Izomsejtek intracelluláris kalciumraktárainak szerepe a kalciumhomeosztázis szabályozásában fiziológiás és kóros körülmények között = The role of intracellular calcium stores in the regulation of calcium homeostasis in muscle cells under physiological and pathological conditions

Vizsgáltuk a vázizom Ca2+-homeosztázisának szabályozásában részt vevő Trisk 95 (a triadin 95 kDa molekulasúlyú izoformája) fehérje szerepét. Liposzóma és adenovírus mediált overexpressziót, valamint RNS interferencia módszert alkalmaztunk a Trisk 95 fehérje expressziójának módosítására. A stabil overexpresszió csökkentette az elemi Ca2+ események amplitúdóját, frekvenciáját és a depolarizáció által kiváltott Ca2+-tranzienseket. Az endogén triadin expresszió shRNS-sel történő gátlása követően a sejteken a koffeinnel kiváltott Ca2+-tranziensek csökkentek, ugyanakkor az elemi Ca2+-felszabadulási események jelentősen nagyobb gyakoriságot mutattak. Így feltételezhető, hogy a Trisk 95 fehérje negatívan szabályozza a rianodin receptor működését, csökkentve a lokalizált, elemi Ca2+-felszabadulási eseményeket, valamint a globális Ca2+-tranzienseket vázizomtenyészetekben. Tanulmányoztuk a szívinfarktust követő vázizomgyengeséget okozó folyamatok hátterét állatmodellen. A posztinfarktusos állatokból származó izomrostokban csökkent a Ca2+ felszabadulás elemi eseményeinek amplitúdója, amivel szinkron megnőtt a hosszuk és a komplexitásuk, utalva a szarkoplazmatikus retikulum (SR) kalcium telítettségének csökkenésére, valamint a rianodin receptor és a SR membrán kapcsolatának megváltozására utal a csatorna vezetőképességének növekedése. Eredményeink alátámasztják a Ca2+-homeosztázisban szerepet játszó fehérjék gén- és sejtszintű szabályozásának módosulását szívinfarktust követően. | We studied how the overexpression or the interference with the expression of Trisk 95 (95 kDa triadin) would affect Ca2+ homeostasis of the skeletal muscle cell. Liposome or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of Ca2+ sparks, the frequency of embers and the depolarisation-evoked Ca2+-transients. On primary cultures of rat skeletal muscle cells transfection with specific shRNA sequence the caffeine-evoked Ca2+-transients were reduced and elementary calcium release events appeared with higher frequency. These results suggest that Trisk 95 negatively regulates ryanodine receptor (RyR) function by suppressing localized Ca2+-release events and global Ca2+- signals in cultured muscle cells. To understand the weakness of skeletal muscles after chronic heart failure excitation-contraction coupling of rats that underwent a left anterior coronary artery occlusion were studied. Calcium release from the sarcoplasmic reticulum was considerably suppressed. Elementary calcium release events showed altered morphology Isolated ryanodine receptors displayed an unusual rectification with increased single channel conductance. These data shows decreased number of channels that open during a calcium release event and altered RyR function thus calcium release is suppressed following a myocardial infarction

A transzmembrán jelátvitel integrált szabályozása: Ca2+-, K+-csatornák és interleukin-receptorok közötti interakciók = An integrated regulation of transmembrane signalling: interactions between Ca2+, K+ channels and interleukin receptors

Projektünk célkitűzése a transzmembrán jelátvitelben résztvevő egyes receptorok és ioncsatornák működésének multidiszciplináris megközelítése volt. Kimutattuk a TASK-3 K+-csatorna expresszióját és döntően mitokondriális lokalizációját melanoma malignum, ill. malignus transzformációt nem mutató sejteken egyaránt. Jellemeztük a dendritikus sejtek (DS) érése és ioncsatorna expressziója közötti kapcsolatot. Feltártuk, hogy az elsődleges Ca2+ jelátviteli utat a CRAC csatornák biztosítják, függetlenül a DS-ek differenciáltsági fokától. Tanulmányoztuk az ic. Ca2+ homeosztázis és a purinerg jelátvitel kapcsolatát ingerlékeny és nem-ingerlékeny sejteken és karakterizáltuk a folyamatban szerepet játszó purinoreceptorok típusát. Kimutattuk, hogy az SR-ban kifejeződő Trisk 95 fehérje negatívan szabályozza a RyR funkcióját; így mind a lokális, mind pedig a globális Ca2+-jelek kialakulását gátolja. Rámutattunk, hogy a közös gamma receptorcsaládba tartozó IL-2/15 és -9 receptorok sejtfelszíni szerveződését feltehetően azonos rendező elvek alakítják ki. Tanulmányoztuk a T sejtek membránpotenciálját meghatározó Kv1.3 K+-csatorna immunszinapszis-beli lokalizációjának funkcionális következményeit. Kidolgoztunk egy újfajta FRET mérési módszert, amivel az IL-2 útvonal által aktivált transzkripciós faktorok konformációját határoztuk meg. Eredményeink hozzásegítenek a T-sejtes immunválasz mechanizmusának megértéséhez. | The main aim of our project was to characterize selected receptors and ion channels in transmembrane signaling processes using a multidisciplinary approach. We detected expression and mainly mitochondrial localization of TASK-3 K+-channels both in malignant melanomas and benign melanocytes. We described the relationship between maturation and ion channel expression in dendritic cells (DCs). We showed that the primary Ca2+ signaling pathway is mediated by CRAC channels, independent of the degree of maturation. We studied the connection between Ca2+ homeostasis and purinerg signaling in excitable and non-excitable cells, and characterized the type of purinerg receptors involved in this process. We demonstrated that the Trisk 95 protein expressed in the SR negatively regulates RYR function; thus it inhibits the generation of both local and global Ca2+ signals. We revealed that the cell surface organization of the members of the common gamma chain family, IL-2/15 and 9 receptors, is probably organized by the same general principles. We studied the functional consequences of the localization of Kv1.3 K+ channels in the immune synapse. We elaborated a new FRET method, by which we described the conformation of transcription factors induced by the IL-2 pathway. Our results contribute to understanding the mechanism of T cell mediated immune responses

Laser induced calcium oscillations in fluorescent calcium imaging

Phototoxicity is the most common problem investigators may encounter when performing live cell imaging. It develops due to excess laser exposure of cells loaded with fluorophores and can lead to often overlooked but significant artifacts, such as massive increase of intracellular Ca2+ concentration, which would make data interpretation problematic. Because information about laser- and dye-related changes in cytoplasmic calcium concentration is very limited, we aimed to describe this phenomenon to help investigators using laser scanning confocal microscopy in a non-invasive way. Therefore, in the present study we evaluated fluorescent fluctuations, which evolved in Fluo- 3/4/8 loaded mouse pancreatic acinar cells during very low intensity laser excitation. We demonstrate that after standard loading procedure (2 μM Fluo-3/4/8-AM, 30 min @ room temperature), applying 488 nm laser at as low as ca. 10 μW incident laser power (0.18 μW/μm2) at 1 Hz caused repetitive, 2-3 fold elevations of the resting intracellular fluorescence. The first latency and the pattern of the fluorescence fluctuations were laser power dependent and were related to Ca2+-release from intracellular stores, as they were abolished by BAPTA-AM treatment in Ca2+- free medium, but were not diminished by the ROS scavenger DMPO. Worryingly enough, the qualitative and quantitative features of the Ca2+-waves were practically indistinguishable from the responses evoked by secretagogue stimulation. Since using similar imaging conditions, a number of other cell types were reported to display spontaneous Ca2+ oscillations, we propose strategies to distinguish the real signals from artefacts

A triadin szerepe a szarkoplazmatikus retikulumból történő kalcium-felszabadulás folyamatában harántcsíkolt izmon = The role of surface membrane and sarcoplasmic reticular proteins in acquired and hereditary muscle disorders

A szarkoplazmatikus retikulum (SR) kalcium csatornája (RyR) több fehérjével is kölcsönhatásban áll. Ide tartozik a 95 kDa-os molekulasúlyú triadint (Trisk 95), melynek alternatív splicingjával többféle molekulasúlyú izoformája jöhet létre. A Trisk 95 funkciójának felderítésére létrehozott triadin knockout (KO) egerek izmai gyengébbek és a kalciumhomeosztázisban résztvevő fehérjék expressziós szintje változott. Eredményeink szerint a triadin hiánya izom myopathiához vezethet és az általunk létrehozott állatmodellel ez jól tanulmányozható. A Trisk 95 további szerepét C2C12 izomsejteken és primer vázizomtenyészeteken tanulmányoztuk. A fehérje stabil overexpressziója csökkentette az elemi kalcium felszabadulási események (ECRE) amplitúdóját és frekvenciáját, és a depolarizáció által kiváltott Ca2+-tranzienseket. Ugyanakkor az endogén triadinexpresszió shRNS-sel történő gátlása után az ECRE-k jelentősen gyakoribbak voltak. Így feltételezhető, hogy a Trisk 95 fehérje negatívan szabályozza a RyR működését. A 32 kDa-os molekulasúlyú triadint (Trisk 32) L6.G8 myoblastokban termeltettük túl. A transzfekció nem befolyásolta a sejtek életképességét, ugyanakkor a sejtek proliferációs képessége lecsökkent. Kimutattuk a Trisk 32 és az IP3R kolokalizációját és közvetlen fizikai kapcsolatát. Funkcionális kapcsolatukat is bizonyítottuk, mivel a Trisk 32 overexpressziója szignifikánsan nagyobb amplitúdójú és felszálló meredekségű Ca2+-tranzienseket eredményezett az IP3 útvonalon keresztül. | The calcium release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum (SR) is associated with different proteins. One of them is the 95 kDa molecular weight triadin (Trisk 95) which has various isoforms with different molecular weights, all of them are splice variants of the same gene. To explore the function of Trisk 95 we generated a triadin knockout (KO) mouse which is characterized by a weaker skeletal muscle force than the control animals. Also, in case of the KO animals, the expression levels of the proteins which play role in calcium homeostasis, was changed. These results indicate that the lack of triadin can cause muscle myopathy and our animal model is suitable to investigate it. To further study the role of Trisk 95, the protein was investigated in C2C12 skeletal muscle cells and in primary skeletal muscle cultures. The constant overexpression of the protein decreased the amplitude and frequency of the elementary calcium release events (ECRE). Furthermore, the silencing of endogenous triadin expression with shRNA led to more frequent ECREs. These results suggest that Trisk 95 is a negative regulator of RyR function. Finally the 32 kDa molecular weight triadin (Trisk 32) was overexpressed in L6.G8 myoblasts. We showed the colocalization and direct physical interaction of Trisk 32 and IP3 receptor. Their functional interaction was also proved as the overexpression of Trisk 32 caused greater Ca2+-transients

Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

: During muscle cell differentiation, the alternatively spliced, acidic β-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the β-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid β-domain variants, as well as by a disordered β-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the β-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that β-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process

REASONS FOR AND OBSTACLES TO CYCLING IN OPINIONS OF RESIDENTS OF DEBRECEN, HUNGARY

It is a basic aim of the European Union that due to the developments in 2014-2020 the bicycle would become one of the most often used transportation, touristic, and sports equipment. We were interested to see to what extent is bicycling present in the transportation system of Debrecen and what are the most important reasons for its residents to use the bicycles. The dedication of Debrecen to promote cycling is clearly proven by the number of newly built or resurfaced bike paths and by the fact that the University of Debrecen has introduced – alone in the region – UniBike which is a bicycle renting system brought forth by the need of its students. Here we present the developments that took place in the North Plain Region in the past few years. We have also analyzed the national and European strategies and reports on bicycling. A survey was conducted among the youth of Debrecen to explore their cycling habits. The data were evaluated using the EvaSys program. Until the end of 2011 with the help of different funds 862 km of bike paths had been built in Hungary. In the North Plain Region due to funds totaling 777 million HUF 15.7 km long bike paths had been constructed until 2015. The development of tourism in this direction is promoted by the web-pages and brochures offering bicycle-tours around Debrecen. Nevertheless, bicycling in the neighboring townships is present not as an instrument for sports and/or tourism, rather as a mean of transportation. It is a clear goal in Europe and thus in Hungary to have bike paths that can provide the means of safe cycling. In parallel, it is also important to promote the benefits of bicycling, including positive physiological effects, cost-effectiveness, and environment-friendliness to increase the proportion of those who select bicycling as an alternative

Reasons for and obstacles to cycling in opinions of residents of Debrecen, Hungary

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Reasons for and obstacles to cycling in opinions of residents of Debrecen, Hungary

It is a basic aim of the European Union that due to the developments in 2014-2020 the bicycle would become one of the most often used transportation, touristic, and sports equipment. We were interested to see to what extent is bicycling present in the transportation system of Debrecen and what are the most important reasons for its residents to use the bicycles. The dedication of Debrecen to promote cycling is clearly proven by the number of newly built or resurfaced bike paths and by the fact that the University of Debrecen has introduced – alone in the region – UniBike which is a bicycle renting system brought forth by the need of its students. Here we present the developments that took place in the North Plain Region in the past few years. We have also analyzed the national and European strategies and reports on bicycling. A survey was conducted among the youth of Debrecen to explore their cycling habits. The data were evaluated using the EvaSys program. Until the end of 2011 with the help of different funds 862 km of bike paths had been built in Hungary. In the North Plain Region due to funds totaling 777 million HUF 15.7 km long bike paths had been constructed until 2015. The development of tourism in this direction is promoted by the web-pages and brochures offering bicycle-tours around Debrecen. Nevertheless, bicycling in the neighboring townships is present not as an instrument for sports and/or tourism, rather as a mean of transportation. It is a clear goal in Europe and thus in Hungary to have bike paths that can provide the means of safe cycling. In parallel, it is also important to promote the benefits of bicycling, including positive physiological effects, cost-effectiveness, and environment-friendliness to increase the proportion of those who select bicycling as an alternative. JEL Code: I1