A spermium/petesejt együttműködés genetikai boncolása. = Genetic dissection of sperm and egg cell cooperation.

Munkánk célja az, hogy a Drosophila HimcaD, HorkaD valamint KompoltD domináns nőstény-steril mutációiból kiindulva molekuláris szinten ismerjük meg az ép Himca, Horka és Kompolt gének szerepét a megtermékenyülésben, az embriógenezis elkezdődésében. Megtudtuk, hogy (i) a Himca gén terméke az inozitol-1,4,5-trifoszfát (ITF) kináz enzim, amely a Ca2+ ionok belső raktárakból történő felszabadulásáért felelős. Úgy tűnik, hogy ITF hiányában a Ca2+ ion-hullámok elmaradása okozza a megtermékenyüléssel, a spermiumfarok elhelyezkedésével, illetve az embriógenezis elkezdődésével kapcsolatos problémákat. (ii) A Horka gén terméke a transzkripció-terminációs-faktor-2 (TTF2). A TTF2 úgy okoz abortív transzkripciót, hogy eltávolítja az RNS polimeráz-II-t a DNS-ről. A DNS-csip, valamint a Q-RT-PCR technika módszereivel arra derítettünk fény, hogy TTF2 hiányában - a HorkaD/+ és a horkanull/? hemizigóta nőstények petéiben - 54 olyan ép gén terméke bukkan fel, amelyek a vadtípusú petékben nincsenek jelen. A szokatlan géntermékek megmérgezik az embriókat. A HorkaD mutációból kiindulva a nőstény-sterilitás eddig ismeretlen típusára derítettünk fényt. (iii) Kompolt gén terméke egy olyan transzmembrán fehérje, amely többféle, több kópiában meglevő, és ismert domént tartalmaz. A doménoknak a spermium adhézióban, a sejt-sejt kölcsönhatásban, valamint a szingnáltranszdukcióban van szerepe. | We aimed to reveal molecular function of the Himca, Horka és Kompolt genes in fertilization and in the commencement of embryogenesis in Drosophila. The genes have been previously identified by the HimcaD, HorkaD and the KompoltD dominant female-sterile mutations. It has turned out that (i) product of the Himca gene is the inozitol-1,4,5-triphosphate (ITF) kinase enzyme. ITF is required for release of Ca2+ from the store sites. It appears that Ca2+ waves do not develop in absence of ITF that leads subsequently to abnormal sperm tail positioning in the egg cytoplasm, aberrant fertilization and the lack of commencement of embryogenesis. (ii) The Horka gene encodes the formation of transcription-termination-factor-2 (TTF2). TTF2 has been known to cause abortive transcription through the removal of RNA polymerase-II from the DNA. Making use of the DNA chip technology and the Q-RT-PCR technique we showed that in absence of TTF2 - in eggs of the HorkaD/+ and the horkanull/? hemizygous females - products of 54 genes appear in the egg cytoplasm that are missing in the wild type eggs. The unusual gene products poison the embryos. Starting from the HorkaD mutation we revealed a thus far unknown type of female sterility. (iii) Product of the Kompolt gene is a giant transmembrane protein that contains several copies of several known domains. The domains play role in sperm adhesion, cell-to-cell interaction and signal transduction

Expression of Tight Junction Components in Hepatocyte-Like Cells Differentiated from Human Embryonic Stem Cells

Human embryonic stem cells can be differentiated in vitro into a wide variety of progeny cells by addition of different morphogens and growth factors. Our aim was to monitor the expression pattern of tight junction (TJ) components and various cellular markers during differentiation of stem cell lines toward the hepatic lineage. Human embryonic stem cell lines (HUES1, HUES9) were differentiated into endoderm-like cells, and further differentiated to hepatocyte-like cells. Gene expressions of Oct3/4, Nanog, alpha-fetoprotein, albumin, cytokeratins (CK-7, CK-8, CK-18, CK-19), ATP-binding cassette (ABC) transporters (ABCC2, ABCC7, ABCG2), and various TJ components, including claudin-1, claudin-4, claudin-5, claudin-7, and tricellulin, as well as an extracellular matrix component, agrin were monitored during hepatic differentiation by real-time quantitative PCR. The differentiated cells exhibit epithelial morphology and functional assessments similar to that of hepatocytes. The expression level of stem cell marker genes (Oct3/4 and Nanog) significantly and gradually decreased, while liver-associated genes (alpha-fetoprotein, albumin) reached their highest expression at the end of the differentiation. The endoderm-like cells expressed claudin-1, which declined eventually. The expression levels of cholangiocyte markers including claudin-4, CK-7, CK-19, and agrin gradually increased and reached their highest level at the final stage of differentiation. In contrast, these cells did not express notable level of claudin-7, CK-8 and tricellulin. The marker set used for monitoring differentiation revealed both hepatocyte and cholangiocyte characteristics of the differentiated cells at the final stage. This is the first report describing the expression level changes of various TJ components, and underlining their importance in hepatic differentiation. © 2015 Arányi Lajos Foundatio

Excision efficiency is not strongly coupled to transgenic rate: cell type dependent transposition efficiency of Sleeping Beauty and piggyBac DNA transposons

The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, “overproduction inhibition,” a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own “in-house” platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2–3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future

Cellular Processing of the ABCG2 Transporter-Potential Effects on Gout and Drug Metabolism.

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking

The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation

Introduction: Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug exporters and the Solute Carrier transporters, with various transport directions and mechanisms.Areas covered: In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges.Expert opinion: The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of stemness and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation. © 2015 Taylor & Francis

Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives

Background: ATP-binding Cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins. Methods: Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized cell types were separated mechanically. Cellular features regarding pluripotency and tissue identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy and qPCR-based low-density arrays. Results: Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells and mesenchymal stem cells) were distinguished by morphology, immunostaining markers and selected mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug and lipid transporter ABC protein expression was examined by using well characterized specific antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded well to the mRNA expression results. Moreover, the cellular localization of these important human ABC transporter proteins could be established in the pluripotent and differentiated hESC derived samples. Conclusions: These studies provide valuable information regarding ABC protein expression in human stem cells and their differentiated offspring. The results may also help to obtain further information concerning the specialized cellular functions of selected ABC transporter

Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

<p>Abstract</p> <p>Background</p> <p>The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The <it>Sleeping Beauty </it>(SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo.</p> <p>Results</p> <p>We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene <it>RPPH1 </it>and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle.</p> <p>Conclusions</p> <p>We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.</p