Az alvó agy egyedspecifikus vonatkozásai - az intelligencia és az alvó agy

Jelen tanulmány a kognitív képességek és az agy alvás közben feltárulkozó összefüggéseivel foglalkozik. Célunk bemutatni a témában összegyűlt fontosabb felfedezéseket és újításokat, különös tekintettel az alvási orsózás és intelligencia kapcsolatára. Az alvási EEG (elektro-enkefalográfia) orsószerű képletei ujjlenyomatszerűen jellemzik az egyént, mely jelenség főként a NREM alvás 2. stádiumában észlehető. Az alvási orsó egyedspecifikussága révén kapcsolatban állhat egyéb kognitív területekkel, mint például a tanulás különböző fajtáival, a memória konszolidációjával és az intelligenciával. Bemutatjuk a SE Magatartástudományi Intézetében működő alváskutató csoport alvás EEG-t feltáró munkáját és eredményeit, melyeket az intelligencia és az alvási orsózás relációjában kapott, és amely eredmények rávilágíthatnak az intelligencia viselkedéses és személyiségbeli változóira

Az alvó agy egyedspecifikus vonatkozásai - az intelligencia és az alvó agy

Jelen tanulmány a kognitív képességek és az agy alvás közben feltárulkozó összefüggéseivel foglalkozik. Célunk bemutatni a témában összegyűlt fontosabb felfedezéseket és újításokat, különös tekintettel az alvási orsózás és intelligencia kapcsolatára. Az alvási EEG (elektro-enkefalográfia) orsószerű képletei ujjlenyomatszerűen jellemzik az egyént, mely jelenség főként a NREM alvás 2. stádiumában észlehető. Az alvási orsó egyedspecifikussága révén kapcsolatban állhat egyéb kognitív területekkel, mint például a tanulás különböző fajtáival, a memória konszolidációjával és az intelligenciával. Bemutatjuk a SE Magatartástudományi Intézetében működő alváskutató csoport alvás EEG-t feltáró munkáját és eredményeit, melyeket az intelligencia és az alvási orsózás relációjában kapott, és amely eredmények rávilágíthatnak az intelligencia viselkedéses és személyiségbeli változóira

Az ABCG2 multidrog transzporter fehérje szerkezetének és működésének vizsgálata = Structure and function of the multidrug transporter ABCG2

Az ABCG2 multidrog transzporternek fontos szerepe van mind a daganatok kemoterápia-rezisztenciájában, mind a fiziológiás xenobiotikum transzportban. A projektben előállítottuk az ABCG2 fehérje különböző mutáns és polimorf változatait, elvégeztük ezek részletes funkcionális vizsgálatát. Egy sejtfelszínen reagáló, konformáció-érzékeny anti-ABCG2 monoklonális antitest alkalmazásával felderítettük a transzporter funkcionális állapotait, kémiai módosítások és mutációk segítségével elvégeztük az epitópok jellemzését és molekuláris szintű modellezését. Részletesen elemeztük az ABCG2 transzporter és a membrán lipidek kölcsönhatásait, megállapítottuk a membrán koleszterin jelentős szabályozó szerepét. Az ABCG2 transzporter és célzott hatású rákellenes vegyületek kölcsönhatásainak vizsgálata során klinikailag is alkalmazott gyógyszerekre vonatkozóan kaptunk új információkat. Új módszereket fejlesztettünk ki az ABCG2 szabályozásának, lokalizációjának és funkciójának vizsgálatára, elemeztük a transzporter expresszióját humán embrionális őssejtekben. Több, magas impakt faktorú nemzetközi folyóiratban közöltünk a témáról review cikkeket. | The human ABCG2 multidrug transporter plays a key role in the chemotherapy resistance of malignant tumors, as well as in the physiological elimination of xenobiotics. In this project we have prepared and expressed various mutant and polymorphic variants of the transporter, performed their detailed functional characterization. By using a cell-surface reacting, conformation-sensitive monoclonal antibody against ABCG2, we mapped the functional states of the transporter. In these experiments we applied specific chemical modifications and generated site-directed mutations to characterize the extracellular loop epitope region of ABCG2 and constructed a molecular model for this part of the transporter. We have investigated the modulation of ABCG2 by membrane lipids and found a major role for cholesterol in regulating the transport activity of this protein. By examining a number of new targeted anticancer agents we found that ABCG2 interacts with several of these compounds and may be involved in the resistance against clinically applied molecules. We have developed new methods for studying the regulation, localization and function of the ABCG2 protein, examined the expression profile of this transporter in human embryonic stem cells. During this project we have published several review articles in high-impact international journals

Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1.

Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions

Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1.

Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions

Cloning and characterization of a novel functional organic anion transporting polypeptide 3A1 isoform highly expressed in the human brain and testis

Organic anion transporting polypeptide 3A1 (OATP3A1, encoded by the SLCO3A1 gene) is a prostaglandin, oligopeptide, and steroid/thyroid hormone transporter with wide tissue distribution, expressed, e.g., in the human brain and testis. Although the physiological importance of OATP3A1 has not yet been clarified, based on its expression pattern, substrate recognition, and evolutionary conservation, OATP3A1 is a potential pharmacological target. Previously, two isoforms of OATP3A1, termed as V1 and V2, have been characterized. Here, we describe the cloning and functional characterization of a third isoform, OATP3A1_V3. The mRNA of isoform V3 is formed by alternative splicing and results in an OATP3A1 protein with an altered C-terminus compared to isoforms V1 and V2. Based on quantitative PCR, we demonstrate the widespread expression of SLCO3A1_V3 mRNA in human organs, with the highest expression in the brain and testis. By generation of an isoform V3-specific antibody and immunostaining, we show that the encoded protein is expressed in the human choroid plexus, neurons, and both germ and Sertoli cells of the testis. Moreover, we demonstrate that in contrast to isoform V1, OATP3A1_V3 localizes to the apical membrane of polarized MDCKII cells. Using HEK-293 cells engineered to overexpress OATP3A1_V3, we verify the protein’s functionality and identify dehydroepiandrosterone sulfate as a novel OATP3A1 substrate. Based on their distinct expression patterns but overlapping functions, OATP3A1 isoforms may contribute to transcellular (neuro)steroid transport in the central nervous system

A novel fluorescence-based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third-generation P-gp inhibitors and OATP1A2

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies

Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate

Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16min, and t1/2(fluorescein) 6.71 and 5.58min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53μM, and Km(fluorescein) 25.73 and 38.55μM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-β-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening