13 and D15. The results indicated that D13 and D15 have great affinity to displace EB from the EB-DNA complex through intercalation [K sv = (3.7 ± 0.1) and (3.4 ± 0.1) × 10 3 M −1 , respectively], an intercalative mode also confirmed through viscosity measurements. K a values, obtained as result of fluorescence titration of BSA with D13 and D15 [K a = (4.2 ± 0.2) and (2.6 ± 0.2) × 10 5 M, respectively], support the fact that a sign

In order to make a progress in discovering a new agents for chemotherapy with improved properties and bearing in mind the fact that substituted 3-hydroxy-3-pyrrolin-2-ones belong to a class of biologically active compounds, series of novel 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones were synthesized and characterized by spectral (UV–Vis, IR, NMR, ESI-MS), X-ray and elemental analysis. All compounds were examined for their cytotoxic effect on human cancer cell lines HeLa and MDA-MB 231 and normal fibroblasts (MRC-5). Four compounds, 3-hydroxy-1-(p-tolyl)-4-(2-thienylcarbonyl)-5-(4-chlorophenyl)-2,5-dihydro-1H-pyrrol-2-one (D10), 3-hydroxy-1-(3-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D13), 3-hydroxy-1-(4-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D14), and 3-hydroxy-1-(4-chlorophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D15), that showed the highest cytotoxicity against malignant cells and the best selectivity towards normal cells were selected for further experiments. Results obtained by investigating mechanisms of cytotoxic activity suggest that selected 3-hydroxy-3-pyrrolin-2-one derivatives in HeLa cells induce apoptosis that is associated with S phase arrest (D13, D15, and D10) or unrelated to cell cycle distribution (D14). Additionally, to better understand their suitability for potential use as anticancer medicaments we studied the interactions between biomacromolecules (DNA or BSA) and D13 and D15. The results indicated that D13 and D15 have great affinity to displace EB from the EB-DNA complex through intercalation [K sv = (3.7 ± 0.1) and (3.4 ± 0.1) × 10 3 M −1 , respectively], an intercalative mode also confirmed through viscosity measurements. K a values, obtained as result of fluorescence titration of BSA with D13 and D15 [K a = (4.2 ± 0.2) and (2.6 ± 0.2) × 10 5 M, respectively], support the fact that a sign

U ovoj doktorskoj disertaciji ispitivano je fotodinamičko citotoksično dejstvo grafenskih kvantnih tačaka (GKT) na ćelije humanog glioma, kao i njihov antibakterijski efekat na bakterije rezistentne na konvencionalnu antibiotsku terapiju. Iako odlične fotoluminiscentne karakteristike GKT omogućavaju njihovu široku primenu u biomedicini, njihova citotoksičnost, kao i antibakterijsko dejstvo do sada nisu ispitivani. Gliomi su najčešći i najletalniji tumori mozga otporni na konvencionalnu terapiju i često inoperabilni, pa su stoga neophodni novi terapijski pristupi u njihovom lečenju. GKT, ovalne ugljenične dvodimenzionalne nanočestice, sintetisane su elektrohemijskom oksidacijom grafita i karakterisane su metodama UV-Vis spektroskopije, mikroskopijom atomskih sila i transmisionom elektronskom mikroskopijom. Nakon fotoekscitacije plavom svetlošću (470 nm, 1 W) GKT su produkovale reaktivne kiseonične vrste, uključujući i singletni kiseonik (1O2), što je dovelo do oksidativnog stresa i smrti U251 ćelija humanog glioma. Ćelijska smrt indukovana fotoekscitiranim GKT pokazivala je morfološke i biohemijske karakteristike kako apoptoze (eksternalizacija fosfatidil-serina, aktivacija kaspaza, DNK fragmentacija), tako i autofagije (formiranje autofagnih vezikula, konverzija LC3-I u LC3-II, degradacija autofagnog kargo receptora SQSTM1/p62). Genetska inaktivacija ključnog autofagnog proteina LC3B smanjila je fotodinamičku citotoksičnost GKT. Otkrivanje novih, efikasnijih metoda lečenja bakterijskih infekcija postalo je neophodno usled porasta učestalosti infekcija uzrokovanih sojevima bakterija koje pokazuju rezistenciju na veliki broj antibiotika. Iz tih razloga je ispitivan uticaj fotoekscitiranih GKT na Gram-pozitivne i Gram-negativne bakterije, meticilinrezistentni Staphylococcus aureus i Escherichia coli, koje spadaju u grupu najznačajnijih nozokomijalnih patogena, ali se sa velikom učestalošću izoluju i kao izazivači infekcija u vanbolničkoj sredini..., In this doctoral dissertation the photodynamic cytotoxic effect of graphene quantum dots (GQD) was investigated in human glioma cells and antibiotic-resistant bacteria. The excellent physicochemical properties of GQD make them suitable candidates for biomedical applications, but their cytotoxicity towards cancer cells or bacteria has not been extensively studied thus far. Gliomas are among the most frequent and letal brain tumors, often inoperable and highly refractive to the standard chemotherapy protocols, thus there is a pressing need for the development of new therapy approaches. GQD, the two-dimensional carbon nanoparticles, were synthesized by the electrochemical oxidation of graphite and characterized by UV-Vis, atomic force microscopy, and transmission electronic microscopy. It was shown that GQD irradiated with the blue light (470 nm, 1 W) produced reactive oxygen species, including singlet oxygen (1O2), which killed U251 human glioma cells by causing oxidative stress. The cell death induced by the photoexcited GQD displayed morphological and/or biochemical characteristics of both apoptosis (phosphatidylserine externalization, caspase activation, DNA fragmentation) and autophagy (formation of autophagic vesicles, LC3-I/LC3-II conversion, degradation of autophagic cargo receptor SQSTM1/p62). Moreover, genetic inactivation of autophagy-essential LC3B protein partly abrogated the photodynamic cytotoxicity of GQD. Searching for new treatments for bacterial infections has become necessary, because of a dramatic increase in the frequency of infections caused by drug-resistant bacteria. Therefore, we examined potential antimicrobial effect of photoexcited GQD on the Gram-positive and Gram-negative bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli, which are leading nosocomial pathogens...

Autofagija, proces programirane ćelijske razgradnje unutarćelijskog sadržaja, je uključena u regulaciju preživljavanja i smrti ćelija kancera. U ovoj doktorskoj disertaciji je po prvi put ispitivana sposobnost antileukemijskih lekova citarabina i idarubicina da indukuju autofagiju u različitim humanim leukemijskim ćelijskim linijama i mononuklearnim ćelijama periferne krvi (MNPK) pacijenata obolelih od leukemije in vitro. Takođe, ispitivani su unutarćelijski mehanizmi odgovorni za indukciju autofagije, kao i uloga autofagije u citotoksičnosti ovih lekova. Vijabilitet REH, HL-60, K562 leukemijskih ćelijskih linija, MNPK pacijenata obolelih od leukemije i MNPK zdravih kontrola je određivan merenjem aktivnosti kisele fosfataze i mitohondrijalnih dehidrogenaza. Protočna citofluorimetrija je korišćena za detekciju apoptoze i zakišeljavanja citoplazme. Indukcija autofagije je ispitivana fluorescentnom mikroskopijom (detekcija unutarćelijskih kiselih vezikula obojenih akridin oranžom), transmisionom elektronskom mikroskopijom (posmatranje autofagnih vezikula) i imunoblot analizom (konverzija LC3-I u LC3-II, degradacija SQSTM1/p62). Aktivacija signalnih puteva koji učestvuju u regulaciji autofagije je analizirana imunoblot metodom. Uloga autofagije u citotoksičnosti citarabina i idarubicina je ispitivana primenom farmakološke inhibicije bafilomicinom A1, hlorokinom, vortmaninom i amonijum hloridom, kao i genetske inaktivacije ekspresije beklina-1, LC3β i SQSTM1 transfekcijom odgovarajućim malim interferirajućim RNK. Citarabin i idarubicin su izazvali povećanje unutarćelijske kiselosti i pojavu autofagnih vezikula sa delimično razgrađenim ćelijskim sadržajem u leukemijskim ćelijskim linijama. Antileukemijski lekovi su stimulisali razgradnju supstrata autofagije SQSTM1 i povećali konverziju LC3-I u LC3-II formu asociranu autofagozomima u odsustvu ili prisustvu inhibitora proteolize, ukazujući tako na povećanje autofagnog fluksa. Oba leka su smanjila fosforilaciju mTOR kinaze, Autophagy, a process of programmed cellular self-digestion, has been implicated in regulation of cancer cell survival and death. The present study investigated for the first time the ability of antileukemic drugs cytarabine and idarubicin to induce autophagy in different human leukemic cell lines and peripheral blood mononuclear cells (PBMC) from leukemia patients in vitro. Intracellular mechanisms responsible for the induction of autophagy, as well as the role of autophagy in cytotoxicity of these drugs were also investigated. Cell viability of REH, HL-60, K562 leukemic cell lines, and PBMC from leukemic patients and healthy controls was determined by measuring the acid phosphatase and mitochondrial succinate dehydrogenase activity. Flow cytometry was used for the detection of apoptosis and intracellular acidification. Autophagy induction was assessed by fluorescent microscopy (detection of acridine orange stained intracellular acidic vesicles), by transmission electron microscopy (observation of autophagic vacuoles), as well as by immunoblot analysis of LC3 conversion and SQSTM1/p62 proteolysis. Activation of autophagy-regulating signaling pathways was analyzed by immunoblotting. Pharmacological inhibition of autophagy with bafilomycin A1, chloroquine, wortmannin, and NH4Cl or RNA interference-mediated knockdown of beclin-1, LC3β and SQSTM1 were used to determine the role of the autophagy in cytotoxicity of antileukemic drugs. Cytarabine and idarubicin induced an increase in intracellular acidification and appearance of autophagic vesicles with partially digested cellular components in leukemic cell lines. Antileukemic drugs stimulated the degradation of autophagic target SQSTM1 and enhanced the conversion of LC3-I to autophagosome-associated LC3-II in the absence or presence of proteolysis inhibitors, thus indicating the increase in autophagic flux...

Autofagija, proces programirane ćelijske razgradnje unutarćelijskog sadržaja, je uključena u regulaciju preživljavanja i smrti ćelija kancera. U ovoj doktorskoj disertaciji je po prvi put ispitivana sposobnost antileukemijskih lekova citarabina i idarubicina da indukuju autofagiju u različitim humanim leukemijskim ćelijskim linijama i mononuklearnim ćelijama periferne krvi (MNPK) pacijenata obolelih od leukemije in vitro. Takođe, ispitivani su unutarćelijski mehanizmi odgovorni za indukciju autofagije, kao i uloga autofagije u citotoksičnosti ovih lekova. Vijabilitet REH, HL-60, K562 leukemijskih ćelijskih linija, MNPK pacijenata obolelih od leukemije i MNPK zdravih kontrola je određivan merenjem aktivnosti kisele fosfataze i mitohondrijalnih dehidrogenaza. Protočna citofluorimetrija je korišćena za detekciju apoptoze i zakišeljavanja citoplazme. Indukcija autofagije je ispitivana fluorescentnom mikroskopijom (detekcija unutarćelijskih kiselih vezikula obojenih akridin oranžom), transmisionom elektronskom mikroskopijom (posmatranje autofagnih vezikula) i imunoblot analizom (konverzija LC3-I u LC3-II, degradacija SQSTM1/p62). Aktivacija signalnih puteva koji učestvuju u regulaciji autofagije je analizirana imunoblot metodom. Uloga autofagije u citotoksičnosti citarabina i idarubicina je ispitivana primenom farmakološke inhibicije bafilomicinom A1, hlorokinom, vortmaninom i amonijum hloridom, kao i genetske inaktivacije ekspresije beklina-1, LC3β i SQSTM1 transfekcijom odgovarajućim malim interferirajućim RNK. Citarabin i idarubicin su izazvali povećanje unutarćelijske kiselosti i pojavu autofagnih vezikula sa delimično razgrađenim ćelijskim sadržajem u leukemijskim ćelijskim linijama. Antileukemijski lekovi su stimulisali razgradnju supstrata autofagije SQSTM1 i povećali konverziju LC3-I u LC3-II formu asociranu autofagozomima u odsustvu ili prisustvu inhibitora proteolize, ukazujući tako na povećanje autofagnog fluksa. Oba leka su smanjila fosforilaciju mTOR kinaze, Autophagy, a process of programmed cellular self-digestion, has been implicated in regulation of cancer cell survival and death. The present study investigated for the first time the ability of antileukemic drugs cytarabine and idarubicin to induce autophagy in different human leukemic cell lines and peripheral blood mononuclear cells (PBMC) from leukemia patients in vitro. Intracellular mechanisms responsible for the induction of autophagy, as well as the role of autophagy in cytotoxicity of these drugs were also investigated. Cell viability of REH, HL-60, K562 leukemic cell lines, and PBMC from leukemic patients and healthy controls was determined by measuring the acid phosphatase and mitochondrial succinate dehydrogenase activity. Flow cytometry was used for the detection of apoptosis and intracellular acidification. Autophagy induction was assessed by fluorescent microscopy (detection of acridine orange stained intracellular acidic vesicles), by transmission electron microscopy (observation of autophagic vacuoles), as well as by immunoblot analysis of LC3 conversion and SQSTM1/p62 proteolysis. Activation of autophagy-regulating signaling pathways was analyzed by immunoblotting. Pharmacological inhibition of autophagy with bafilomycin A1, chloroquine, wortmannin, and NH4Cl or RNA interference-mediated knockdown of beclin-1, LC3β and SQSTM1 were used to determine the role of the autophagy in cytotoxicity of antileukemic drugs. Cytarabine and idarubicin induced an increase in intracellular acidification and appearance of autophagic vesicles with partially digested cellular components in leukemic cell lines. Antileukemic drugs stimulated the degradation of autophagic target SQSTM1 and enhanced the conversion of LC3-I to autophagosome-associated LC3-II in the absence or presence of proteolysis inhibitors, thus indicating the increase in autophagic flux...

A small series of 1-acetyl-2-(4-alkoxy-3-methoxyphenyl)cyclopropanes was prepared, starting from dehydrozingerone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and its O-alkyl derivatives. Their microbiological activities toward some strains of bacteria and fungi were tested, as well as their in vitro cytotoxic activity against some cancer cell lines (HeLa, LS174 and A549). All synthesized compounds showed significant antimicrobial activity and expressed cytotoxic activity against tested carcinoma cell lines, but they showed no significant influence on normal cell line (MRC5). Butyl derivative is the most active on HeLa cells (IC50 = 8.63 m), while benzyl one is active against LS174 and A549 cell lines (IC50 = 10.17 and 12.15 m, respectively).

Uvod: Glukokortikoidni hormoni (GH) su mimo svog anti-inflamatornog dejstva i jedni od najpoznatijih inhibitora zarastanja rana. Nedavno je pokazano da farnezil pirofosfat (FPP), ključni međuprodukt mevalonatskog puta sinteze holesterola i farnezilacije proteina može delovati i kao agonista za nekoliko nuklearnih hormonskih receptora uključujući glukokortikodni receptor (GR). Interesantno je da statini, lekovi koji se koriste u terapiji ateroskleroze, svoje dejstvo ostvaruju upravo inhibirajući ovaj sintetski put. Međutim, statini imaju i dodatne plejotropne efekte, koji se ostvaruju nezavisno od sniženja koncentracije holesterola u plazmi i značajno doprinose njihovom korisnom dejstvu u različitim bolestima. Jedan od dobro dokumentovanih plejotropnih efekata je poboljšana reparacija tkiva uključujući i zarastanje rana. Zanimljivo je da se upravo inhibicija farnezilacije signalnih molekula, usled sniženja nivo ćelijskog FPP-a, smatrala do sada ključnim mehanizmom kojim statini ostvaruju ove svoje dodatne efekte. Međutim otkriće da FPP može delovati kao ligand za GR otvara vrata za drugačiju interpretaciju mehanizma dejstva plejotropnih efekata statina i novu primenu ovih lekova. Ciljevi: U ovoj disertaciji dokazivano je postojanje novog mehanizma dejstva statina, kojim oni mogu pospešiti zarastanje rana. Ovaj efekat se zasniva na sniženju koncentracije endogenog FPP-a u ćelijama kože, putem inhibicije HMG-CoA reduktaze Osnovna hipoteza je da, s obzirom da statini svoje dejstvo ostvaruju upravo inhibirajući ovaj sintetski put, modifikuju FPP-GR signalizaciju i tako učestvuju u stimulaciji zarastanje rana. Prvi cilj istraživanja je bio aktivacije glukokortikoidnog signalnog puta i stimulacije transkripcije gena. Potom je ispitatano da li povećanje nivoa endogenog FPP-a putem inhibicije enzima skvalen sintetaze i farnezil transferaze ima isti efekat na aktivaciju glukokortikoidnog receptora kao i dodavanje egzogenog FPP-a..., Introduction: Glucocorticoid hormones, in addition to their anti-immflamatory effect, are well known inhibitors of wound healing. Recent studies have shown that farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway of the cholesterol synthesis and protein farnesylation, can act as an agonist for several nuclear hormone receptors including glucocorticoid receptor (GR). Interestingly, mechanism of action of statins, drugs used in therapy of atherosclerosis, is based on inhibition of mevalonath pathway. However, statins might exert also additional pleiotropic, noncholesterol lowering effects which significantly contribute to their therapeutic action. One of the well known pleiotropic effects of the statins is stimulation of tissue repair, including wound healing. Interestingly, mechanism of pleiotropic effects is traditionally explained by statin mediated inhibition of the farensylation of signaling molecules. By inhibiting the enzime HMG-CoA reductase, statins prevent the synthesis of substrate for reaction, farnesylpyrophosphate. Given that is recently shown that FPP can act as a ligand for GR, it is not surprising that statins might have additional mechanisms of action beyond inhibition of the farnesylation. Better understanding of this mechanism may contribute to novel therapeutic roles for this drugs. Objectives: In this study we are exploring new mechanism of action of statins that can contribute to improvement of wound healing. This effect is based on decreasing the levels of endogenus FPP in keratinocytes by inhibition of the HMG-CoA reductase. We postulate that statins, acting as an inhibitors of mevalonate pathway, stimulate wound healing through modulation of FPP-GR signal. The first step in in our study is to prove that the exogenous FPP, acting as an agonist for GR, can activate the glucocorticoid signaling pathway and regulate the transcription of the target genes. Second, will explore the effects of ZGA and B581, which both can incre

Uvod. Langerhansove ćelije (LĆ) predstavljaju subpopulaciju dendritskih ćelija (DĆ) koja ima ulogu u započinjanju imunskog odgovora na antigene koji su dospeli u epidermis ili sluzokože. DĆ vezuju molekulske obrasce patogena (engl. Pathogen-associated molecular patterns, PAMP) i oštećenih ćelija (engl. Damage-associated molecular patterns, DAMP) pomoću membranskih i citosolnih receptora za prepoznavanje molekulskih obrazaca (engl. Pattern recognition receptors, PRR). Među PRR su najviše proučavani receptori slični Tollu (engl. Toll-like receptors, TLR) i lektinski receptori C-tipa (engl. C-type lectin receptors, CLR). Uspostavljanje kontakta između DĆ i PAMP i DAMP je ključni događaj kojim se povezuju urođena i stečena imunost, što za posledicu ima eradikaciju antigena i nastanak imunološke memorije ili izazivanje periferne tolerancije. Nakon internalizacije i obrade PAMP i DAMP u proinflamacijskom okruženju, DĆ započinju migraciju u T-zavisne zone regionalnih limfnih čvorova u toku koje fenotipski i funkcionalno sazrevaju. Aktivacija CD40 receptora na DĆ nakon vezivanja liganda ovog molekula (CD40L) ispoljenog na površini aktivisanih T-limfocita dovodi do okončanja maturacije DĆ. Tada one postaju potpuno efikasne antigen-prezentujuće ćelije sposobne da polarizuju imunski odgovor CD4+ T-limfocita u Th1 smeru i da indukuju citotoksičnu efektorsku funkciju CD8+ T-limfocita. Konvencionalne DĆ, sa napred navedenim funkcionalnim karakteristikama, su našle primenu u prevenciji i terapiji infektivnih bolesti i maligniteta u obliku DĆ vakcina. Iako je za in vivo ili in vitro sazrevanje DĆ često dovoljna stimulacija samo jednim agonistom TLR ili CLR, fenotipski stabilniji i funkcionalno potentniji imunski odgovor u eksperimentalnim i kliničkim ispitivanjima se najčešće dobija tek kombinovanom primenom njihovih analoga, što je posledica specifične integracije različitih, međusobno nezavisnih, unutarćelijskih signala. Nakon nedavne potvrde da LĆ imaju sposobnost unakrsne preze, Introduction. Langerhans cells (LCs) represent a subpopulation of dendritic cells (DCs) whose function is to initiate immune responses against the antigens from epidermis and mucoses. DCs bind pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) via membrane and cytosolic pattern recognition receptors (PRRs). The most frequently studied PRRs are Toll-like receptors (TLRs) and C-type lectine receptors (CLRs). Similarly to conventional DCs, the contact established between LC’s PRRs and PAMPs and DAMPs is the key event that links innate and adaptive immunity and leads to eradication of the antigen, initiation of the peripheral tolerance and introduction of the immunological memory. After LCs internalise and process PAMPs and DAMPs in a proinflammatory environment, they start to migrate toward T-dependent regions of the regional lymph nodes and, at the same time, become phenotipically and functionally mature. The activation of the CD40 molecule on LCs with a CD40 ligand (CD40L) expressed on the surface of activated T-lymphocytes brings the maturation of LCs to its end, when they become fully efficient antigen-presenting cells, capable of inducing the Th1 polarisation of CD4+ T-lymphocytes and citotoxic effector functions of CD8+ T-lymphocytes. Conventional DCs with the aforementioned functional characteristics are used in the prevention and therapy of infectious or malignant diseases as DC-vaccines. Although DCs require stimulation with a single TLR or CLR agonist to become mature in vitro and in vivo conditions, phenotipically stable and functionally more potent immune response of these cells in experimental and clinical studies is usually achieved only after combined treatment with analoges of these receptors because of the specific integration of the various, mutually independent, intracellular signals. LCs became candidates for DC-vaccines after recent confirmation that they are capable to cross-present antigens. Until now

Akutna HCV infekcija u većini slučajeva (>80%) protiče kao oligosimptomatska ili asimptomatska bolest. Nakon akutne faze, spontana rezolucija HCV infekcije događa se u samo 20 % obolelih, dok kod najvećeg broja inficiranih virus perzistira i bolest prelazi u hroničnu fazu. Pretpostavlja sa da oko 180 miliona ljudi širom sveta, ima hroničnu HCV infekciju. Patogeneza HCV infekcije je rezultat multiplih interakcija između imunskog sistema domaćina i virusnih faktora. Snažan HCV specifičan CD4+ i CD8+ T ćelijski odgovor usmeren na multiple HCV epitope u nekim slučajevima omogućava spontanu eliminaciju virusa u akutnoj fazi. Međutim, HCV virus poseduje različite mehanizme kojima, tokom rane faze akutne infekcije, može da izbegne odbrambene snage domaćina, što omogućava dalju progresiju bolesti u hroničnu fazu. Supresija koju izazivaju Treg indukovane HCV virusom može da snizi imunski odgovor T ćelija u ranim fazama infekcije i tako onemogući otklanjanje virusa, ali se time može smanjiti i zapaljenski infiltrat u jetri i sprečiti masivno oštećenje tkiva. Cilj ove studije je da se utvrdi prisustvo i odredi apsolutni i relativni broj CD4+, CD4+/Foxp3+ (Treg), CD8+ i CD20+ limfocita u jetri i perifernoj krvi pacijenata sa hroničnom HCV infekcijom, ispita citokinski profil imunskog odgovora u hroničnoj HCV infekciji kao i postojanje eventualne povezanosti ovih parametara, virusne replikacije i nivoa oštećenja jetrinog parenhima. Rezultati naše studije ukazuju da su pacijenti sa hroničnom HCV infekcijomom češće muškog pola, da je najčešći put infekcije IVUPAS, a najčešći genotip virusa genotip 1. Povoljan odgovor na terapiju je zabeležen kod tri četvrtine ispitanika. Metode logističke regresije su pokazale da su starost ispitanika i genotip značajni prediktivni faktori za nastanak fibroze, dok se kao najznačajniji prediktivni faktori povoljnog odgovora na terapiju izdvajaju starost ispitanika, put infekcije i stadijum fibroze. Ispitivanje citokinskog profila imunskog odgovora, In up to 80% of patients acute form of hepatitis C is asymptomatic. After the acute phase, the spontaneous resolution of HCV infection occurs in only 20% of cases, while most of infected patients develop a chronic desease. It is assumed that about 180 million people worldwide, have chronic HCV infection. The pathogenesis of HCV infection is the result of multiple interactions between the host's immune system and viral factors. Strong HCV-specific CD4+ and CD8+ T cell response directed to multiple epitopes of HCV in some cases eliminate the virus spontaneously in the acute phase. However, HCV has mechanisms to escape humoral and cellular response, which allows progression of the disease to the chronic phase. A subset of regulatory T cells contributes to failure of T cell-mediate immunity in the early stages of infection and thus prevent the removal of the virus, but also can reduce the inflammatory infiltrate in the liver and help prevent massive tissue damage. The aim of this study was to determine the presence and absolute and relative number of CD4+, CD4+/Foxp3+ (Treg), CD8+ and CD20+ cells in the liver and peripheral blood of patients with chronic HCV infection, to assess sera cytokine profile and to find possible correlations of these parameters, viral replication and liver histopathology. The results of our study showed that chronically infected patients were mostly males, IVUPAS was most frequent route of infection and genotype 1 was dominant. About 77% of patients achieved sustained virologic response. The logistic regression showed that an age of the patients and genotype are significant predictive factors for the development of fibrosis, while the most important predictors of a favorable response to therapy are age of the patient, route of infection and the stage of fibrosis. Cytokine profiling has shown predominance of inflammatory cytokines in patients without fibrosis and tendency of decrease in cytokine levels with progression of liver injury. In F3 sta

Multipla skleroza (MS) je hronična, zapaljenska, demijelinizaciona i neurodegenerativna bolest centralnog nervnog sistema (CNS). Smatra se da je MS autoimunska bolest kod koje autoreaktivni pomoćnički T-limfociti (Th) specifični za antigene mijelina pokreću proces oštećenja direktno i aktivacijom drugih imunskih i neimunskih ćelija. Njihovo dejstvo bar delom antagonizuju regulatorni T-limfociti (Treg). Prva linija terapije za najčešću formu MS, relapsno-remitentnu (RR), je interferon-β koji svoje efekte ostvaruje dejstvom na ekspresiju različitih gena. Problem koji se nameće u lečenju ovim lekom je relativno visoka učestalost slabog terapijskog odgovora. U ovoj doktorskoj disertaciji prospektivno je praćena relativna genska ekspresija iRNK u mononuklearnim ćelijama periferne krvi kod bolesnika sa RRMS. Mereni su markeri biološkog odgovora na IFN-β, marker T-limfocita, Th1/17 − polarišući citokini, njihovi receptori, transkripcioni faktori Th1/17 i Treg, efektorski citokinii endogeni IFN-β pre početka terapije sa IFN-β1b i nakon 6, 12, 24 i 36 meseci trajanja terapije. Pored toga, praćena je kinetika koncentracije odabranih proinflamatornih citokina u plazmi. Analiziran je potencijal ovih medijatora kao biomarkera kliničkog odgovora na terapiju. Tokom IFN-β terapije su se značajno menjali nivoi ekspresije svih ispitivanih gena, i to na isti način kod pacijenata koji dobro odgovaraju na terapiju i kod pacijenata koji suboptimalno odgovaraju na terapiju. IFN-β terapija nije uticala na koncentracije merenih proinflamatornih citokina u plazmi bolesnika sa RRMS. Nije postojala povezanost nivoa RNK, kao ni koncentracije citokina u plazmi sa kliničkim i demografskim parametrima obolelih. Međutim, kada su podaci analizirani u zavisnosti od pojave relapsa, nađena je povezanost niže ekspresije β2 subjedinice receptora za IL-12 (IL-12Rβ2) sa višim rizikom za pojavu relapsa. Relativna genska ekspresija IL-12Rβ2 ima potencijal kao biomarker odgovora na terapiju sa IFN-β1b. Dalja

Uvod. Langerhansove ćelije (LĆ) predstavljaju subpopulaciju dendritskih ćelija (DĆ) koja ima ulogu u započinjanju imunskog odgovora na antigene koji su dospeli u epidermis ili sluzokože. DĆ vezuju molekulske obrasce patogena (engl. Pathogen-associated molecular patterns, PAMP) i oštećenih ćelija (engl. Damage-associated molecular patterns, DAMP) pomoću membranskih i citosolnih receptora za prepoznavanje molekulskih obrazaca (engl. Pattern recognition receptors, PRR). Među PRR su najviše proučavani receptori slični Tollu (engl. Toll-like receptors, TLR) i lektinski receptori C-tipa (engl. C-type lectin receptors, CLR). Uspostavljanje kontakta između DĆ i PAMP i DAMP je ključni događaj kojim se povezuju urođena i stečena imunost, što za posledicu ima eradikaciju antigena i nastanak imunološke memorije ili izazivanje periferne tolerancije. Nakon internalizacije i obrade PAMP i DAMP u proinflamacijskom okruženju, DĆ započinju migraciju u T-zavisne zone regionalnih limfnih čvorova u toku koje fenotipski i funkcionalno sazrevaju. Aktivacija CD40 receptora na DĆ nakon vezivanja liganda ovog molekula (CD40L) ispoljenog na površini aktivisanih T-limfocita dovodi do okončanja maturacije DĆ. Tada one postaju potpuno efikasne antigen-prezentujuće ćelije sposobne da polarizuju imunski odgovor CD4+ T-limfocita u Th1 smeru i da indukuju citotoksičnu efektorsku funkciju CD8+ T-limfocita. Konvencionalne DĆ, sa napred navedenim funkcionalnim karakteristikama, su našle primenu u prevenciji i terapiji infektivnih bolesti i maligniteta u obliku DĆ vakcina. Iako je za in vivo ili in vitro sazrevanje DĆ često dovoljna stimulacija samo jednim agonistom TLR ili CLR, fenotipski stabilniji i funkcionalno potentniji imunski odgovor u eksperimentalnim i kliničkim ispitivanjima se najčešće dobija tek kombinovanom primenom njihovih analoga, što je posledica specifične integracije različitih, međusobno nezavisnih, unutarćelijskih signala. Nakon nedavne potvrde da LĆ imaju sposobnost unakrsne preze, Introduction. Langerhans cells (LCs) represent a subpopulation of dendritic cells (DCs) whose function is to initiate immune responses against the antigens from epidermis and mucoses. DCs bind pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) via membrane and cytosolic pattern recognition receptors (PRRs). The most frequently studied PRRs are Toll-like receptors (TLRs) and C-type lectine receptors (CLRs). Similarly to conventional DCs, the contact established between LC’s PRRs and PAMPs and DAMPs is the key event that links innate and adaptive immunity and leads to eradication of the antigen, initiation of the peripheral tolerance and introduction of the immunological memory. After LCs internalise and process PAMPs and DAMPs in a proinflammatory environment, they start to migrate toward T-dependent regions of the regional lymph nodes and, at the same time, become phenotipically and functionally mature. The activation of the CD40 molecule on LCs with a CD40 ligand (CD40L) expressed on the surface of activated T-lymphocytes brings the maturation of LCs to its end, when they become fully efficient antigen-presenting cells, capable of inducing the Th1 polarisation of CD4+ T-lymphocytes and citotoxic effector functions of CD8+ T-lymphocytes. Conventional DCs with the aforementioned functional characteristics are used in the prevention and therapy of infectious or malignant diseases as DC-vaccines. Although DCs require stimulation with a single TLR or CLR agonist to become mature in vitro and in vivo conditions, phenotipically stable and functionally more potent immune response of these cells in experimental and clinical studies is usually achieved only after combined treatment with analoges of these receptors because of the specific integration of the various, mutually independent, intracellular signals. LCs became candidates for DC-vaccines after recent confirmation that they are capable to cross-present antigens. Until now

Upotreba biljaka u lečenju i ishrani stara je koliko i sama ID istorija ljudskog roda. Veština lečenjem biljem razvijala se kod svih naroda sveta i sačuvala se manje ili više sve do današnjih dana uz tendeciju porasta primene fitoterapije u ekonomski razvijenim zemljama poslednjih godina. Naučni interes za lekovitost belog luka počinje u drugoj polovini XIX veka kada je Luj Paster utvrdio da ova biljka sprečava rast i razmnožavanje određenih sojeva bakterijsih vrsta i od tada je predmet stalnih naučnih istraživanja. U ovom radu ispitivana je antimikrobna aktivnost vodenog ekstrakta belog luka gajenog sa područja Šumadije, glavnog aktivnog principa alicina kao i nekih organosumpornih jedinjenja konstituenasa biljke Alium Sativum L.,rastvornih u vodi derivata cisteina, na gram-negativne bakterije Eserihia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027 i gram-pozitivne bakterije Staphylococcus aureus ATCC 43300, Bacillus subtilis ATCC 11774. Merenja i kvantifikovanje antimikrobnog dejstva su bila određena korišćenjem površinskog disk difuzionog metoda na Mueller Hinton agarnoj podlozi uz primenu aplikativnog softvera Motic images plus 2.0 ML na čistim bakterijskim kulturama i referentnom supstancom antibiotikom gentamicinom korišćenog u svim eksperimentima kao kontrola. Očekivanu antimikrobnu aktivnost je ispoljio ekstrakt belog luka i najači aktivni princip alicin, ali je 112 istovremeno i konstituens N-acetil-L-cistein takođe ispoljio efekat na sve u radu ispitivane bakterijske sojeve. N-acetil-L- cistein je veći antimikrobni efekat pokazao na ispitivane gram- pozitivne nego na gram-negativne bakterijske vrste. Pretpostavka za dobijeni antimikrobni efekat kod gram negativnih bakterija bi bila da tiolna grupa reaguje prvo sa lipopolisaharidnim slojem pa tek nakon toga sa kapsulnim polisaharidima poznatih kao glikokaliks, odnosno da razlike u strukturi ćelijskog zida direktno utiču na dobijene vrednosti zone inhibicije. Dobijeni rezultati naše studije upravo bi mogl, The use of plants in medical treatment and nutrition is as old as AB human history itself. The skills for treatment using plants have been developed in all nations of the world and have been maintained more or less up to the present day. Developed countries show the tendency to increase use of phototherapy in recent years. Scientific interest in the curative properties of garlic begins in the second half of the nineteenth century, when Louis Pasteur found out that this herb inhibits the growth and reproduction of certain strains of bacteria. The garlic has been subject to ongoing scientific research since then. This study examined the antimicrobial activity of aqueous extract of garlic cultivated in Šumadija region and its main active principle of allicin and some organosulfur compound constituents of plants Alium sativum L., soluble in water derivatives of cysteine on gram- negative bacteria Eserihia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027 and gram- positive bacteria Staphylococcus aureus ATCC 43300, Bacillus subtilis ATCC 11774. Measurement and quantification of antimicrobial activity were determined using surface disk diffusion method on Mueller Hinton agar substrate with the use of Motic Images Plus 2.0 ML software on pure bacterial cultures. The 115 antibiotic gentamicin is reference substance and is used in all experiments as a control. The garlic extract and its strongest active constituent of allicin has showed the expected antimicrobial activity, but also the constituent N-acetyl-L-cysteine has showed the effect on all bacterial strains used for this study. N-acetyl-L-cysteine demonstrated greater antimicrobial effect on gram-positive bacteria tested than gram- negative bacteria. The presumption of resulting antimicrobial effect of gram negative bacteria would be that the thiol group reacts first with lipopolysaccharide layer and only then with the capsule polysaccharides known as glycocalyx and differences in the structure of the cell wall direct