Određivanj prisutnosti ekotoksikološki važnih Pgp i MRP-proteina u šarankama na genskoj i proteinskoj razini

One of the most intriguing defence strategies which aquatic organisms developed through evolution is multixenobiotic resistance (MXR). The key mediators of MXR activity are ATP-binding cassette (ABC) transport proteins. They provide resistance of aquatic organisms by binding xenobiotics and extruding them from cells in an energy-dependent manner. Since Cyprinid fi sh species are common target in freshwater biomonitoring programs, we have studied the presence of two main MDR/MXR effl ux transporters Pglycoprotein (Pgp, Abcb1) and MRP-like protein(s) (Abcc) in the liver of fi ve Cyprinid species: common carp, European chub, sneep, barbel, and silver prussian carp. Their presence was evaluated on the mRNA and protein level. Various pairs of primers were designed to clone homologous fragments of MXR-related genes. At the protein level, we used Western blotting with specifi c monoclonal antibodies against human Pgp (Abcb1, Ab C219), MRP1 (Abcc1; Ab MRPm6) or MRP2 (Abcc2; Ab M2I-4). Transcripts of both key types of MXR transporters were identifi ed in all species examined and here we provide the phylogenetic analysis of new partial sequences. Immunochemical determinations with mammalian antibodies failed to identify the presence of MRP(s), but Pgp expression was found in all fi ve Cyprinid species. These results support that MXR is a defence system mediated by both Pgp and MRP types of ABC transport proteins.Mehanizam multiksenobiotičke otpornosti (MXR) stanični je obrambeni sustav odgovoran za svojstvo istovremene otpornosti na različite ksenobiotike, koje se očituje smanjenjem akumulacije, odnosno povećanjem izbacivanja potencijalno toksičnih tvari iz stanica vodenih organizama. MXR-mehanizam pokazuje odlike analogne fenomenu istovremene otpornosti na različite lijekove (engl. multidrug resistance, MDR) prvi put dokazanom u tumorskim stanicama. Posredovan je istim ABC transportnim proteinima kao i MDR. Istraživanja vezana uz MXR vodenih životinja uglavnom su imala težište na određivanju prisutnosti i funkcije P-glikoproteina (Pgp). Budući da se ribe iz reda šaranki često rabe u biomonitoringu slatkovodnih voda, cilj ovog istraživanja bilo je određivanje prisutnosti dvaju glavnih MDR/MXR-tipova proteina - Pgp i MRP-tip proteina - u jetri iz pet vrsta šaranki; šarana (Cyprinus carpio), klena (Sqalius cephalus), mrene (Barbus barbus), babuške (Carassius auratus gibelio) i podusta (Chondrostoma nasus). Njihova prisutnost utvrđena je na razini mRNA te na proteinskoj razini. Različiti parovi početnica dizajnirani su kako bi se identifi cirali homologni fragmenti gena sličnih MXR-u. Detekcija na razini proteina napravljena je putem Western blot analize s pomoću specifi čnih monoklonskih protutijela proizvedenih da prepoznaju konzervirane epitope; Pgp (C219), MRP1 (MRPm6) ili MRP2 (M2I-4) u sisavaca. Transkripti obaju ključnih MXR-transportera identifi cirani su u svim jedinkama, a napravljena je i fi logenetska analiza dobivenih sekvenci. Imunokemijskom detekcijom s protutijelima sisavaca nismo uspjeli detektirati prisutnost MRP-proteina, dok je Pgp-ekspresija potvrđena u svih pet vrsta šaranki. Nove spoznaje da je za MXRmehanizam zaslužno više transportnih proteina zasigurno će pridonijeti potpunijoj karakterizaciji MXR-a kao integralnog dijela detoksikacijskog, odnosno temeljnog obrambenog sustava vodenih organizama te njegovoj znanstvenoj afi rmaciji kao vjerodostojnog pokazatelja kvalitete okoliša

DIGI4Teach - Handbook

One of the important outputs of our DIGI4Teach consortium is this Handbook, which consists of two parts. Part A contains an analysis of the most important descriptive research results conducted within the DIGI4Teach Erasmus+ project regarding the use of digital technology in teaching economic disciplines in partner countries. Part B contains twelve case studies from different areas of economics and business (accounting, finance, marketing, tourism and trade) that were prepared using various digital tools and they can be freely used in classes or other forms of education