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Arthrospira (Spirulina), fotosintetska filamentozna cijanobakterija, koristi se u ishrani čovjeka od davnina. To je jedan od najbogatijih izvora proteina i esencijalnih amino kiselina, kao i izvanredan izvor vitamina, makro- i mikro-elemenata, esencijalnih masnih kiselina, pigmenata, glikolipida i polisaharida. C-fikocijanin (C-PC), najzastupljeniji protein u Spirulini, je u vodi rastvoran heterodimerni molekul koji se odlikuje intezivnom fluorescencijom. Njegova plava boja potiče od kovalentno vezane (tioetarskom vezom) tetrapirolne hromofore fikocijanobilina. Jedan molekul PCB-a je vezan za α subjedinicu preko cisteinskog ostatka 84, dok β subjedinica vezuje dva molekula hromofore preko cisteinskih ostataka 82 i 153. Brojne studije su pokazale da C-PC ima značajne antoksidativne, anti-inflamatorne, imunomodulatorne i antikancerogene efekte. Štaviše, PCB per se ispoljava različite biološke aktivnosti koje mogu poboljšati zdravlje ljudi. U literaturi ne postoje podaci o strukturi i bioaktivnosti digestijom oslobođenih peptida sa vezanom hromoforom (hromopeptida), kao ni informacije o vezivanju PCB-a za humani (HSA) i goveđi (BSA) serum albumin, glavne proteine plazme i model sisteme za proučavanje protein-ligand interakcija.Prvi dio ove doktorske disertacije imao je za zadatak ispitivanje digestibilnosti (C-PC-a) pepsinom, kao i određivanje strukture i ispitivanje bioaktivnih svojstava hromopeptida dobijenih nakon digestije C-PC-a, izolovanog i prečiščenog iz komercijalnog praha Arthrospira platensis. SDS-PAGE pod redukujućim uslovima je pokazala da se hromoprotein brzo digestuje pepsinom u simuliranoj želudačnoj tečnosti. HPLC analiza digesta je pokazala postojanje pet dominantnih hromopeptidnih frakcija. Struktura oslobođenih hromopeptida je određena primjenom tandemske masene spektrometrije i peptidi, sa varijacijom u veličini od 2 do 13 aminokiselinska ostatka, su identifikovani u obje subjedinice C-PC-a. Utvrđeno je da sve hromopeptidne frakcije ispoljavaju odličnu antioksidativnu (ORAC esej i test ukupne redukujuće moći) i Cu2+-helirajuću aktivnost (studija gašenja fluorescencije), kao i značajne citotoksične efekte (MTT esej vijabilnosti) na ćelijskim linijama humanog cervikalnog adenokarcinoma i epitelnog kancera kolona. Zaključeno je da digestija pepsinom oslobađa biološki aktivne hromopeptide iz C-PC-a, čija je aktivnost u najvećoj mjeri zasnovana na njihovom antioksidativnom potencijalu.U drugom dijelu ove disertacije, najpre je okarakterisano vezivanje (prethodno izolovanog) PCB-a za HSA, a zatim je ispitan uticaj liganda na stabilnost proteina... Arthrospira (Spirulina), photosynthetic, filamentous cyanobacteria, has been used as food for centuries. It is one of the the richest known natural source of proteins and essential amino acids, excellent source of vitamins, macro- and micro-elements, pigments, essential fatty acids, glycolipids, and polysaccharides. C-phycocyanin (C-PC), the most abundant protein of Spirulina, is highly fluorescent and water-soluble heterodimeric protein. Its blue color arises from covalently attached (via tioether bond) tetrapyrrole chromophore phycocyanobilin (PCB). One PCB molecule is attached to α-subunit via Cys 84, while β-subunit binds two molecules of PCB via cysteines 82 and 153. Numerous studies have shown C-PC exhibit significant antioxidative and free radical-scavenging properties, anti-inflammatory, immunomodulatory and anti-cancer effects. Furthermore, PCB itself exhibits various strong health-promoting activities. There are no literature data evaluating structure and bioactivities of peptides with bound chromophore (chromopeptides) released in C-PC digest, nor information on the PCB binding to HSA and BSA, major plasma proteins and model systems for studying protein-ligand interactions.First part of this thesis examined the structures and bioactivities of chromopeptides obtained by pepsin digestion of C-PC, isolated from commercial Arthrospira platensis powder. SDS-PAGE under reducing conditions has shown that chromoprotein is rapidly digested by pepsin in simulated gastric fluid. HPLC analyses of digest revealed five major chromopeptide fractions. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-PC. It was shown that all chromopeptide fractions have excellent antioxidant (ORAC and reducing power test) and Cu2+-chelating (fluorescence quenching study) activities, and show significant cytotoxic effect (MTT viability assay) on human cervical adenocarcinoma and epithelial colonic cancer cell lines. Therefore, obtained results demonstrate that digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore.In the seacond part, binding of PCB to HSA was studied, as well as effects of ligand binding on protein stability. Based on a computational approach (molecular docking), we demonstrated two putative high-affinity binding pockets on HSA for PCB chromophore...

Vanbolnička pneumonija se definiše kao potencijalno ozbiljna bolest sa visokom incidencom i značajnim ekonomskim uticajem. Ova studija se bavila utvrđivanjem prosečnog troška lečenja vanbolničke pneumonije u bolničkim uslovima, po pacijentu, u KC Kragujevac, uz prikaz i analizu svih determinanti koje utiču na njegov ukupan iznos i upoređenje dobijenih podataka sa rezultatima dobijenih iz sličnih studija u Evropi i svetu. Planirana je i sprovedena kao prospektivna studija. Procena težine bolesti i rizik mortaliteta su procenjivani uz pomoć indeksa težine pneumonije (pneumonia severity index - PSI) i CURB-65 (Confusion – Urea - Respiratory rate - Blood pressure - age ≥ 65 years) skale. Uzorak se sastojao od 95 pacijenata hospitalizovanih zbog dijagnostikovane vanbolničke pneumonije. Analiza troškova je sprovedena iz društvene perspektive, sa vremenskom odrednicom od jedne godine. Direktni i indirektni troškovi po pacijentu lečenom od vanbolničke pneumonije su iznosili 696 ± 531 i 410 ± 283 evra, koji su zajedno sačinjavali ukupne troškove u iznosu od 1,106 ± 657 evra. Ukupni ekonomski uticaj čitave kohorte pacijenata je iznosio 105,087 evra (66,109 direktnih i 38,979 evra indirektnih troškova). Najznačajnije stavke u ukupnoj strukturi troškova su bile: troškovi apsentizma, troškovi dijagnoze i lečenja komorbiditeta, lekovi, kiseonik i derivati krvi. Vrednosti CURB-65 i PSI skora su bili u korelaciji sa indirektnim troškovima lečenja vanbolničke pneumonije. Vrednost PSI skora je bila u pozitivnoj korelaciji sa ukupnim troškovima lečenja vanbolničke pneumonije. Nijedan skor nije pokazao jasnu povezanost sa direktnim troškovima lečenja vanbolničke pneumonije. Procena težine bolesti na prijemu ne pokazuje povezanost sa troškovima lečenja bolesti. Ova činjenica se najverovatnije može dovesti u vezu sa nepotrebnim prijemima na bolničko lečenje pacijenata sa vanbolničkom pneumonijom koji se mogu lečiti ambulantno ili dugim trajanjem bolničkog lečenja, kao i sa prekomernim propisivanjem antibiotika. Pružanje više konkretnih dokaza lekarima u pogledu ekonomski efikasnih medicinskih intervencija sa ciljem lečenja pneumonije bi značajno poboljšalo kliničke ishode i indukovalo uštede, pa se samim tim usvajanje dugoročnih strategija sa ciljem preoblikovanja načina razmišljanja medicinskog osoblja nameće kao neophodnost. Community-acquired pneumonia is defined as a potentially severe illness with high incidence and significant economic impact. The objective of this study was to assess the direct and indirect costs of diagnosing and treating community-acquired pneumonia (CAP), correlating those costs with CAP severity at diagnosis and identifying the major cost drivers which determinate average cost of illness, per patient, in Clinical center Kragujevac, with comparison of results obtained with results from similar trials conducted in Europe and worldwide. This was a prospective cost analysis study using bottom-up costing. Clinical severity and mortality risk were assessed with the pneumonia severity index (PSI) and the mental Confusion-Urea-Respiratory rate-Blood pressure-age ≥ 65 years (CURB-65) scale, respectively. The sample comprised 95 inpatients hospitalized for newly diagnosed CAP. The analysis was run from a societal perspective with a time horizon of one year. Expressed as mean ± standard deviation, in Euros, the direct and indirect medical costs per CAP patient were 696 ± 531 and 410 ± 283, respectively, the total per-patient cost therefore being 1,106 ± 657. The combined budget impact of our patient cohort, in Euros, was 105,087 (66,109 and 38,979 in direct and indirect costs, respectively). The major cost drivers, in descending order, were the opportunity cost (lost productivity); diagnosis and treatment of comorbidities; and administration of medications, oxygen, and blood derivatives. The CURB-65 and PSI scores both correlated with the indirect costs of CAP treatment. The PSI score correlated positively with the overall frequency of use of health care services. Neither score showed any clear relationship with the direct costs of CAP treatment. Clinical severity at admission appears to be unrelated to the costs of CAP treatment. This is mostly attributable to unwarranted hospital admission (or unnecessarily long hospital stays) in cases of mild pneumonia, as well as to over-prescription of antibiotics. Authorities should strive to improve adherence to guidelines and promote cost-effective prescribing practices among physicians. Providing more concrete evidence to medical workers aiming at economically efficient medical interventions with the purpose of treating pneumonia would improve clinical outcomes significantly and induce savings.

Kontrakcije uterusa su važne za regulaciju brojnih reproduktivnihfunkcija. Neregularna i neadekvatna aktivnost uterusa povezana je sa brojnimpatološkim stanjima: neplodnost, neadekavtna implantacija, dismenoreja, kasnetrudnoće i prevremeni porođaji. Kontraktilnost uterusa regulisana jekompleksnom elektrofiziološkom mrežom koja može biti modulirana različitimfarmakološkim i signalnim molekulima.Uterus se karakteriše izuzetnom osobinom, brzom adaptacijom iz stanjamirovanja u oscilatorne cikluse jakih kontrakcija. Ovo je omogućeno zahvaljujućiaktivnosti ćelija koje su sposobne da remodeliraju svoje signalne puteve.Endogeno-sintetisani vodonik-sulfid (H2S), koji je zajedno sa azotmonoksidomi ugljen-monoksidom prepoznat kao gasoviti transmiter, imasignalnu ulogu i u uterusu. Vodonik-sulfid redukuje kontrakcije uterusa iprepoznat je kao agens velikog potencijala u tretmanu oboljenja uterusa. Do sadanisu urađene detaljnije studije, koje su dale uvid u mehanizme njegovogrelaksantnog dejstva na uterusu, te je to od značajnog interesa. Nije poznatoda li subiološki efekti sulfida posredovani njegovim molekulskim oblikom (H2S) ilitiolatnim anjonom (HS−).Rezultati ove disertacije implicirali su da molekulska i anjonska forma nemogu imati identične farmakološke efekte, uključujući i regulaciju konraktilnosti.Efekti H2S/HS− na relaksaciju uterusa upoređeni su sa efektom metantiol (CH3SH,koji „imitira“ H2S). Uočene razlike mogu biti posljedica specifične aktivnosti HS−jona. Ujedno, ovo upoređivanje može biti jedan od načina za razlikovanje efekataH2S i HS−. Metabolizam CH3SH je povezan sa sulfidom, budući da se H2S metiluje doCH3SH i ova metilacija može biti novi način modulacije H2S efekata in vivo.Ispitivano je učešće jonskih kanala (K+, Ca2+, Cl−), kao posrednika u relaksacijiuterusa indukovanoj sa H2S/HS−. Nađeno je da sulfid izaziva reverzibilnukoncentracijski-zavisnu relaksaciju koja je posredovana DIDS-osjetljivim Cl−kanalom. Dodatno, pokazano je da je DIDS-osjetljivi CaCC kanal (engl. Ca2+-... Uterine contractions are important in many reproductive functions.Improper or irregular uterine activity may underlie the common pathologicaldisorders such as infertility, improper implantation, dysmenorrhea, weak uterinecontraction during labor and preterm labor. The contractile activity of the uterus isregulated by the complex electrophysiologic network which is highly sensitive tovarious pharmacological and signaling molecules. Remarkably, uterus is able toswitch from quiescence to oscillatory cycles of strong contractions. This isdependent on the activity of the cells being able to remodel their signallingsystems.Endogenously produced hydrogen sulphide (H2S), which, together withNO and CO, is a recognized gasotransmitter, also appears to be a signallingmolecule in rat uterus. Hydrogen sulphide reduces uterine contractility, and it isrecognized as a promising treatment for uterine disorders. There were no studiesabout mechanistic insights of its relaxatory effect, done so far, and thus is ofpotential interest. It is not currently known whether the biological effects of H2Sare mediated directly by H2S or by its derivatives, the most important being thethiolate anion HS−. The results presented in this thesis imply that it appearsunlikely that molecular and anionic forms have pharmacologically identicalactions, including the regulation of contractility. Pharmacological effects ofH2S/HS− were compared to effects of methanethiol (CH3SH, mimicks H2S), andobserved differences might be a consequence of the specific actions of HS−. Thiscomparison may represent a useful tool for discrimination between differentactions of H2S and HS−. The metabolism of CH3SH is intertwined with hydrogensulfide (H2S), since the later is methylated to CH3SH and this methylation may be anew way of modulation of H2S effects in vivo.We investigated the involvement of ion channels (K+, Ca2+, Cl−) in therelaxation of rat uteri induced by H2S/HS−. We found that sulphide induces...

Struktura ekonomskog rasuđivanja zdravstvenih stručnjaka (ERZS) je još uvek nepoznata. CILjEVI: Procenjena je konstrukcona validnost i pouzdanost upitnika ERZS. Procenjena je njegova diskriminaciona validnost prema tipu zdravstene ustanove, nivou pružanja zdravstvene zaštite i uticaju sistemskih ekonomskih ograničavanja zdravstvenih ustanova. METOD: Sprovedena je retrospektivno-prospektivna, multicentrična, psihometrijska studija procene uticaja zdravstveno ekonomske intervencije na činioce ERZS. Studija je sprovedena u 60 ustanova. Anketirani su zdravstveni stručnjaci, zaposleni u državnom i privatnom sektoru zdravstva Republike Srbije. Upitnik ERZS sačinjen je od 29 pitanja. Period pre intervencije bio je od početka 2010. do sredine 2011. godine. Period posle intervencije bio je od sredine 2012. do kraja 2013. godine. REZULTATI: Definisano je sedam faktora ERZS: (1) zanemarivanje troškova kvaliteta od strane nemedicinskih organa uprave; (2) razmatranje tržišne cene i zdravstvene dobiti; (3) tržišna orijentisanost u pogledu zahteva pacijenta; (4) prepoznavanje neadekvatne alokacije sredstava nemedicinskih organa uprave; (5) stav o preusmeravanju profita iz državnog u privatni sektor zdravstva; (6) prepoznavanje neopravdane potrošnje zdravstvenih profesionalaca i (7) odnos lekar – pacijent. Postoji negativna korelacija između perioda posle intervencije sa ukupnim stepenom ERZS, u odnosu na period pre intervencije. ZAKLjUČAK: Upitnik ERZS ima visoku konstrukcionu i diskriminacionu validnost i pouzdanost u merenju komponenti ERZS. The structure of health professionals’ economic reasoning (HPER) is still unknown. OBJECTIVES: The validity and reliability of the HPER questionnaire has been estimated. Its discriminant validity of the HPER questionnaire has also been estimated according to the type of healthcare facilities, the healthcare provision level and the impact of systemic economic restrictions in the healthcare facilities. METHODS: We performed a retrospective-prospective, multicentric, psychometric study of the impact assessment of health economic interventions on the HPER factors. The study was conducted in 60 facilities. Respondents were health professionals, employees of public and private health sector of the Republic of Serbia. The HPER questionnaire was composed of 29 items. The period before the intervention lasted from the beginning of 2010th until mid-2011. The period after the intervention lasted from the middle of 2012th until the end of 2013. RESULTS: We defined seven HPER factors: (1) the neglect of quality costs by non-medical administrative authorities (2) consideration of the market value and health benefits; (3) market orientation with respect to the patient's needs; (4) recognition of inadequate allocation of resources from non-medical administrative authorities (5) the attitude about diverting profits from the state to the private health sector; (6) identification of unjustified consumption of health professionals, and (7) doctor - patient relationship. There is a negative correlation between the period after intervention with the total HPER degree, compared to the period before intervention. CONCLUSION: The HPER questionnaire has high constructional and discriminatory validity and reliability in measuring HPER components.

ATP i drugi purinski nukleotidi su parakrini signalni molekuli koji vezivanjem za purinske receptore (P1 i P2) na ćelijskoj membrani učestvuju u kontroli kontrakcija i modulaciji imunog odgovora u uterusu sisara. Poremećaji kontraktilnosti uterusa mogu imati važnu ulogu u nastanku neplodnosti, dismenoreje i prevremenog porođaja. U ovom radu ispitano je prisustvo i kinetičke osobine ektoenzima koji hidrolizuju vanćelijski ATP i ADP u uterusu pacova (Wistar albino). Takođe, ispitan je in vitro uticaj teških metala (kadmijuma, žive i bakra) kao i zaštitni efekat EDTA i L-cisteina na aktivnost ektoenzima u prisustvu jona metala. Rezultati ove studije pokazali su da hidrolizu vanćelijskog ATP i ADP u uterusu pacova katalizuju enzimi iz familije E-NTPDaza. To potvrđuju rezultati: neosetljivosti na inhibitore drugih membranskih ATPaza (oligomicin, ouabain, teofiln i levamisol); pH optimum za maksimalnu aktivnost enzima na alkalnom pH (8.0-8.5) za oba supstrata; zavisnost enzimske aktivnosti od dvovalentnih jona i inhibicija suraminom. Izmerana aktivnost E-NTPDaza u uterusu je veoma visoka u odnosu na ostala tkiva pacova, što se vidi iz dobijenih vrednosti kinetičkih parametara za hidrolizu ATP (Vmax (app)=3.973±0.280 μmol Pi/min/mg, Km (app)=0.506±0.062 mM), odnosno ADP (Vmax (app)=2.854±0.080 μmol Pi/min/mg, Km (app)=0.639±0.031 mM). Slične Km vrednosti za hidrolizu ATP i ADP, odnos početnih brzina hidrolize ATP/ADP (1:0.57), Chevillard-ova kriva kompeticije ATP i ADP, kao i inhibicija milimolarnim koncentracijama natrijum-azida (5-10 mM) ukazuju da je E-NTPDaza 1 dominantni enzim koji hidrolizuje vanćelijske nukleotide u uterusu pacova. RT-PCR i Western blot analizom pokazana je genska i proteinska ekspresija NTPDaze 1 i 2 u uterusu pacova. U daljem toku istraživanja ispitan je efekat i mehanizma delovanja dvovalentnih jona kadmijuma, žive(II) i bakra(II) na aktivnost E-NTPDaza iz uterusa pacova... ATP and other purine nucleotides are paracrine signaling molecules. Acting via purine receptors (P1 and P2) on the cell membrane, extracellular nucleotides participate in the control of contraction and modulation of the immune response in mammalian uterus. Disorder of uterine contractility may play an important role in the development of infertility, dysmenorrhea and premature labor. In this study, we examined the presence and kinetic properties of ectoenzymes that hydrolyze extracellular ATP and ADP in rat uterus (Wistar albino). Also, effects of heavy metal ions (cadmium, mercury, and copper) on the activity of ectoenzymes from rat uterus were studied in vitro, in the absence and presence of EDTA and L-cysteine. This study showed that hydrolysis of extracellular ATP and ADP in rat uterus is catalyzed by enzymes classified as the E-NTPDase family. The following results confirmed common properties characteristic for E-NTPDases: insensitivity to inhibitors of other membrane ATPase (oligomycin, ouabain, theophylline and levamisole), an alkaline pH optimum for both substrates (8.0-8.5), divalent cations dependence and significant inhibition by suramine. E-NTPDase activity in rat uterus appeared to be very high compared to other rat tissues. The apparent Km values were 0.506±0.062 and 0.639±0.031 mM, with a calculated Vmax (app) of 3.973±0.280 and 2.854±0.080 μmol Pi/min/mg for ATP and ADP, respectively. According to similar apparent Km values for both substrates, the ATP/ADP hydrolysis ratio (1:0:57), Chevillard competition plot, and inhibition by high milimolar concentrations of sodium-azide (5-10 mM), E-NTPDase 1 is dominant ATP/ADP hydrolyzing enzyme in uterine cell membranes. RT-PCR and Western blot analysis revealed gene and protein expression of NTPDaze 1 and 2 in rat uterus. In the further course of research, the effects and mechanism of action of divalent ions (mercury, cadmium and copper) on the activity of E-NTPDases from rat uterus were examined...

ATP and other purine nucleotides are paracrine signaling molecules. Acting via purine receptors (P1 and P2) on the cell membrane, extracellular nucleotides participate in the control of contraction and modulation of the immune response in mammalian uterus. Disorder of uterine contractility may play an important role in the development of infertility, dysmenorrhea and premature labor. In this study, we examined the presence and kinetic properties of ectoenzymes that hydrolyze extracellular ATP and ADP in rat uterus (Wistar albino). Also, effects of heavy metal ions (cadmium, mercury, and copper) on the activity of ectoenzymes from rat uterus were studied in vitro, in the absence and presence of EDTA and L-cysteine. This study showed that hydrolysis of extracellular ATP and ADP in rat uterus is catalyzed by enzymes classified as the E-NTPDase family. The following results confirmed common properties characteristic for E-NTPDases: insensitivity to inhibitors of other membrane ATPase (oligomycin, ouabain, theophylline and levamisole), an alkaline pH optimum for both substrates (8.0-8.5), divalent cations dependence and significant inhibition by suramine. E-NTPDase activity in rat uterus appeared to be very high compared to other rat tissues. The apparent Km values were 0.506±0.062 and 0.639±0.031 mM, with a calculated Vmax (app) of 3.973±0.280 and 2.854±0.080 μmol Pi/min/mg for ATP and ADP, respectively. According to similar apparent Km values for both substrates, the ATP/ADP hydrolysis ratio (1:0:57), Chevillard competition plot, and inhibition by high milimolar concentrations of sodium-azide (5-10 mM), E-NTPDase 1 is dominant ATP/ADP hydrolyzing enzyme in uterine cell membranes. RT-PCR and Western blot analysis revealed gene and protein expression of NTPDaze 1 and 2 in rat uterus. In the further course of research, the effects and mechanism of action of divalent ions (mercury, cadmium and copper) on the activity of E-NTPDases from rat uterus were examined... ATP i drugi purinski nukleotidi su parakrini signalni molekuli koji vezivanjem za purinske receptore (P1 i P2) na ćelijskoj membrani učestvuju u kontroli kontrakcija i modulaciji imunog odgovora u uterusu sisara. Poremećaji kontraktilnosti uterusa mogu imati važnu ulogu u nastanku neplodnosti, dismenoreje i prevremenog porođaja. U ovom radu ispitano je prisustvo i kinetičke osobine ektoenzima koji hidrolizuju vanćelijski ATP i ADP u uterusu pacova (Wistar albino). Takođe, ispitan je in vitro uticaj teških metala (kadmijuma, žive i bakra) kao i zaštitni efekat EDTA i L-cisteina na aktivnost ektoenzima u prisustvu jona metala. Rezultati ove studije pokazali su da hidrolizu vanćelijskog ATP i ADP u uterusu pacova katalizuju enzimi iz familije E-NTPDaza. To potvrđuju rezultati: neosetljivosti na inhibitore drugih membranskih ATPaza (oligomicin, ouabain, teofiln i levamisol); pH optimum za maksimalnu aktivnost enzima na alkalnom pH (8.0-8.5) za oba supstrata; zavisnost enzimske aktivnosti od dvovalentnih jona i inhibicija suraminom. Izmerana aktivnost E-NTPDaza u uterusu je veoma visoka u odnosu na ostala tkiva pacova, što se vidi iz dobijenih vrednosti kinetičkih parametara za hidrolizu ATP (Vmax (app)=3.973±0.280 μmol Pi/min/mg, Km (app)=0.506±0.062 mM), odnosno ADP (Vmax (app)=2.854±0.080 μmol Pi/min/mg, Km (app)=0.639±0.031 mM). Slične Km vrednosti za hidrolizu ATP i ADP, odnos početnih brzina hidrolize ATP/ADP (1:0.57), Chevillard-ova kriva kompeticije ATP i ADP, kao i inhibicija milimolarnim koncentracijama natrijum-azida (5-10 mM) ukazuju da je E-NTPDaza 1 dominantni enzim koji hidrolizuje vanćelijske nukleotide u uterusu pacova. RT-PCR i Western blot analizom pokazana je genska i proteinska ekspresija NTPDaze 1 i 2 u uterusu pacova. U daljem toku istraživanja ispitan je efekat i mehanizma delovanja dvovalentnih jona kadmijuma, žive(II) i bakra(II) na aktivnost E-NTPDaza iz uterusa pacova...

α-Dikarbonilna jedinjenja su veoma reaktivna, reaguju sa nukleofilnim grupama bocnih ostataka Lys i Arg, N-terminalnom amino-grupom i tiol-grupom ostatka Cys na površini molekula proteina i tako ih modifikuju. U disertaciji je ispitan doprinos tiolgrupe modifikaciji proteina metilglioksalom (MG), kinetika i konkurentnost ove reakcije u odnosu na reakcije amino- i guanidino-grupe. Nađeno je da i pored male zastupljenosti tiol-grupe na površni molekula proteina (kod HSA oko 80 puta manja u odnosu na ukupan broj amino- i guanidino-grupa) ona reaguje u velikom procentu (do 65 %), ucestvuje u umrežavanju molekula proteina (stvaranju dimera i oligomera) preko nagrađenih HSA-MG (HSA-amino-MG ili HSA-SH-MG) intermedijera sa udelom od 4% u slucaju nedovoljne kolicine metilglioksala Povecanje koncentracije metilglioksala utice na brzinu reakcije sa tiol-grupom, na vreme uspostavljanja ravnoteže, na doprinos tiol-grupe modifikaciji molekula proteina, ali ne utiče na procenat izreagovanih grupa u stanju ravnoteže (60 %), što omogucava ekstrapolaciju dobijenih rezultata na niže, fiziološke koncentracije metilglioksala. U okviru teze su razvijene spektrofotometrijske metode zasnovane na reakciji amino-grupa sa p-benzohinonom i guanidino-grupa sa reagensom koji sadrži timol i natrijum-hipobromit. Metode su precizne (RSD u opsegu 1.2-1.8 %, odnosno 0.9–2.0 %, resp.) i tačne (100.6 ± 1.2 %, 99.8 ± 0.8 %, redom), što ih cini pogodnim za kvantifikaciju ovih grupa za vreme karbonilovanja in vitro. Uticaj mikrookoline tiol-grupe na njenu reakciju sa metilglioksalom ispitan je sa aminokiselinama (Cys, NAcCys i CMC), peptidom (GSH) i proteinom (HSA). Utvrđeno je da mikrookolina –SH grupe utice na njenu reaktivnost i na prinos proizvoda reakcije. Reaktivnost tiol-grupe opada u nizu Cys>GSH>NAcCys, za sve ispitivane odnose koncentracija reaktanata. CMC ne reaguje sa metilglioksalom. Prisustvo α- amino-β-merkapto-etanske grupe u tiol-jedinjenju povecava reaktivnost tiol-grupe. Predožen je reakcioni put za reakciju Cys i metilglioksala po kome slobodna α- amino-grupa molekula Cys (cija je tiol-grupa u reakciji sa metilglioksalom vec nagradila hemitioacetal), reaguje sa slobodnom acetil-karbonilnom grupom hemitioacetala. Stvara se tiazolidinski intermedijer, koji dalje prelazi u stabilniji proizvod Cys-MG-glikozilamin... α-Dicarbonyles are reactive compounds that react with the nucleophilic groups of Lys and Arg side chains, thiol group of Cys and N-terminal amino on the protein surface, modifying them. In this thesis, the contribution of thiol group to protein modification with methylglyoxal (MG), kinetics and competitivness of this reaction with those of amino and guanidino groups reactions were inestigated. Although less abundant, thiol groups present on the protein surface (approx. 80 times lower then total number of amino and guanidino groups in HSA), are found to be reactive in considerable percentage (up to 65 %) and are participated in protein cross-linking (dimer and oligomer production), through HSA-MG intermediates (HSA-amino-MG or HSA-SH-MG), with 4% in case of unsufficient amount of MG. Increase in methylglyoxal concentration influences the reaction rate with thiol group, the time for the reaction to reach equilibrium and the contribution of thiol group to modification of proteins, yet it has no influence on the percent of reacted groups (60 %) when chemical equilibrium is reached, enabling extrapolation of the obtained results to lower, physiological concentrations of methylglyoxal. Spectrophotometric methods, based on the reaction of amino groups with pbenzoquinone and guanidino groups with thimol and sodium hypobromite reagent, have been developed. The methods are precise (RSD 1.2-1.8 % and 0.9–2.0 %, respectively) and accurate (100.6±1.2 %; 99.8±0.8 %, resp.), and therefore suitable for quantification of these groups during carbonylation process in vitro. The influence of microenviroment of thiol group on its reactivity with MG has been examined using amino acids (Cys, NAcCys and CMC), peptide (GSH) and protein (HSA). It has been shown that microenviroment of thiol group influences its reactivity and the product yield. The reactivity of thiol group decreases in the order Cys>GSH>NAcCys, for all concentration ratios examined. CMC doesn’t react with MG. The presence of α-amino-β-mercapto ethane group in thiol compound increases the reactivity of thiol group. The reaction pathway for Cys and MG reaction was proposed, in which the free α-amino group of Cys (whose thiol group is already in the form of hemithioacetale with MG) reacts with the free acetil carbonyl group of hemithioacetale...

α-Dikarbonilna jedinjenja su veoma reaktivna, reaguju sa nukleofilnim grupamabocnih ostataka Lys i Arg, N-terminalnom amino-grupom i tiol-grupom ostatka Cys napovršini molekula proteina i tako ih modifikuju. U disertaciji je ispitan doprinos tiolgrupemodifikaciji proteina metilglioksalom (MG), kinetika i konkurentnost overeakcije u odnosu na reakcije amino- i guanidino-grupe. Nađeno je da i pored malezastupljenosti tiol-grupe na površni molekula proteina (kod HSA oko 80 puta manja uodnosu na ukupan broj amino- i guanidino-grupa) ona reaguje u velikom procentu (do65 %), ucestvuje u umrežavanju molekula proteina (stvaranju dimera i oligomera) prekonagrađenih HSA-MG (HSA-amino-MG ili HSA-SH-MG) intermedijera sa udelom od4% u slucaju nedovoljne kolicine metilglioksala Povecanje koncentracije metilglioksalautice na brzinu reakcije sa tiol-grupom, na vreme uspostavljanja ravnoteže, na doprinostiol-grupe modifikaciji molekula proteina, ali ne utiče na procenat izreagovanih grupa ustanju ravnoteže (60 %), što omogucava ekstrapolaciju dobijenih rezultata na niže,fiziološke koncentracije metilglioksala.U okviru teze su razvijene spektrofotometrijske metode zasnovane na reakcijiamino-grupa sa p-benzohinonom i guanidino-grupa sa reagensom koji sadrži timol inatrijum-hipobromit. Metode su precizne (RSD u opsegu 1.2-1.8 %, odnosno 0.9–2.0%, resp.) i tačne (100.6 ± 1.2 %, 99.8 ± 0.8 %, redom), što ih cini pogodnim zakvantifikaciju ovih grupa za vreme karbonilovanja in vitro.Uticaj mikrookoline tiol-grupe na njenu reakciju sa metilglioksalom ispitan je saaminokiselinama (Cys, NAcCys i CMC), peptidom (GSH) i proteinom (HSA).Utvrđeno je da mikrookolina –SH grupe utice na njenu reaktivnost i na prinos proizvodareakcije. Reaktivnost tiol-grupe opada u nizu Cys>GSH>NAcCys, za sve ispitivaneodnose koncentracija reaktanata. CMC ne reaguje sa metilglioksalom. Prisustvo α-amino-β-merkapto-etanske grupe u tiol-jedinjenju povecava reaktivnost tiol-grupe.Predožen je reakcioni put za reakciju Cys i metilglioksala po kome slobodna α-amino-grupa molekula Cys (cija je tiol-grupa u reakciji sa metilglioksalom vecnagradila hemitioacetal), reaguje sa slobodnom acetil-karbonilnom grupomhemitioacetala. Stvara se tiazolidinski intermedijer, koji dalje prelazi u stabilniji proizvod Cys-MG-glikozilamin... α-Dicarbonyles are reactive compounds that react with the nucleophilic groupsof Lys and Arg side chains, thiol group of Cys and N-terminal amino on the proteinsurface, modifying them. In this thesis, the contribution of thiol group to proteinmodification with methylglyoxal (MG), kinetics and competitivness of this reactionwith those of amino and guanidino groups reactions were inestigated. Although lessabundant, thiol groups present on the protein surface (approx. 80 times lower then totalnumber of amino and guanidino groups in HSA), are found to be reactive inconsiderable percentage (up to 65 %) and are participated in protein cross-linking(dimer and oligomer production), through HSA-MG intermediates (HSA-amino-MG orHSA-SH-MG), with 4% in case of unsufficient amount of MG. Increase inmethylglyoxal concentration influences the reaction rate with thiol group, the time forthe reaction to reach equilibrium and the contribution of thiol group to modification ofproteins, yet it has no influence on the percent of reacted groups (60 %) when chemicalequilibrium is reached, enabling extrapolation of the obtained results to lower,physiological concentrations of methylglyoxal.Spectrophotometric methods, based on the reaction of amino groups with pbenzoquinoneand guanidino groups with thimol and sodium hypobromite reagent, havebeen developed. The methods are precise (RSD 1.2-1.8 % and 0.9–2.0 %, respectively)and accurate (100.6±1.2 %; 99.8±0.8 %, resp.), and therefore suitable for quantificationof these groups during carbonylation process in vitro.The influence of microenviroment of thiol group on its reactivity with MG hasbeen examined using amino acids (Cys, NAcCys and CMC), peptide (GSH) and protein(HSA). It has been shown that microenviroment of thiol group influences its reactivityand the product yield. The reactivity of thiol group decreases in the orderCys>GSH>NAcCys, for all concentration ratios examined. CMC doesn’t react withMG. The presence of α-amino-β-mercapto ethane group in thiol compound increases thereactivity of thiol group.The reaction pathway for Cys and MG reaction was proposed, in which the freeα-amino group of Cys (whose thiol group is already in the form of hemithioacetale with MG) reacts with the free acetil carbonyl group of hemithioacetale...

Polyunsaturated fatty acids, especially eicosapentanoic acid (EPA) and dosahexanoic acid (DHA) which are the content of fish oil and belong to the n-3 fatty acid family. n-3 are known to have effect on physiological parameters. The balance of the n-6/n-3 ratio in phospholipids, as well as the balance between reactive oxygen species and reactive nitric species on the one side and on the anti oxidative defense on the other is important for the normal function of organism. Wistar rats (3 and 22 months) were supplemented with fish oil (EPA+DHA and vitamin E) over the period of 6 weeks. Biochemical parameters in plasma, fatty acid phospholipids profiles and the parameters of oxidative stress in blood and liver were examined. In young rats plasma concentrations of cholesterol, HDL-cholesterol and triglycerides in plasma were increased compared to aged rats. Fish oil treatment increased HDL-cholesterol, uric acid and LDL-cholesterol in both groups of animals (3 and 22 months). Treatment decreased triglycerides concentrations in young rats and decreased plasma cholesterol in aged rats. Senescence increased LA, AA and n-6 FA. Fish oil treatment increased EPA, DPA, decreased AA in plasma phospholipids in both group of animals. In young rats treatment increased oleic acid while in aged it increased DGLA. Senescence decreased n-3 FA and the n-6/n-3 ratio in liver phospholipids. Treatment increased EPA, DPA, n-3 and decreased AA in liver phospholipids in both group of animals. In young rats, treatment increased MUFA while in aged rats it increased SFA and PUFA. Senescence also increased lipid peroxidation in liver cells, as well as SOD activity and nitrite concentration while the activity of PON1 in plasma is increased in young rats compared to aged Wistar rats. Fish oil treatment in both groups of rats (3 and 22 months) decreased lipid peroxidation, concentration of plasma nitrite and LDH5 and increased SOD, CAT, PON1 activity in blood. Treatment decreased lipid peroxidation, nitrite concentration in liver of both groups of rats. In young rats treatment also decreased CAT activity and increased PON1. In aged rats treatment increased CAT, SOD activity, concentration of SH groups while PON activity decreased compared to control... Polinezasićene masne kiseline, posebno eikozapentaenska (EPA) i dokozaheksaenska (DHA) masna kiselina koje ulaze u sastav ribljeg ulja i pripadaju n-3 familiji masnih kiselina, imaju efekte na čitav niz fizioloških parametara. Značaj balansa odnosa n-6/n-3 masnih kiselina u fosfolipidima kao i balans izmeñu produkcije reaktivnih kiseoničnih vrsta i reaktivnih azotnih vrsta s jedne i antoksidativne zaštite s druge strane je od važnosti za normalno funkcionisanje organizma. Suplementirali smo pacove Wistar soja dve starosne dobi (3 i 22 meseca) u trajanju od 6 nedelja sa ribljim uljem (EPA+DHA i vitamin E). Ispitivani su biohemijski parametri plazme, masnokiselinski profili fosfolipida plazme i jetre kao i parametri oksidativnog stresa u krvi i jetri pomenutih životinja. Dobijeni rezultati su pokazali više vrednosti holesterola, HDL- holesterola i triglicerida u plazmi mladih pacova u odnosu na stare. Tretman ribljim uljem doveo je do povišenja HDL-holesterola, koncentracije mokraćne kiseline i sniženja LDL-holesterola kod obe grupe tretiranih životinja dok je kod mladih tretman izazvao sniženje koncentracije triglicerida a kod starih sniženje holesterola. Starenjem, povećava se sadržaj linolne, arahidonske kiseline kao i ukupnih n-6 MK. Tretman ribljim uljem, doveo je do povećanja sadržaja EPA, DPA, sniženja koncentracije arahidonske kiseline u fosfolipidima plazme u obe starosne grupe životinja. Sadržaj oleinske kiseline je kod mladih pacova tretmanom povižen, dok je kod starih pacova došlo do povećanja dihomo-γ- linolenske kiseline. Starenjem, snižava se i sadržaj ukupnih n-3 MK i odnos n-6/n-3 u fosfolipidima jetre. Tretman u životinja obe starosne dobi doveo je do povećanja sadržaja EPA, DPA, ukupnih n-3, smanjenja sadržaja arahidonske kiseline u fosfolipidima jetre. Kod mladih tretman je povećao sadržaj MUFA, a kod starih SFA i PUFA. Starenjem, povećava se lipidna peroksidacija u hepatocitima, kao i aktivnost SOD i koncentracija nitrita, dok je aktivnost PON1 u plazmi veća kod mladih pacova što je u korelaciji sa višim vrednostima HDL-holesterola u odnosu na stare pacove. Tretman ribljim uljem kod pacova obe starosne dobi doveo je do sniženja lipidne peroksidacije, koncentracije plazma nitrita i zastupljenosti LDH5 izoenzimskog oblika a do povišenja aktivnosti SOD, CAT i PON1 u krvi tretiranih životinja u odnosu na kontrolnu grupu...

Sisаri su izlоžеni еndоgеnim i еgzоgеnim izvоrimа аzоt mоnоksidа (NO). U biоlоškој srеdini NO pоdlеžе kоmplеksnim rеаkciјаmа u kојimа nаstајu vrstе rеаktivniје оd sаmоg аzоt mоnоksidа (RNOC), kоје su uklјučеnе u mnоgе fiziоlоškе i (nаrоčitо) pаtоfiziоlоškе prоcеsе. Оvе rеаkciје оbuhvаtајu i kоnvеrziјu NO u njеgоvе rеаktivnе srоdnikе, nitrоzоniјum јоn (NO+) i nitrоksilni (HNO/NO-) аnјоn, kојi mоgu rеаgоvаti sа rаznim cilјnim mоlеkulimа u biоlоškim sistеmimа štо imа vаžnu ulоgu u NO pоsrеdоvаnim prоcеsimа. Prеdlоžеnо је dа u biоlоškim sistеmimа NO prеlаzi u NO+ ili HNO/NO- vrstе оksidаciјоm оdnоsnо rеdukciјоm NO pоmоću mеtаlnih јоnа. Мi smо pоstаvili hipоtеzu dа kаtаlizоm pоmоću SOD i "slоbоdnоg" gvоžđа NO mоžе dа dismutuје (2NO → NO+ + HNO/NO-) dајući оbе vrstе. U prvоm dеlu rаdа ispitivаn је еfеkаt NO nа аktivnоst svе tri klаsе SOD: gоvеđu Cu/ZnSOD, MnSOD (E.coli), FeSOD (E.coli) i FeSOD (P.leiognathi), а dеtаlјnо su ispitаnе mоdifikаciје MnSOD nаkоn trеtirаnjа sа NO. Nаđеnо је dа је Cu/ZnSOD stаbilnа pri inkubirаnju sа NO: čаk i pri prоdužеnоm inkubirаnju niје dоšlо dо znаčајnih prоmеnа u strukturi i аktivnоsti еnzimа. Inkubirаnjе FeSOD i MnSOD sа NO dоvоdi dо brzе i znаtnе inаktivаciје еnzimа. Pоkаzаnо је dа inаktivаciја MnSOD nаstаје kао pоslеdicа еkstеnzivnih mоdifikаciја mоlеkulа еnzimа: frаgmеntаciје pоlipеptidnоg nizа nа оstаcimа histidinа u аktivnоm cеntru, mоdifikаciја аminо grupа, nitrоvаnjа i оksidаciје оstаtаkа tirоzinа. Dоkumеntоvаnо је dа FeSOD i MnSOD kаtаlizuјu dismutаciјu NO u NO+ i NO- vrstе kоје izаzivајu mоdifikаciјu i inаktivаciјu еnzimа. U drugоm dеlu rаdа ispitivаnа је spоsоbnоst trаgоvа јоnа gvоžđа nа dismutаciјu NO. Nаstајаnjе NO+ vrstа rеzultuје u sintеzi S-nitrоzоtiоlа (RSNO) i nitritа, а HNO/NO- u nаstајаnju hidrоksilаminа. Hidrоksilаmin је dеtеktоvаn u rаstvоrimа јоnа gvоžđа inkubirаnim sа NO u prisustvu cistеinа, аli nе i glutаtiоnа (GSH). Pоkаzаnо је dа dоdаtаk urаtа, dоminаntnоg аgеnsа zа vеzivаnjе "slоbоdnоg" gvоžđа, u rаstvоr glutаtiоnа i јоnа gvоžđа prе njihоvоg inkubirаnjа sа NO, dоvоdi dо pоvеćаnе sintеzе GSNO i nаstајаnjа hidrоksilаminа, uz gubitаk urаtа kојi prеlаzi u nitrоzо/nitrо prоizvоdе kојi nisu zа sаdа idеntifikоvаni. Utvrđеnо је dа GSH štiti urаt оd rаspаdаnjа; еfеkаt zаvisi оd оdnоsа GSH:urаt. U nеutrаlnim rаstvоrimа prоtеinа i јоnа gvоžđа inkubirаnih sа NO u prisustvu (аli nе i u оdsustvu) urаtа dеtеktоvаn је hidrоksilаmin i S-nitrоzоvаnjе prоtеinа. RNOS gеnеrisаnе kао pоslеdicа dismutаciје NO u rаstvоrimа tirоzinа i јоnа gvоžđа izаzivајu nitrоvаnjе i оksidаciјu tirоzinа. U trеćеm dеlu је u in vitro еkspеrimеntimа ispitivаnа dismutаciја NO kаtаlizоvаnа "slоbоdnim" gvоžđеm u cеrеbrоspinаlnој tеčnоsti (CSF) zdrаvih оsоbа i оsоbа оbоlеlih оd аmiоtrоfičnе lаtеrаlnе sklеrоzе (SALS). Nivо аntiоksidаnаtа, prоtеinskih SH grupа, аskоrbаtа i urаtа, kојi prеdstаvlјајu ligаndе zа dinitrоzil-gvоžđе kоmplеksе (DNIC) sа "slоbоdnim" gvоžđеm i/ili hvаtаčе NO+ i HNO/NO- vrstа је prоmеnjеn kоd SALS pаciјеnаtа. Dа "slоbоdnо" gvоžđе kаtаlizuје dismutаciјu NO је dоkаzаnо nаstајаnjеm RSNO i hidrоksilаminа u CSF uzоrcimа inkubirаnim sа NO in vitro, оdnоsnо pоtpunоm inhibiciјоm оvih rеаkciја pоmоću о-fеnаntrоlinа, hеlаtоrа zа јоnе gvоžđа. Pоkаzаnо је dа RNOS gеnеrisаnе dismutаciјоm NO rеаguјu sа аskоrbаtоm i urаtоm, štо dоvоdi dо njihоvоg gubitkа, dа prisustvо urаtа pоvеćаvа kаpаcitеt "slоbоdnоg" gvоžđа dа kаtаlizuје NO dismutаciјu, dоk је аskоrbаt bоlјi hvаtаč оd urаtа zа gеnеrisаnе RNOS. Vеći prinоs RSNO, nitritа, hidrоksilаminа i 3-nitrоtirоzinа u CSF uzоrcimа SALS pаciјеnаtа inkubirаnih sа NO u оdnоsu nа kоličinе nаđеnе u kоntrоlnim CSF uzоrcimа mоžе dа sе оbјаsni pоvеćаnim nivооm urаtа i smаnjеnim nivооm аskоrbаtа u CSF uzоrcimа SALS pаciјеnаtа. Rеzultаti prеzеntirаni u оvоm rаdu ubеdlјivо pоkаzuјu dа mеtаl-pоsrеdоvаnа dismutаciја NO u biоlоškim uslоvimа nе sаmо dа је mоgućа nеgо је i fiziоlоški rеlеvаntnа. Zbоg tоgа fiziоlоški znаčај mеtаl pоsrеdоvаnе dismutаciје NO zаslužuје dаlја istrаživаnjа. Mammalian systems are exposed to nitric oxide (NO) through both exogenous and endogenous sources. Once formed, NO undergoes a complex series of reactions which lead to formation of several species more reactive than NO itself. Reactive nitrogen species (RNOS) derived from NO have been implicated in numerous physiological and (particularly) pathophysiological processes. These include NO conversion into its reactive congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, which both could react with various target molecules in biological systems with important consequences for NO mediated processes. It was suggested that in a biological system NO may be converted either to NO+ or HNO/NO- species by metal-catalyzed oxidation or reduction of NO, respectively. We hypothesized that the conversion of NO into both NO+ and HNO/NO- species may occur through the metal assisted NO dismutation (2NO → NO+ + HNO/NO-) catalyzed by SODs and "free" iron. The effect of NO treatment in vitro on structural and functional alterations of Cu/Zn, Mn, and Fe type of SODs was studied. Significant difference in response to NO of Cu/ZnSOD compared to the Mn and Fe types was demonstrated. Cu/ZnSOD was shown to be stable with respect to NO: even on prolonged exposure, NO produced negligible effect on its structure and activity. In contrast, exposure to NO led to fast and extensive inactivation of both Mn and Fe types. Inactivation of MnSOD was demonstrated to be due to the extensive structural alterations, including the cleavage of enzyme polypeptide chains, presumably at His residues of the enzyme metal binding sites, amino groups modifications, tyrosine residue nitration and oxidation. The generation of nitrosonium and nitroxyl ions in NO treated Mn and FeSODs, which produce enzyme modifications and inactivation, was demonstrated. Dinitrosyl iron complex (DNIC) initiated iron ion catalyzed NO dismutation into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) and nitrite, and into HNO/NO- to produce hydroxylamine was demonstrated. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione. The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. NO treatment of a neutral solution containing proteins and iron ions in the presence of urate, but not in its absence, resulted in hydroxylamine formation and protein S-nitrosation. The consequences of NO exposure in vitro were tested on cerebrospinal fluid (CSF), system containing "free" iron and constituents such as protein-SH groups, ascorbate and urate which represent ligands and/or scavengers of both NO+ and HNO/NO- species. The results demonstrate that "free" iron in CSF can catalyze NO dismutation as evidenced from the formation of both RSNO and hydroxylamine in CSF exposed to NO in vitro which was inhibited upon addition of iron ion chelator o-phenanthroline. NO treatment caused loss of CSF ascorbate and urate. Urate and ascorbate levels were found to affect both "free" iron ion catalyzed NO dismutation as well as the reactions of protein -SH groups with NO+ and HNO/NO- species. Results described in this work suggest that metal/assisted NO dismutation into NO+ and HNO/NO- species in biological systems may be feasible and possibly physiologically relevant.

Sisаri su izlоžеni еndоgеnim i еgzоgеnim izvоrimа аzоt mоnоksidа (NO). U biоlоškој srеdini NO pоdlеžе kоmplеksnim rеаkciјаmа u kојimа nаstајu vrstе rеаktivniје оd sаmоg аzоt mоnоksidа (RNOC), kоје su uklјučеnе u mnоgе fiziоlоškе i (nаrоčitо) pаtоfiziоlоškе prоcеsе. Оvе rеаkciје оbuhvаtајu i kоnvеrziјu NO u njеgоvе rеаktivnе srоdnikе, nitrоzоniјum јоn (NO+) i nitrоksilni (HNO/NO-) аnјоn, kојi mоgu rеаgоvаti sа rаznim cilјnim mоlеkulimа u biоlоškim sistеmimа štо imа vаžnu ulоgu u NO pоsrеdоvаnim prоcеsimа. Prеdlоžеnо је dа u biоlоškim sistеmimа NO prеlаzi u NO+ ili HNO/NO- vrstе оksidаciјоm оdnоsnо rеdukciјоm NO pоmоću mеtаlnih јоnа. Мi smо pоstаvili hipоtеzu dа kаtаlizоm pоmоću SOD i "slоbоdnоg" gvоžđа NO mоžе dа dismutuје (2NO → NO+ + HNO/NO-) dајući оbе vrstе. U prvоm dеlu rаdа ispitivаn је еfеkаt NO nа аktivnоst svе tri klаsе SOD: gоvеđu Cu/ZnSOD, MnSOD (E.coli), FeSOD (E.coli) i FeSOD (P.leiognathi), а dеtаlјnо su ispitаnе mоdifikаciје MnSOD nаkоn trеtirаnjа sа NO. Nаđеnо је dа је Cu/ZnSOD stаbilnа pri inkubirаnju sа NO: čаk i pri prоdužеnоm inkubirаnju niје dоšlо dо znаčајnih prоmеnа u strukturi i аktivnоsti еnzimа. Inkubirаnjе FeSOD i MnSOD sа NO dоvоdi dо brzе i znаtnе inаktivаciје еnzimа. Pоkаzаnо је dа inаktivаciја MnSOD nаstаје kао pоslеdicа еkstеnzivnih mоdifikаciја mоlеkulа еnzimа: frаgmеntаciје pоlipеptidnоg nizа nа оstаcimа histidinа u аktivnоm cеntru, mоdifikаciја аminо grupа, nitrоvаnjа i оksidаciје оstаtаkа tirоzinа. Dоkumеntоvаnо је dа FeSOD i MnSOD kаtаlizuјu dismutаciјu NO u NO+ i NO- vrstе kоје izаzivајu mоdifikаciјu i inаktivаciјu еnzimа. U drugоm dеlu rаdа ispitivаnа је spоsоbnоst trаgоvа јоnа gvоžđа nа dismutаciјu NO. Nаstајаnjе NO+ vrstа rеzultuје u sintеzi S-nitrоzоtiоlа (RSNO) i nitritа, а HNO/NO- u nаstајаnju hidrоksilаminа. Hidrоksilаmin је dеtеktоvаn u rаstvоrimа јоnа gvоžđа inkubirаnim sа NO u prisustvu cistеinа, аli nе i glutаtiоnа (GSH). Pоkаzаnо је dа dоdаtаk urаtа, dоminаntnоg аgеnsа zа vеzivаnjе "slоbоdnоg" gvоžđа, u rаstvоr glutаtiоnа i јоnа gvоžđа prе njihоvоg inkubirаnjа sа NO, dоvоdi dо pоvеćаnе sintеzе GSNO i nаstајаnjа hidrоksilаminа, uz gubitаk urаtа kојi prеlаzi u nitrоzо/nitrо prоizvоdе kојi nisu zа sаdа idеntifikоvаni. Utvrđеnо је dа GSH štiti urаt оd rаspаdаnjа; еfеkаt zаvisi оd оdnоsа GSH:urаt. U nеutrаlnim rаstvоrimа prоtеinа i јоnа gvоžđа inkubirаnih sа NO u prisustvu (аli nе i u оdsustvu) urаtа dеtеktоvаn је hidrоksilаmin i S-nitrоzоvаnjе prоtеinа. RNOS gеnеrisаnе kао pоslеdicа dismutаciје NO u rаstvоrimа tirоzinа i јоnа gvоžđа izаzivајu nitrоvаnjе i оksidаciјu tirоzinа. U trеćеm dеlu је u in vitro еkspеrimеntimа ispitivаnа dismutаciја NO kаtаlizоvаnа "slоbоdnim" gvоžđеm u cеrеbrоspinаlnој tеčnоsti (CSF) zdrаvih оsоbа i оsоbа оbоlеlih оd аmiоtrоfičnе lаtеrаlnе sklеrоzе (SALS). Nivо аntiоksidаnаtа, prоtеinskih SH grupа, аskоrbаtа i urаtа, kојi prеdstаvlјајu ligаndе zа dinitrоzil-gvоžđе kоmplеksе (DNIC) sа "slоbоdnim" gvоžđеm i/ili hvаtаčе NO+ i HNO/NO- vrstа је prоmеnjеn kоd SALS pаciјеnаtа. Dа "slоbоdnо" gvоžđе kаtаlizuје dismutаciјu NO је dоkаzаnо nаstајаnjеm RSNO i hidrоksilаminа u CSF uzоrcimа inkubirаnim sа NO in vitro, оdnоsnо pоtpunоm inhibiciјоm оvih rеаkciја pоmоću о-fеnаntrоlinа, hеlаtоrа zа јоnе gvоžđа. Pоkаzаnо је dа RNOS gеnеrisаnе dismutаciјоm NO rеаguјu sа аskоrbаtоm i urаtоm, štо dоvоdi dо njihоvоg gubitkа, dа prisustvо urаtа pоvеćаvа kаpаcitеt "slоbоdnоg" gvоžđа dа kаtаlizuје NO dismutаciјu, dоk је аskоrbаt bоlјi hvаtаč оd urаtа zа gеnеrisаnе RNOS. Vеći prinоs RSNO, nitritа, hidrоksilаminа i 3-nitrоtirоzinа u CSF uzоrcimа SALS pаciјеnаtа inkubirаnih sа NO u оdnоsu nа kоličinе nаđеnе u kоntrоlnim CSF uzоrcimа mоžе dа sе оbјаsni pоvеćаnim nivооm urаtа i smаnjеnim nivооm аskоrbаtа u CSF uzоrcimа SALS pаciјеnаtа. Rеzultаti prеzеntirаni u оvоm rаdu ubеdlјivо pоkаzuјu dа mеtаl-pоsrеdоvаnа dismutаciја NO u biоlоškim uslоvimа nе sаmо dа је mоgućа nеgо је i fiziоlоški rеlеvаntnа. Zbоg tоgа fiziоlоški znаčај mеtаl pоsrеdоvаnе dismutаciје NO zаslužuје dаlја istrаživаnjа. Mammalian systems are exposed to nitric oxide (NO) through both exogenous and endogenous sources. Once formed, NO undergoes a complex series of reactions which lead to formation of several species more reactive than NO itself. Reactive nitrogen species (RNOS) derived from NO have been implicated in numerous physiological and (particularly) pathophysiological processes. These include NO conversion into its reactive congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, which both could react with various target molecules in biological systems with important consequences for NO mediated processes. It was suggested that in a biological system NO may be converted either to NO+ or HNO/NO- species by metal-catalyzed oxidation or reduction of NO, respectively. We hypothesized that the conversion of NO into both NO+ and HNO/NO- species may occur through the metal assisted NO dismutation (2NO → NO+ + HNO/NO-) catalyzed by SODs and "free" iron. The effect of NO treatment in vitro on structural and functional alterations of Cu/Zn, Mn, and Fe type of SODs was studied. Significant difference in response to NO of Cu/ZnSOD compared to the Mn and Fe types was demonstrated. Cu/ZnSOD was shown to be stable with respect to NO: even on prolonged exposure, NO produced negligible effect on its structure and activity. In contrast, exposure to NO led to fast and extensive inactivation of both Mn and Fe types. Inactivation of MnSOD was demonstrated to be due to the extensive structural alterations, including the cleavage of enzyme polypeptide chains, presumably at His residues of the enzyme metal binding sites, amino groups modifications, tyrosine residue nitration and oxidation. The generation of nitrosonium and nitroxyl ions in NO treated Mn and FeSODs, which produce enzyme modifications and inactivation, was demonstrated. Dinitrosyl iron complex (DNIC) initiated iron ion catalyzed NO dismutation into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) and nitrite, and into HNO/NO- to produce hydroxylamine was demonstrated. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione. The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. NO treatment of a neutral solution containing proteins and iron ions in the presence of urate, but not in its absence, resulted in hydroxylamine formation and protein S-nitrosation. The consequences of NO exposure in vitro were tested on cerebrospinal fluid (CSF), system containing "free" iron and constituents such as protein-SH groups, ascorbate and urate which represent ligands and/or scavengers of both NO+ and HNO/NO- species. The results demonstrate that "free" iron in CSF can catalyze NO dismutation as evidenced from the formation of both RSNO and hydroxylamine in CSF exposed to NO in vitro which was inhibited upon addition of iron ion chelator o-phenanthroline. NO treatment caused loss of CSF ascorbate and urate. Urate and ascorbate levels were found to affect both "free" iron ion catalyzed NO dismutation as well as the reactions of protein -SH groups with NO+ and HNO/NO- species. Results described in this work suggest that metal/assisted NO dismutation into NO+ and HNO/NO- species in biological systems may be feasible and possibly physiologically relevant.