Fehérje-kölcsönhatások szerkezeti alapjainak feltárása és a kölcsönhatás szelektív gátlása irányított evolúciós eljárásokkal = Deciphering the structural basis of protein-protein interactions and their selective inhibition via directed evolutionary methods

Fő célom az volt, hogy meghonosítsam az irányított fehérjeevolúció szemléletét, és legsikeresebb technológiáját, a fágbemutatást. Egyrészt azt kutattuk, mi szabja meg a proteáz inhibitorok affinitását és specifitását. Három inhibitort vizsgáltunk: a kispeptid SFTI-t (Sunflower Trypsin Inhibitor), a kis fehérje SGPI-t (Schistocerca Gregaria Protease Inhibitor) és a homodimer Ecotint (E. Coli Trypsin Inhibitor). Másrészt inhibitorokat fejlesztettünk olyan humán proteázok ellen, amelyeknek nem volt ismert szelektív gátlószere. Mindhárom inhibitort sikerült evolválnunk. Feltártuk, hogy az SFTI mely csoportjai fontosak a molekula stabilitása és a proteázzal létesített specifikus kölcsönhatás szempontjából. Az SGPI családban feltártuk, hogy a hidrofób mag összetétele kihat a felszíni csoportokra és így a proteázzal alkotott komplex stabilitására. Egy rekord-felbontású SGPI variáns-tripszin komplex szerkezet által finomítottuk a szerin proteázok működési modelljét. Az Ecotint egyláncú formában fejeztük ki fágon, így a két monomer rész külön-külön evolválható lett. Szelektív inhibitorokat fejlesztettünk a hasnyálmirigy enzimek fő szabályzó proteáza, a kimotripszin C ellen, és a komplement rendszer lektin útjának MASP-1 és MASP-2 proteázai ellen. A MASP-gátlókkal létrehoztuk az első lektin út szelektív inhibitorokat, és korrigáltuk az útvonal-aktiválódás korábban hibásan leírt mechanizmusát. A projekt során 11 cikk, 3 szabadalom, 3 PhD dolgozat és tucatnyi tudományos díj született. | A major project goal was to establish in Hungary the principle and technology of directed protein evolution. Via phage display I aimed to decipher fundamental rules governing affinity and specificity of protease inhibitors focusing on 3 inhibitors: the small peptide SFTI (Sunflower Trypsin Inhibitor), the small protein SGPI (Schistocerca Gregaria Trypsin Inhibitor) and the homodimer Ecotin (E. Coli Trypsin Inhibitor). I also aimed to evolve selective inhibitors against human proteases lacking such blockers. We identified SFTI residues providing inhibitor stability and those involved in specific protease-binding. In the SGPI family we revealed that the hydrophobic inhibitor core greatly affects the function of surface residues and thereby the stability of the protease-inhibitor complex. Based on an ultrahigh resolution SGPI variant – trypsin structure we refined the model of serine protease catalysis. We displayed Ecotin on phage in a single-chain format such that the two monomers could be evolved independently. We developed selective inhibitors against a major regulatory pancreatic protease, human chymotrypsin C. We also evolved mono-specific inhibitors against the lectin pathway complement proteases MASP-1 and MASP-2. With these we developed the first lectin pathway selective complement inhibitors and corrected the hitherto incorrect pathway activation model. Altogether 11 papers, 3 patents, 3 PhD thesis and a dozen scientific awards indicate the success of the project

Role of direct bioautographic method for detection of antistaphylococcal activity of essential oils.

The aim of the present study was the chemical characterization of some traditionally used and therapeutically relevant essential oils (thyme, eucalyptus, cinnamon bark, clove, and tea tree) and the optimized microbiological investigation of the effect of these oils on clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). The chemical composition of the oils was analyzed by TLC, and controlled by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The antibacterial effect was investigated using a TLC-bioautographic method. Antibacterial activity of thyme, clove and cinnamon oils, as well as their main components (thymol, carvacrol, eugenol, and cinnamic aldehyde) was observed against all the bacterial strains used in this study. The essential oils of eucalyptus and tea tree showed weak activity in the bioautographic system. On the whole, the antibacterial activity of the essential oils could be related to their most abundant components, but the effect of the minor components should also be taken into consideration. Direct bioautography is more cost-effective and better in comparison with traditional microbiological laboratory methods (e.g. disc-diffusion, agar-plate technique)

Az MBL-hez kapcsolódó szerin proteázok szubsztrát specificitása és fiziológiai jelentősége = Substrate specificity and physiological relevance of MBL-associated serine proteases

A komplement rendszer aktiválódásának lektin útja az egyik első védelmi vonalnak tekinthető a szervezet fertőzések elleni védekezésében. A mannóz kötő lektin (MBL) baktérium felszínhez való kötődése után szerin proteáz zimogének (MASP= MBL-kötött szerin proteáz) aktiválódnak, melyek többféle mechanizmus révén járulnak hozzá az idegen mikroorganizmus megsemmisítéséhez ill. eltávolításához. Munkánk során felderítettük, a proteolitikus kaszkádrendszer beindításáért felelős MASP-2 enzim autoaktiválódásásnak mechanizmusát atomi szinten. Felfedeztük a MASP-2 egy eddig ismeretlen biológiai funkcióját, amely kapcsolatot teremt a véralvadási és a komplement kaszkád között. A MASP-2 hasítja és aktiválja a protrombint. Ugyancsak részletesen tanulmányoztuk a MASP-1 trombin-szerű aktivitását is. Ezek az eredmények arra utalnak, hogy a vérben lévő két proteolitikus kaszkádrendszer szoros evolúciós és funkcionális kapcsolatban van egymással, a komplement lektin útja által indukált limitált koaguláció az immunvédekezés egy ősi formájának tekinthető. | The lectin pathway of the complement system forms one of the first defence lines against the infections in our body. Upon MBL (mannose-binding lectin) binds to the bacterial surface serine protease zymogens (MASP=MBL-associated serine protease) become activated, and the active MASPs contribute to the inactivation and elimination of the foreign microorganism in several ways. In the course of our work we revealed the detailed atomic mechanism of the autoactivation of MASP-2 that is responsible for the initiation of the complement cascade. We discovered a new biological function of MASP-2 which makes contact between the complement and the coagulation cascades. MASP-2 cleaves and activates prothrombin. We also studied the thrombin-like activity of MASP-1 in detail. These results suggest that the two proteolytic cascade systems are in close evolutionary and functional relationship. The limited coagulation induced by the lectin pathway of the complement system can be regarded as an ancient form of immunity

Development of Polypropylene-based Thermoplastic Elastomers with Crumb Rubber by Dynamic Vulcanization: A Potential Route for Rubber Recycling

In our current paper the preparation and properties of thermoplastic elastomer produced by dynamic vulcanization is presented and discussed. We dynamically vulcanized natural and styrene butadiene rubber (NR/SBR) phase by continuous extrusion. Dispersion and in-situ vulcanization of the rubber phase occurred simultaneously in a co-rotating twin screw extruder. We used a random polypropylene copolymer (rPP) as the thermoplastic matrix and untreated crumb rubber (CR) to partially substitute the neat fresh rubber in order to check whether this is a potential recycling route for waste rubber products. We studied the effect of various rubber formulations, various processing conditions (screw speed and configuration) and various CR particle size distributions by characterizing the mechanical performance of the thermoplastic dynamic vulcanizates (TDVs) with tensile and hardness tests and their morphology by evaluating SEM micrographs taken from the fracture surfaces of the tensile specimens. The results showed that increasing screw speed and more high-shear elements in the screw setup led to a finer dispersion of the rubber phase, resulting in improved mechanical properties. The ultimate tensile properties of the best TDVs reached 20.5 MPa in tensile strength and 550 % in strain at break. However, partial replacement of the fresh rubber with untreated CR caused a significant deterioration in mechanical properties, due to poor adhesion between the CR particles and the matrix and rubber. This suggests that some kind of pre-treatment (e.g. by microwave or other devulcanization techniques) is necessary to enhance the surface activity of the CR particles

Antibacterial activity evaluation of selected essential oils in liquid and vapor phase on respiratory tract pathogens.

BACKGROUND: The increasing number of multidrug-resistant bacteria and the fact of antibiotic resistance is leading to a continuous need for discovering alternative treatments against infections, e.g. in the case of respiratory tract diseases. Essential oils (EOs), because of their volatility, can easily reach both the upper and lower parts of the respiratory tract via inhalation. Therefore, the aim of the present study was the antibacterial evaluation of clove, cinnamon bark, eucalyptus, thyme, scots pine, peppermint, and citronella EOs against respiratory tract pathogens such as Streptococcus pneumoniae, S. mutans, S. pyogenes, Haemophilus influenzae, H. parainfluenzae, and Moraxella catarrhalis. Furthermore, we wanted to compare the antibacterial effect of these EOs in two different test systems to provide data for the development of an appropriate product formulation. METHODS: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with in vitro vapor phase test (VPT) and broth macrodilution test (BDT). The chemical and percentage compositions of the EOs were determined by GC-MS and GC-FID analysis. RESULTS: Among the EOs, thyme was the most effective against S. mutans (MIC: 0.04 mg/mL in BDT, but cinnamon bark and clove oils also presented high inhibition in liquid medium with MIC values of 0.06 mg/mL and 0.1 mg/mL against S. pneumoniae and S. pyogenes, respectively. M. catarrhalis was the most sensitive to thyme EO (MIC: 0.09 mg/mL). Cinnamon bark EO was the most effective against Haemophilus spp. (MIC: 0.06 mg/mL). In the VPT, cinnamon bark was the most effective oil against all investigated pathogens with MIC values in the range of 15.62-90 mul/L. Surprisingly, the eucalyptus and scots pine showed weak activity against the test bacteria in both test systems. CONCLUSIONS: The EO of thyme, clove and cinnamon bark may provide promising antibacterial activity against respiratory tract pathogens either in liquid medium or in vapor phase. However, their effect is lower than that of the reference antibiotics. The combination of EOs and antibiotics may be beneficial in the alternative treatment of respiratory tract diseases. In vivo studies are necessary to calculate the effective dose of EOs in patients and determine their possible side effects and toxicity

Study of basic taste recognition among the tourism and catering management students of the Budapest Business School

The role of sensory analyses in the food industry and in gastronomy has increased significantly in the last decade. The regulation of both quality and certification is of paramount importance in this area as well. In this paper presenting our series of analyses, we endeavored to find out to what extent tourism and catering management students are able to recognize the four basic tastes (sweet, salty, sour, bitter) when using all of their senses, and whether their abilities are affected by the exclusion of sight and smell. In the first part of the study, tasters could use all their sense organs. In this case, sweet and salty tastes were properly recognized by more than 75% of the students. However, sour and bitter tastes were correctly identified by only 30% of the students, many mixed them up, and a quarter of the students did not even attempt to identify the taste. In terms of sensing sweet, salty and sour tastes, there was no difference between men and women, but the bitter taste was properly identified by more than 40% of men, and only 30% of women. False identification of bitter and sour tastes to such a large extent can be explained by significant consumption of coffee, tea or cigarettes, or by an inappropriate diet [7]. During smoking, substances in the tobacco smoke can greatly impair the functioning of taste buds. Consumption of energy drinks and tea can also add to the flavor reducing effect of coffee and cigarettes, so these factors together could be responsible for the large number of false identification of the bitter taste