Katedra za medicinsku biologiju i genetiku, Medicinski fakultet u Osijeku Karcinom vrata maternice najčešći je karcinom ženskog spolnog sustava u svijetu. Tijekom imunološkog odgovora na infekciju ...humanim papiloma virusom dolazi do premještanja T-staničnog receptora unutar lipidnih splavi limfocita T, u kojima gangliozidi imaju aktivnu ulogu u oblikovanju, stabilizaciji i biološkoj funkciji. Svrha istraživanja je utvrditi postoji li povezanost pojedinih polimorfizama u genima za biosintezu gangliozida i gangliozidnoga sastava membrana limfocita T s pojavom karcinoma vrata maternice. Genotipizacija polimorfizama gena za biosintezu gangliozida učinjena je primjenom metodologije Taqman. Sve ispitanice bile su homozigoti i imale su divlji tip alela kod svih ispitivanih gena (CC za gen B4GALT5, AA za gen ST3GAL5, AA za gen ST8SIA1 i CC za gen B4GALNT1). Analiza gangliozidnoga sastava staničnih membrana limfocita T provedena je korištenjem dvostrukog označavanja protutijelima na gangliozid GM1 i na antigene pojedinih loza limfocita. Nismo našli razliku u gangliozidnom sastavu membrana limfocita T između istraživanih skupina.
Department of Medical Biology and Genetics, Faculty of Medicine, University of Osijek Cervical cancer is the most common gynaecological cancer in women. Cell mediated immunity plays a significiant role in progression or regression of neoplastic cervical lesions caused by human papilloma virus infection. Engagement of antigen-specific T cell receptors is a prerequisite for T cell activation. The initial events of T cell activation involves movement of the T cell receptor into specialized microdomains known as lipid rafts. Gangliosides play an active role in the formation, stabilization and biological functions of lipid rafts. This study aims to determine whether polymorphisms in the genes involved in the biosynthesis of gangliosides and gangliosides expression in T cell membrane represent risk factor for cervical cancer. Taqman methods for single nucleotide polymorphism genotyping was used. All subjects carried the homozygous wild-type genotypes for all analyzed genes (CC for gene B4GALT5, AA for gene ST3GAL5, AA for gene ST8SIA1 and CC for gene B4GALNT1). Gangliosides expression in T lymphocytes membranes was analyzed using double labelling antibodies on ganglioside GM1. We have not found difference in gangliosides expression in T lymphocytes membranes between the two groups.
Hlapljivi anestetici se godinama primjenjuju u kliničkoj praksi, iako mehanizma njihovog djelovanja još nije dovoljno poznat. Brojne hipoteze ukazuju na mjesta njihovog djelovanja. Uz do sada ...ispitivane mogućnosti uključenosti GABA i NMDA receptora u novije vrijeme se vjeruje kako hlapljivi anestetici djeluju i na lipidne signalne domene. Lipidne splavi su lokalizirane lipidne građevine sa visokim sadržajem kolesterola i glikosfingolipida. Lipidne splavi kao signalne platforme, uklopljene u fluidne membrane, sudjeluju u predaji signala unutar stanice. U radu je sposobnost hlapljivih anestetika da djeluju na signalne domene ispitana in vivo na modelu miša B4galnt1 koji imaju promijenjenu sposobnost stvaranja lipidnih domena uslijed bloka u sintezi kompleksnih gangliozida, WT miševima i WT miševima hranjenim kelatorom bakra kuprizonom. Miševi su podvrgnuti kognitivnim i motoričkim testovima prije i nakon anestezije izofluranom i sevofluranom. Mjereno je vrijeme potrebno za uvod u anesteziju i buđenje iz anestezije. B4galnt1 miševi imaju značajno kraće vrijeme uvodau anesteziju izofluranom (p<0,001) i sevofluranom (p<0,001) od WT miševa hranjenih kuprizonom. WT miševi hranjeni kuprizonom postižu značajno duže vrijeme održanja na rotirajućoj osovini od B4galnt1 miševa prije indukcije anestezije (p<0,001). U testu pasivnog izbjegavanja zadatka, 84.6 % B4galnt1 miševa i 25 % miševa hranjenih kuprizonom odgodilo je ulazak u tamnu prostoriju. B4galnt1 miševi imaju značajno manje horizontalnih pokreta u 1. (p=0,002) i 4. (p<0,001) minuti te značajno manje vertikalnih pokreta u 1. (p<0,001) i 4. (p<0,001) minuti prije uvoda u anesteziju od WT miševa hranjenih kuprizonom. Razlike izmjerene u kognitivnim testovima i testovima motorike prije i poslije anestezije prije su posljedica su promijenjenog sastava lipida u B4galnt1 mišu, a ne hipomijelinizacije koja postoji i kod kuprizonom tretiranih miševa i kod B4galnt1.
Volatile anaesthetics have been in use for years in clinical practice, although mechanism of their action is not yet fully understud. There are many hypotheses suggesting the site of their action. With so far investigated possible involvements of GABA and NMDA receptors, recent researches indicate possible action of volatile anaesthetics on lipid signalling domains. Lipid rafts are localized lipid structures with large quantity of cholesterol and glycosphingolipids. Lipid rafts function as signalling platforms, incorporated in the fluid membrane, in the transmission of signals inside the cell. This disertation presents a research on the ability of volatile anaesthetics to act on lipid rafts signalling domains. Tests were conducted in vivo, using the B4galnt1 mouse model. This mouse model has a modified ability to produce lipid domains due to the blocked synthesis of complex gangliosides. Tests included both mice strains, C57BL/6 and C57BL/6J fed with Cuprizone. Mice were exposed to different tests before and after anaesthesia with sevoflurane and isoflurane. Tests included Visual placing response, Hindlimb extension reflex, Forelimb grip strength, RotaRod, Activity cage, modified Lashley III maze, and passive avoidance task. The induction and recovery times from anaesthesia were observed and measured. The research results suggest that B4galnt1 mice have significantly shorter induction time of anaesthesia using isoflurane (p<0.001) and sevoflurane (p<0.001), compared to the WT mice fed with Cuprizone. WT mice fed with Cuprizone had significantly longer time in maintaining RotaRod, than B4galnt1 mice before induction of anaesthesia (p<0.001). Under Passive Avoidance test, 84.6% of B4galnt1 mice and 25% of Cuprizone fed mice models postponed entrance into the dark room. B4galnt1 mice have significantly less horizontal movement in the first (p = 0.002) and fourth (p<0.001) minute, as well as significantly less vertical movement in the first (p <0.001) and fourth (p <0.001) minute before induction time of anaesthesia compared to WT mice fed with Cuprizone. The differences in the results of cognitive and motor skills tests before and after anaesthesia were caused by changes in composition of lipid domains, and not by hypomyelinisation.
Cilj istraživanja: Prvenstveni cilj ovoga rada bila je karakterizacija konstitutivne internalizacije konformiranih i nekonformiranih MHC molekula I razreda, odnosno karakterizacija njihove ...endocitoze, recikliranja i degradacije. Poznato je da razlika u konformaciji MHC-I molekula uzrokuje njihovo razdvajanje u sekretornom putu. Stoga su u ovom radu istražena načela njihova razdvajanja tijekom endocitoze i putovanja kroz endosomalne odjeljke. Na temelju eksperimentalnih mjerenja cilj nam je bio i uspostavljanje matematičkog modela za kinetičku analizu endosomalnih putova.
Materijal i metode: Istraživanja smo proveli na mišjim fibroblastima J26 transficiranim s genom koji kodira molekule HLA-B7 (J26-B7), HLA-Cw6 (J26-Cw6) ili HLA-G1 (J26-G1), na humanoj tumorskoj staničnoj liniji HeLa koja konstitutivno izražava sve klasične HLA molekule I razreda, Balb 3T3 mišjim fibroblastima, te L-Ld stanicama (mišji fibroblasti transficirani genom za molekule H2-Ld). Za praćenje MHC molekula I razreda koristili smo monoklonska protutijela (mPt): mPt W6/32, koje prepoznaje sve klasične konformirane HLA molekule i konformirane HLA-G1 molekule, mPt HC-10, koje prepoznaje sve klasične nekonformirane HLA molekule, mPt MEM-G/1 koje prepoznaje nekonformirane molekule HLA-G1, mPt 30-5-7 koje prepoznaje konformirane H2-Ld molekule, te mPt 64-3-7 koje prepoznaje nekonformirane H2-Ld molekule. Transferinski receptor (TfR) smo pratili pomoću mPt R17, a molekule GM1 pomoću obilježene podjedinice B kolera toksina. Neionski detergent Triton X-100 nam je poslužio za ispitivanje nazočnosti molekula u lipidnim splavima. Oponašanjem uvjeta endosomalnog pH izvan stanice, utvrdili smo promjenu konformacije MHC molekula I razreda u zadanim uvjetima. Kako bismo utvrdili koji putovi su uključeni u endocitozu, endosomalno putovanje, recikliranje i degradaciju ovih molekula, koristili smo različite kemijske inhibitore. Kinetiku internalizacije pratili smo protočnom citometrijom, a unutarstanično putovanje konfokalnom mikroskopijom koristeći princip površinskog vezivanja odgovarajućeg monoklonskog protutijela i njegovog praćenja nakon određene kinetike internalizacije. Internalizirane molekule kolokalizirali smo međusobno, te s određenim endosomalnim markerima. Recikliranje molekula pratili smo modificirajući tri različita protokola iz literature i to protočnom citometrijom i konfokalnom mikroskopijom. U cilju praćenja kinetike degradacije koristili smo metodu površinske biotinilacije, te imunoprecipitacije, elektroforeze na poliakrilamidnim gelovima (SDS-PAGE), Western-blota i kemiluminiscencije. Za matematičko modeliranje i simulacije endocitoznih procesa koristili smo programski paket Mathematica (Wolfram Research Europe Ltd.). Rezultati: Konformirane MHC-I molekule dobro su izražene na staničnoj površini za razliku od nekonformiranih molekula. Djelovanjem kiselog pH (fiziološke i nefiziološke vrijednosti) izražaj konformiranih MHC-I molekula pada, dok izražaj nekonformiranih raste. Različite konformacije MHC-I molekula u ustaljenom stanju samo djelomično se kolokaliziraju unutar stanice i to prvenstveno u ranim endosomalnim odjeljcima. Niti jedan od ispitivanih kemijskih inhibitora (klorpromazin, Dynasore, filipin i AlF4-) nije blokirao internalizaciju MHC-I molekula. Nekonformirane MHC-I molekule se znatno brže internaliziraju i degradiraju od konformiranih MHC-I molekula što je najvjerojatnije posljedica činjenice da se konformirane MHC-I molekule usmjeravaju u reciklirajuće odjeljke, a nekonformirane MHC-I molekule u degradacijski put. Tome u prilog govori rezultat da se konformirane MHC-I molekule nakon internalizacije nalaze prvenstveno u jukstanuklearnom području zajedno s Tf/TfR i Rab11 GTPazom, dobro poznatim biljezima jukstanuklearnih reciklirajućih endosoma. Internalizirane konformirane MHC-I recikliraju s učinkovitošću od oko 30%, znatno manje od TfR/Tf koji recikliraju s učinkovitošću od 85-99%, što ukazuje da konformirane MHC-I molekule ulaze u zasebne reciklirajuće odjeljke. Ubrzo nakon internalizacije konformirane i nekonformirane MHC-I se kolokaliziraju s biljegom ranih endosoma (EEA-1), kao i međusobno, ali nedugo zatim stupanj kolokalizacije se smanjuje što znači da se razmjerno brzo razdvajaju. To razdvajanje blokira LY294002 (blokator PI3K), kao i konkanamicin A (blokator endosomalne H+ crpke). Djelovanjem Tx-ispiru se konformirane MHC-I molekula i TfR sa staničnih membrana, dok se nekonformirane MHC-I molekule i GM1 ne ispiru. Matematičkim modeliranjem potvrđeni su eksperimentalni rezultati, te izračunati slijedeći parametri: stopa endocitoze, stopa recikliranja i stopa degradacije.
Zaključak: Konformirane MHC-I molekule kao i TfR nalaze se u detergent-osjetljivim membranskim mikrodomenama za razliku od nekonformiranih MHC-I molekula i GM1, kako na staničnoj površini tako i u unutrašnjosti stanice. Internalizacija MHC-I molekula nije ovisna o klatrinu i dinaminu, već najvjerojatnije o funkciji malih GTPaza (Cdc42 i Arf6). Sortiranje, odnosno odvajanje endosomalnih putova konformiranih i nekonformiranih MHC-I molekula odvija se na razini ranih endosoma i ovisno je o pH. Nakon sortiranja konformirane MHC-I molekule se prvenstveno usmjeravaju u recikliranje, a nekonformirane u degradaciju.
Objectives: The aim of this study was to characterize constitutive internalization of conformed and non-conformed MHC class I molecules, with respect to their endocytosis, recycling and degradation. Conformational difference is known to segregate MHC-I molecules in secretory pathway. Therefore, we investigated endocytic pathway and putative segregation principles of these molecules during endocytic trafficking. Additionally, based on experimental measurements our goal was to establish a mathematical model for kinetics analysis of endosomal transport.
Material and methods: Experiments were performed on murine fibroblasts J26 transfected with HLA-B7 (J26-B7), HLA-Cw6 (J26-Cw6) or HLA-G1 molecules (J26-G1), on human HeLa cell line that constitutively expresses all classical HLA class I molecules, Balb 3T3 murine fibroblasts, and murine L cells stably transfected with Ld molecules (L-Ld). In order to follow MHC class I molecules, we used the following monoclonal antibodies (mAb): mAb W6/32 for all classical conformed HLA-I molecules and for conformed HLA-G1 molecules, mAb HC-10 for all classical non-conformed HLA-I molecules, mAb MEM-G/1 for non-conformed HLA-G1 molecules, mAb 30-5-7 for conformed H2-Ld molecules, and mAb 64-3-7 for non-conformed H2-Ld molecules. Transferrin receptor (TfR) was followed by mAb R17, and GM1 molecules with labeled cholera toxin B subunits. Non-ionic detergent Triton X-100 was used for testing the presence of molecules in lipid rafts. Conditions of endosomal pH were simulated outside the cell and the conformation changes of MHC-I molecules were under given conditions. In order to determine which pathways are involved in endocytosis, endosomal trafficking, recycling and degradation of these molecules, we used different chemical inhibitors. The kinetics of surface molecules was followed by flow citometry and their intracellular trafficking by confocal microscopy after binding of appropriate mAbs. Internalized molecules were colocalized with each other as well as with specific endosomal markers. After rearranging three different protocols from the literature we followed molecules recycling by flow citometry and confocal microscopy. In order to monitor the kinetics of degradation, we used the method of surface biotinilation and imunoprecipitation, electrophoresis on polyacrylamide gels (SDS-PAGE), Western-blot and chemiluminiscence. For mathematical modeling and simulation of endocytic processes we used a software package Mathematica (Wolfram Research Europe Ltd.). Results: Conformed MHC-I molecules are well expressed on the cell surface, unlike non-conformed molecules. Expression of conformed MHC-I molecules decreases due to treatment with acid pH (physiological and non- physiological values), while expression of non-conformed MHC-I molecules increases. Different conformations of MHC-I molecules in steady state conditions are only partially colocalized intracellular, primarily in the early endosomal compartments. Neither of the tested chemical inhibitors (chlorpromazine, Dynasore, filipin and AlF4-) did not block internalization of MHC-I molecules. Kinetics of internalization, as well as degradation of non-conformed MHC-I molecules are significantly faster than kinetics of conformed MHC-I molecules, due to fact that conformed MHC-I molecules recycle, unlike non-conformed molecules. These thesis are supported also by the finding that conformed MHC-I molecules after internalization are directed primarily in juxtanuclear region where they colocalize with Tf/TfR, and with Rab11. Internalized conformed MHC-I molecules recycle with 30% efficiency, while TfR/Tf recycling efficiency is 85-99%. Shortly after internalization conformed and non-conformed MHC-I molecules colocalize with early endosomal marker EEA-1, as well as to each other, but soon thereafter the level of colocalization decreases. This separation is blocked due to the influence of LY294002 (inhibitor of PI3K) or concanamycin A (inhibitor of endosomal H+ pump). Treatment with Tx-100 removes conformed MHC-I molecules and TfR from the cell membranes, while non-conformed MHC-I molecules and GM1 are resistant to the Tx-100 treatment. Experimental results are confirmed by mathematical modeling, by which the following parameters are also calculated: the rate of endocytosis, the rate of recycling and the rate of degradation.
Conclusion: Conformed MHC-I molecules, as well as transferrin receptor are localized in detergent-sensitive membrane micro domains, while non-conformed MHC-I molecules and GM1 are localized in detergent-resistant membrane micro domains, not only on the cell surface, but also intracellularly. Internalization of MHC-I molecules is clathrin- and dynamin-i
