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U okviru ovog rada, pokušali smo da modifikujemo papirnu elektroforezu, agaroznu gel-elektroforezu i PAGE tako da se uz pomoć njih može precizno raditi analiza kiselih mukopolisaharida. Dobijeni uzorak je nanošen u različitim koncentracijama na medijum (papir ili neki od gelova) i elektroforeza je raĎena prema pažljivo adaptiranom protokolu. Finalni ishod je pokazao da je papirna elektroforeza nedovoljno rezolutivna tehnika za analitiku kiselih mukopolisaharida. Agarozna gel-elektroforeza je znatno rezolutivnija od papirne, meĎutim je zbog fragilnosti gela i polisaharidne prirode samog gela manje zahvalna za analitiku MPS, za razliku od PAGE koja je pokazala izuzetan potencijal za analitiku kiselih MPS.

Сумпор-сумпор интеракције су препознате у многим молекулским системима, где су одговорне за молекулску структуру и функцију многих познатих неорганских и органских материјала као и протеина. У овој докторској дисертацији проучаване су геометрије и енергије сумпор-сумпор интеракција анализом података из кристалних структура и применом квантнохемијских прорачуна високог нивоа теорије... Sulfur-sulfur interactions have been recognized in various molecular systems, where they are responsible for the molecular structure and function of many well known inorganic and organic moleculs and proteins. This doctoral study investigates the geometries and energies of sulfur-sulfur interactions by analyzing data obtained from crystal structures and by high-level quantum chemical calculations...

Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical isolates based on its QS inhibitory activity against P. aeruginosa MMA83 clinical isolate. Whole genome sequencing identified this isolate as D. tsuruhatensis and revealed genetic armamentarium of virulence factors and antibiotic resistance determinants. Ethyl acetate extract of D. tsuruhatensis 11304 culture supernatant (QSI extract) prevented biofilm formation of P. aeruginosa MMA83, but was unable to cause biofilm decomposition. QSI extract showed a synergistic effect in combination with meropenem and gentamycin, against P. aeruginosa MMA83. A dose-dependent reduction of the virulence factors: elastase, rhamnolipid and pyocyanin production by P. aeruginosa MMA83 and significant downregulation of lasI, lasR, rhlI, rhlR, pqs and mvfR expression were observed. Matrix-assisted Laser Desorption Ionization (MALDI) mass spectrometry of D. tsuruhatensis 11304 QSI extract revealed the presence of N-acyl homoserine lactones (AHL) with chain lengths of C12 to C18. The main ion peak was identified as N-octadecanoylhomoserine lactone (C18-HSL). Commercial C18-HSL (20 µM) reduced pyocyanin production as well as mRNA level of the lasI gene. A novel AHL species, dihydroxy-N-octadecanoylhomoserine lactone, was also described.

Celobioza – dehidrogenaza poreklom iz Phanerochaete chrysosporium, gljive bele truleži, pripada ekstracelularnim oksidoredukcionim enzimima i katalizuje oksidaciju β – 1,4 – glikozidno vezanih oligosaharida poput celobioze i laktoze. Oksidacijom laktoze dolazi do formiranja laktobionske kiseline koja pronalazi veliku primenu u farmaceutskoj i kozmetičkoj industriji gde se koristi prilikom distribucije lekova i za hidrataciju kože kao sastavni deo različitih krema, gde zajedno sa hijaluronskom kiselinu ima ulogu u smanjenju bora. Celobioza – dehidrogenaza prilikom oksidacije laktoze ili celobioze, kao prirodnih supstrata, katalizuje redukciju jednog dvoelektronskog ili dva jednoelektronska akceptora elektrona. Jedan od najkorišćenijih dvoelektronskih akceptora elektrona je upravo dihlor fenol indofenol (DCIP), dok jednoelektronski akceptori elektrona mogu biti citohrom c, ABTS, ali i Fe3+ i Mn3+ joni. Redukcijom Fe3+ jona u prisustvu molekulskog kiseonika dolazi do formiranja vodonik peroksida i posredstvom Fentonove reakcije do generisanja hidroksil radikala. Polazeći od ove činjenice, iskoristili smo upravo Fentonovu reakciju za razvoj fluorescentnog eseja za visoko efikasnu pretragu biblioteka gena celobioza – dehidrogenaze, baziranog na detekciji proizvedenih hidroksil radikala fluorogenom probom aminofenil – fluoresceinom (APF). Primena celobioza – dehidrogenaze u konstruisanju biosenzora i biogorivnih ćelija leži upravo u njenoj sposobnosti da katalizuje oksidaciju laktoze, celobioze i β – 1,4 – vezanih oligosaharida do odgovarajućih laktona koji potom spontano hidrolizuju do aldonskih kiselina. Enzimi koji nalaze primenu u konstruisanju biosenzora i biogorivnih ćelija, moraju da zadovoljavaju nekoliko kriterijuma, odnosno moraju da imaju veliku osetljivost i supstratnu specifičnost, ali i da pokazuju povećanu stabilnost... Cellobiose dehydrogenase from Phanerochaete chrysosporium, a white rot fungus, belongs to the extracellular oxidoreductive group of enzymes and catalyzes the oxidation of the β – 1,4 – glycoside bond of oligosaccharides such as cellobiose and lactose. During oxidation of lactose, formation of lactobionic acid occurs which has many applications in pharmaceutical and cosmetic industry. Such applications include the distribution of medicine, a component responsible for skin hydration and when combined with hyaluronic acid as an agent against wrinkles. During oxidation of lactose or cellobiose by cellobiose dehydrogenase, reduction catalysis occurs of one two electron or two one electron acceptors. One of the most utilized two electron acceptors is DCIP, while one electron acceptors are usually cytochrome c, ABTS, Fe3+ and Mn3+ ions. During reduction of Fe3+ ions in the presence of molecular oxygen, H2O2 is formed and due to the Fenton reaction formation of hydroxyl radicals occurs. Due to this occurrence we wanted to use the Fenton reaction in order to develop a fluorescent assay based on the production of hydroxyl radicals and the fluorescence of aminophenyl fluorescein (APF). This would allow us to efficiently analyze cellobiose dehydrogenase gene libraries. With this fact in mind, the Fenton reaction was used to develop a fluorescent assay for the high throughput screening of cellobiose dehydrogenase genes, based on the detection of hydroxyl radicals with the fluorescent probe APF. The possible application of cellobiose dehydrogenase in the construction of various biosensors and biofuel cells is due the its ability to catalyze the oxidation of lactose, cellobiose and similar β – 1,4 – oligosaccharides do their corresponding lactones which then spontaneously hydrolyze to aldonic acids. Enzymes used in such applications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity...

Сумпор-сумпор интеракције су препознате у многим молекулским системима, где су одговорне за молекулску структуру и функцију многих познатих неорганских и органских материјала као и протеина. У овој докторској дисертацији проучаване су геометрије и енергије сумпор-сумпор интеракција анализом података из кристалних структура и применом квантнохемијских прорачуна високог нивоа теорије... Sulfur-sulfur interactions have been recognized in various molecular systems, where they are responsible for the molecular structure and function of many well known inorganic and organic moleculs and proteins. This doctoral study investigates the geometries and energies of sulfur-sulfur interactions by analyzing data obtained from crystal structures and by high-level quantum chemical calculations...

Celobioza – dehidrogenaza poreklom iz Phanerochaete chrysosporium, gljive bele truleži, pripada ekstracelularnim oksidoredukcionim enzimima i katalizuje oksidaciju β – 1,4 – glikozidno vezanih oligosaharida poput celobioze i laktoze. Oksidacijom laktoze dolazi do formiranja laktobionske kiseline koja pronalazi veliku primenu u farmaceutskoj i kozmetičkoj industriji gde se koristi prilikom distribucije lekova i za hidrataciju kože kao sastavni deo različitih krema, gde zajedno sa hijaluronskom kiselinu ima ulogu u smanjenju bora.Celobioza – dehidrogenaza prilikom oksidacije laktoze ili celobioze, kao prirodnih supstrata, katalizuje redukciju jednog dvoelektronskog ili dva jednoelektronska akceptora elektrona. Jedan od najkorišćenijih dvoelektronskih akceptora elektrona je upravo dihlor fenol indofenol (DCIP), dok jednoelektronski akceptori elektrona mogu biti citohrom c, ABTS, ali i Fe3+ i Mn3+ joni. Redukcijom Fe3+ jona u prisustvu molekulskog kiseonika dolazi do formiranja vodonik peroksida i posredstvom Fentonove reakcije do generisanja hidroksil radikala.Polazeći od ove činjenice, iskoristili smo upravo Fentonovu reakciju za razvoj fluorescentnog eseja za visoko efikasnu pretragu biblioteka gena celobioza – dehidrogenaze, baziranog na detekciji proizvedenih hidroksil radikala fluorogenom probom aminofenil – fluoresceinom (APF).Primena celobioza – dehidrogenaze u konstruisanju biosenzora i biogorivnih ćelija leži upravo u njenoj sposobnosti da katalizuje oksidaciju laktoze, celobioze i β – 1,4 – vezanih oligosaharida do odgovarajućih laktona koji potom spontano hidrolizuju do aldonskih kiselina. Enzimi koji nalaze primenu u konstruisanju biosenzora i biogorivnih ćelija, moraju da zadovoljavaju nekoliko kriterijuma,odnosno moraju da imaju veliku osetljivost i supstratnu specifičnost, ali i da pokazuju povećanu stabilnost... Cellobiose dehydrogenase from Phanerochaete chrysosporium, a white rot fungus, belongs to the extracellular oxidoreductive group of enzymes and catalyzes the oxidation of the β – 1,4 – glycoside bond of oligosaccharides such as cellobiose and lactose. During oxidation of lactose, formation of lactobionic acid occurs which has many applications in pharmaceutical and cosmetic industry. Such applications include the distribution of medicine, a component responsible for skin hydration and when combined with hyaluronic acid as an agent against wrinkles.During oxidation of lactose or cellobiose by cellobiose dehydrogenase, reduction catalysis occurs of one two electron or two one electron acceptors. One of the most utilized two electron acceptors is DCIP, while one electron acceptors are usually cytochrome c, ABTS, Fe3+ and Mn3+ ions. During reduction of Fe3+ ions in the presence of molecular oxygen, H2O2 is formed and due to the Fenton reaction formation of hydroxyl radicals occurs. Due to this occurrence we wanted to use the Fenton reaction in order to develop a fluorescent assay based on the production of hydroxyl radicals and the fluorescence of aminophenyl fluorescein (APF). This would allow us to efficiently analyze cellobiose dehydrogenase gene libraries.With this fact in mind, the Fenton reaction was used to develop a fluorescent assay for the high throughput screening of cellobiose dehydrogenase genes, based on the detection of hydroxyl radicals with the fluorescent probe APF.The possible application of cellobiose dehydrogenase in the construction of various biosensors and biofuel cells is due the its ability to catalyze the oxidation of lactose, cellobiose and similar β – 1,4 – oligosaccharides do their corresponding lactones which then spontaneously hydrolyze to aldonic acids. Enzymes used in suchapplications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity...

Упркос четвородеценијским студијама, биохемија хром(III) јона и даље остаје права енигма. Тек на основу недавних студија и сазнања, створена је јаснија представа о значају хром(III) јона и његовој улози у метаболизму угљених хидрата и липида. Такође, захваљујући спектроскопским студијама, данас се значајно више зна о структури и улози олигопептида хромодулина, као и о његовим амино-киселинским остацима који координуjу хром(III) јон... Despite four-decade studies, the biochemistry of chromium (III) ion has remained a true enigma. Recent studies and findings have been provided a clearer notion of the importance of chromium (III) ion and its role in the carbohidrate and lipid metabolism. Thanks to spectroscopic studies as well, much more is known today about the structure and role of oligopeptide chromodulin, and its amino-acid residue which cooordinates Chromium (III) ion...

Denaturacija globularnih proteina na niskim temperaturama predstavlja univerzalni fenomen. Narušavanje nativne strukture proteina usled izlaganja niskim temperaturama dešava se primarno kao posledica kolapsa hidrofobnog efekta, entropijskog faktora koji u najvećoj meri doprinosi stabilizaciji nativne strukture. Sekundarno, denaturacija proteina na niskim temperaturama posledica je slabljenja hidrofobnih interakcija u unutrašnjosti nativnog proteina. Ispitivanje stabilnosti proteina na niskim temperaturama ima, pre svega, fundamentalni značaj koji se ogleda u činjenici da bi detaljno razumevanje mehanizma denaturacije proteina na niskim temperaturama moglo značajno da doprinese razjašnjavanju jednog od najvažnijih problema savremene biohemije, problema uvijanja proteina. Ispitivanje stabilnosti proteina na niskim temperaturama započeto je pre oko dve decenije. Međutim, direktnu denaturaciju proteina, koja je posledica niske temperature per se, teško je proučavati rutinski primenjivanim metodama, jer većina proteina ima tačke denaturacije ispod temperature mržnjenja vode. Razvojem savremenih FT-IR instrumenata, kao i primenom istih u određivanju promena sekundarnih struktura proteina, došlo je do povećanog interesovanja za proučavanjem denaturacije proteina izazvane niskim temperaturama. Praktični značaj proučavanja ove problematike ogleda se u tome što razumevanje mehanizama denaturacije može pomoći u pronalaženju optimalnih uslova za skladištenje proteina pri kojima će se produžiti njihov vek trajanja. Poznavanje stabilnosti enzima naročito je važno u slučaju enzima koji se koriste u biotehnologiji, medicini ili nauci, kao što je slučaj sa sve četiri model proteaze koje su predmet istraživanja u okviru ove doktorske disertacije. Najvažniji cilj koji je postavljen u ovoj studiji jeste pronalaženje objašnjenja za gubitak aktivnosti izazvan niskom temperaturom ispitivanih serin- i cistein-proteaza na nivou detaljnih strukturnih promena proteina, odnosno, pokazati da su, zapravo, denaturacija proteaza i strukturni rearanţmani koji prate denaturaciju odgovorni za gubitak aktivnosti u mnogo većoj meri nego autoproteoliza. Dodatni cilj bilo je optimizovanje uslova za skladištenje komercijalno vaţnih proteaza na niskim temperaturama. Praćenje stabilnosti proteina na niskim temperaturama nailazi na metodološka ograničenja kada je reč o primeni uobičajeno korišćenih metoda za ispitivanje stabilnosti proteina, kao što su spektroskopske metode i diferencijalna skenirajuća kalorimetrija. Stoga je u ovoj studiji pribegnuto pristupu izlaganja proteaza uzastopnim ciklusima zamrzavanja/odmrzavanja u cilju praćenja uticaja temperatura ispod nule na strukturu proteaza. Rezultati praćenja promena primarne strukture model proteaza koji su dobijeni kao deo ove disertacije, ukazuju da autoproteoliza ne može biti odgovorna za visoke procente gubitka aktivnosti, (čak do 75% u slučaju papaina, preko 40% u slučaju ficina i oko 60% u slučaju tripsina u kiselim uslovima) nakon šest ciklusa zamrzavanja/odmrzavanja. Praćenje strukturnih perturbacija na nivou sekundarne i tercijarne strukture pokazalo je da nakon šest do sedam uzastopnih ciklusa zamrzavanja/odmrzavanja proteaze gube elemente nativne sekundarne strukture (α- heliks i neuređene strukture) u korist β-pločica (intramolekulskih u slučaju tripsina u kiselim uslovima). Naročito je izražen porast sadržaja intermolekulskih β-pločica (u slučaju papaina i ficina) koje predstavljaju strukturne elemente neophodne za agregiranje. Agregiranje je potvrđeno gel-filtracijom. Opisani trendovi promena sekundarnih struktura detektovani su u literaturi i za druge proteine denaturisane niskim temperaturama, odnosno niskom pH vrednošću, indicirajući da je inaktiviranje papaina, ficina i tripsina (u kiselim uslovima) na niskoj temperaturi posledica denaturacije. U slučaju tripsina za sekvenciranje, proteaze od najvećeg komercijalnog značaja u ovoj studiji, predložen je alternativni način skladištenja na niskoj temperaturi u rastvornom obliku kojim se izbegava denaturacija i ograničava autoproteoliza. Blago alkalna pH vrednost (u blizini optimalne vrednosti za aktivnost tripsina) uz dodatak krioprotektivnih agenasa (glicerola i lizina) za koje je poznato da stabilizuju proteine (mehanizmom preferencijalne potisnutosti sa površine proteina favorizujući nativnu konformaciju), dovela je do efikasnog očuvanja nativne strukture tripsina. Takođe, inhibicijom autoproteolitičke aktivnosti u prisustvu lizina koji okupira vezujuće mesto tripsina, ograničena je i autoproteoliza. Tripsin skladišten na niskoj temperaturi na pH vrednosti od 8,0 uz dodatak glicerola ili lizina, pokazao je efikasnost identičnu netretiranom nativnom tripsinu u metodi identifikovanja BSA peptidnim mapiranjem, što sugeriše da skladištenje tripsina u blago alkalnim uslovima uz dodatak krioprotektanata može da produži njegov vek trajanja. Cold denaturation of globular proteins represents a universal phenomenon. Disruption of native structure at low temperatures primarily happens as a consequence of the collapse of the hydrophobic effect, an entropy parameter which represents a main driving force for protein folding. Secondarily, protein denaturation at low temperatures is a consequence of the weakening of hydrophobic interactions in the interior of a protein's three dimensional structure. Investigation of the cold denaturation of proteins has fundamental importance, because detailed understanding of the mechanism of cold denaturation could contribute to a great extent toward elucidationg one of the most challenging problems of contemporary structural biochemistry: the protein folding problem. Investigation of the cold stability of proteins began two decades ago. Direct cold denaturation, which is a consequence of low temperature per se, is difficult to investigate using routine methods since the majority of proteins have cold denaturation points well below 0 °C. However, the development of modern FT-IR instruments and their application in secondary structure determination has lead to increased interest in the investigation of the cold stability of proteins. The practical importance of this topic reflects the fact that understanding structural rearrangements induced by cold denaturation could help define optimal conditions for protein storage in order to prolong their shelf lives. The cold stability of proteins is especially important for enzymes used in biotechnology, medicine or research: which is the case for all four model proteases investigated in this dissertation. The overall goal of this study was to explain the dramatic loss of serine and cysteine proteases activity induced by low temperature, by showing that cold denaturation and subsequent massive structural rearrangements are responsible for this loss, rather than autoproteolysis. In addition, optimal cold storage conditions were defined for commercially important proteases. In this work, repeated freeze-thaw cycles were used to analyse the influence of sub-zero temperatures on protease structures, due to methodological limitations in monitoring protein stability at sub-zero temperatures by commonly used techniques such as spectroscopy and differential scanning calorimetry. Results from monitoring the primary structure stability of model proteases obtained as a part of this dissertation suggest that autoproteolysis cannot be the cause for the dramatic activity losses (as large as 75% in the case of papain, above 40% in the case of ficin and around 60% in the case of trypsin in acidic conditions) observed after six freeze-thaw cycles. Secondary/tertiary structure perturbations after six-to-seven freeze-thaw cycles suggest that these model proteases lose part of their native secondary structure elements (mainly α-helices and random coils) in favor of a β-sheet conformation (intramolecular in the case of trypsin in acidic conditions). An especially large increase was detected for intermolecular β- sheets (in the case of papain and ficin) which represents a structural element necessary for aggregation. Aggregation of cold denatured cysteine proteases was shown by sizeexclusion chromatography. Similar trends in secondary structure changes were detected in the literature for other proteins denatured by low temperature and/or low pH values, indicating that inactivation of papain, ficin and trypsin (in acidic conditions) was a consequence of cold denaturation. Sequencing grade trypsin, being the most important commercial protease of this study, was chosen for optimization of cold storage conditions. An alternative protocol for cold storage of trypsin in solution is proposed which circumvents cold denaturation and limits autoproteolysis. Slightly alkaline conditions (close to the optimum pH value of trypsin, pH around 8) with the addition of cryoprotectants (glycerol or lysine which are known to stabilise proteins in solution by preferential exclusion from the protein surface favoring the native state) led to complete preservation of native structure. In the case of lysine as a cryoprotectant, autoproteolysis was inhibited as well. After seven cycles of cold storage at pH around 8 with the addition of cryoprotectants, trypsin was as efficient as untreated trypsin in trypsin mass fingerprinting of BSA, suggesting that proposed conditions could prolong its shelf life.

Mlečno-kiselinske bakterije su mikroorganizmi od izuzetnog značaja kako za industriju fermentisanih proizvoda, tako i za medicinu gde kao probiotici dobijaju sve veći značaj. Proteinaze i bakteriocini, molekuli koje ove bakterije sekretuju u ekstraćelijsku sredinu, imaju važnu ulogu u oba ova aspekta. Proteinaze su veliki ekstraćelijski enzimi uključeni u prvi korak digestije proteina iz vanćelijske sredine i neophodne su za efikasan rast bakterija u sredinama siromašnim malim peptidima i slobodnim aminokiselinama. U ovom radu je analizirana proteolitička aktivnost različitih mezofilnih laktobacila, pripadnika Lb. casei i Lb. plantarum grupa prema pet različitih proteina mleka. Utvrđeno je da Lb. zeae LMG17315 poseduje najefikasniju proteolitičku aktivnost, s obzirom da hidrolizuje sve ponuđene supstrate. Takođe pokazano je da se proteinaze ovog soja odvajaju u medijum u aktivnom obliku. Lb. casei ATCC393, soj sa kontroverznim sistematičkim statusom, pokazao je sličnu, ali manje efikasnu proteolitičku aktivnost. Prisustvo različitih proteinaznih gena u ispitivanim sojevima analizirano je DNK-DNK hibridizacijom i PCR metodom. Utvrđeno je da LMG17315 poseduje sekvence gena koje kodiraju katalitičke domene za tri, a ATCC393 za jednu proteinazu. Kod soja LMG17315 je utvrđeno da poseduje prtP-sličan i prtR-sličan gen, dok je kod oba soja pronađen i novi proteinazni gen, označen kao prtP1. S obzirom na dosadašnji problematičan sistematički status ovih sojeva urađena je i njihova molekularna determinacija čime je stečen bolji uvid u njihove filogenetske odnose. Iako je u relevantnoj literaturi predloženo da ATCC393 treba da bude reklasifikovan u vrstu Lb. zeae, dobijeni rezultati ukazuju na značajne razlike između soja ATCC393 i tipskog soja Lb. zeae LMG17315. Lactic acid bacteria are microorganisms of great importance for the industry of fermented products as well as for medicine, where they are gaining great attentionas probiotics. Proteinases and bacteriocins, molecules secreted by these bacteria, have important roles in both of these aspects. Proteinases are large enzymes involved the in first steps of digestion of extracellular proteins and are necessary for efficient bacterial growth in environments with low concentrations of small peptides and free amino acids. The proteolytic activity of different mesophilic lactobacilli, members of Lb. casei and Lb. plantarum groups, was tested against five main milk proteins, in this thesis. It was determined that Lb. zeae LMG17315 possessed the most efficient proteolytic activity, since it hydrolyzed all given substrates. In addition, it was shown that proteinases of this strain could be released from cell wall in active form. Lb. casei ATCC393, a strain with controversial systematics, showed similar, but less efficient proteolytic activity. Presence of different proteinase genes in tested strains was analyzed with DNA-DNA hybridization and PCR. It was determined that strain LMG17315 possessed gene sequences coding for catalytic domains of three proteinases, while ATCC393 possessed only one such sequence. Two of these genes in strain LMG17315 were similar to previously characterized prtP and prtR genes, while the third one was a new proteinase gene, here named prtP1. The same gene was found in the genome of ATCC393. Considering the problematic taxonomic status of these strains, methods of molecular determination were applied for better understanding of their phylogenetic relationship. Although many authors suggest that ATCC393 should be reclassified in taxon Lb. zeae, results obtained in this work highlighted some significant differences between these strains.

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...

Аlergije na hranu se definišu kao nepoželjne imunološke reakcije koje se javljaju svaki put prilikom konzumiranja odrеđene hranljive namirnice. Danas alergije na hranu predstavljaju značajan zdravstveni problem, i smatra se da oko 5% dece i 3-4% odraslih ljudi pati od ovog poremećaja, a uočena je i tendencija rasta broja alergičnih pacijenata. Istovremeno, čak i samo dijagnostifikovanje alergije na hranu predstavlja izazov, s obzirom da se u tu svrhu trenutno upotrebljavaju alergeni ekstrakti, koji su uzrok slabe specifičnosti i osetljivosti dijagnostičkih testova. Rekombinantna DNK tehnologija je razvijena tokom 80-tih godina prošlog veka i pružila je mogućnost proizvodnje rekombinantnih proteina koji bi bili ekvivalentni prirodnim alergenima. Ovi proteini se proizvode kao biohemijski definisani molekuli sa ujednačenim strukturnim i imunološkim osobinama. Do danas je nekoliko stotina različitih alergena klonirano i eksprimirano u obliku rekombinantnih proteina. Ovi rekombinantni alergeni su omogućili znatno detaljniju dijagnostiku senzitizacionih profila alergičnih pacijenata. Iz ovog razloga je proizvodnja dobro okarakterisanih rekombinantnih proteina postala važno pitanje u farmaceutskoj industriji. Banana (Musa acuminata) je veoma popularno voće širom sveta i prisutno je tokom cele godine. Međutim, 1991. godine su prvi put opisani simptomi alergijske reakcije na ovo voće. Do danas je identifikovano 5 proteina koji čine molekulsku osnovu alergije na bananу. Ovi proteini su označeni kao Mus a 1 do 5. Alergen Mus a 5 predstavlja β-1, 3-glukanazu iz PR-2 porodice proteina i učestvuje kako u odbrani biljke, tako i u mnogim drugim raznolikim fiziološkim i razvojnim procesima. Tokom izrade ove disertacije, β-1, 3-glukanaza je proizvedena kao heterologi protein u bakteriji Escherichia coli. Gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). BL21 ćelije transformisane sa GST-Mus a 5 konstruktom su korišćene za proizvodnju proteina. Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h)... Food allergy is defined as an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food. Currently, food allergy is a major health problem, with an estimated prevalence of about 5% in young children and 3-4% in adults, and the prevalence is increasing. Food allergies present a challenge in the field of diagnostics, since the currently employed allergen extracts frequently give rise to poor specificity and sensitivity of tests. Recombinant DNA technology developed in the 1980s has provided the means for producing allergens that are equivalent to their natural counterparts. The proteins are produced as biochemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and they can provide the means for making a very detailed diagnosis of a patient's sensitization profile. The production of recombinant proteins in a well-characterized form has become an important issue in the pharmaceutical industry. Banana (Musa acuminata) is a very popular fruit worldwide. It is available year round and is very present in human nutrition. However, in 1991 symptoms of allergic reaction to banana were described for the first time. The molecular basis of allergic reactions to banana has been ascribed to five proteins, designated Mus a 1 to 5. The Mus a 5 allergen is a β-1, 3-glucanase belonging to the PR-2 family of proteins and is involved not only in plant defense, but also in diverse physiological and developmental processes. In this thesis banana β-1,3-glucanase was produced as a heterologous protein in Escherichia coli. Its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for production of the protein. The conditions for protein expression were optimized by varying the temperature (25, 30 and 37 °C) and duration of protein expression (3, 6 and 12 h)...