神戸大学大学院人文学研究科地域連携センター 福崎町連携事業平成23年度活動報告書 : 共同研究「福崎町の地域歴史遺産掘り起こし及び大庄屋三木家住宅活用案の作成等」

Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases

Eltérő expressziós szintet mutató mikroRNS-ek és a miR-126 antiproliferatív hatása a kissejtes tüdőrákban

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by binding to the 3' untranslated region (3'UTR) of target mRNAs, inducing mRNA degradation or translation repression. Approximately 50% of miRNA genes are in cancer-associated genomic regions, suggesting that miRNAs play a significant role in tumor biology. Small cell lung cancer (SCLC) is a high-grade neuroendocrine tumor characterized by rapid progression and frequent metastasis. We combined microarray and qRT-PCR analyses to identify miRNAs aberrantly expressed in SCLC. The microarray approach alone identified 19 miRNAs that are significantly overexpressed and 35 miRNAs that are downregulated in SCLC cell lines compared to normal lung. RNA samples from SCLC cell lines and a small number of microdissected primary SCLC tumors were analyzed with qRT-PCR as well. At first we identified 16 overexpressed and 8 downregulated miRNAs in primary SCLC tumor samples, as well as in SCLC cell line samples. 7 downregulated and 8 overexpressed miRNAs were selected for further analysis in a larger panel of individual SCLC tumors and SCLC cell lines. qRT-PCR analysis verified that miR-126 is a uniformly downregulated miRNA, while miR-301 and miR-183 are uniformly overexpressed miRNAs in all SCLC sample types. We analyzed DNA copy number changes in primary SCLC tumors for 5 genomic regions with overexpressed miRNAs. We identified one novel amplified region in SCLC: 7q32.2 contains the miR-183/96/182 cluster. In our further work we demonstrated that miR-126 overexpression has a negative effect on SCLC cell proliferation, by delaying cells in the G1 phase of the cell cycle. Importantly, we identified SLC7A5 as a novel target of miR-126 in SCLC cells. miR-126 downregulates the expression of SLC7A5 at the translation level, and reduces mRNA stability simultaneously. We demonstrated that in SCLC cells, similarly to other tumor types, suppression of SLC7A5 expression has an anti-proliferative effect. SCL7A5 suppression or miR-126 overexpression both delay SCLC cells in the G1 phase, suggesting that the effect of miR-126 on the cell cycle is mediated at least in part through SLC7A5. SLC7A5 provides the essential amino acids that act as signal to enhance growth of cancer cells through mammalian target-of-rapamycin (mTOR)-stimulated translation. Through different targets, miR-126 can negatively regulate PI3K/Akt pathway, which is aberrantly active in a large percentage of SCLC tumors. Therefore, miR-126 is an important negative regulator of the growth and proliferation of SCLC cells, which probably fine-tunes the activity of the PI3K/Akt/mTOR network through multiple targets, including SLC7A5. A mikroRNS-ek (miRNS) rövid, nemkódoló RNS molekulák, melyek a génexpresszió szabályozásában vesznek részt azáltal, hogy a target mRNS-hez kapcsolódva gátolják azok transzlációját, illetve bizonyos esetekben az mRNS degradációját váltják ki. A tumorbiológiában betöltött szerepüket alátámasztja az a tény is, hogy az ismert miRNS-ek 50%-a olyan genomi régiókban található, melyek tumorokban amplifikálódnak vagy deletálódnak. Az általunk vizsgált tumorttípus a kissejtes tüdőrák (SCLC), ami egy neuroendokrin eredetű, gyorsan metasztatizáló tumor. Kísérleteinkben microarray és qRT-PCR technikák kombinálásával sikerült meghatározni az SCLC-re jellemző miRNS-ek expressziós mintázatát. MiRNS microarray technikával azonosítottunk 19 magas- és 35 alacsony expressziós szintet mutató miRNS-et az SCLC sejtvonalakban a normál tüdőszövethez viszonyítva. A microarray adatoknak megfelelően qRT-PCR technikával sikerült azonosítani 16 magas- és 8 alacsony expressziós szintet mutató miRNS-t az SCLC sejtvonalakban és primer SCLC tumorokban. A továbbiakban nagyszámú primer SCLC tumorban vizsgáltuk a különbözően expresszálódó miRNS-eket. A qRT-PCR egyöntetűen igazolta a miR-126 alacsony, míg a miR-301 és a miR-183 magas expresszióját az összes SCLC mintában, sejtvonalakban és primer tumorokban egyaránt. Kísérleteinkben 5 magas expressziós szintet mutató miRNS kópiaszám változását is megvizsgáltuk és azonosítottunk egy új, SCLC-ben amplifikálódó régiót, a 7q32.2-t, mely tartalmazza a miR-183/96/182 klasztert. További kísérleteinkben az alacsony szinten expresszálódó miR-126 miRNS szerepét vizsgáltuk SCLC sejtekben. Kimutattuk, hogy a miR-126 az SCLC sejtek proliferációját gátolja, a sejtciklus G1 fázisból S fázisba való átmenetét lassítva. Azonosítottunk egy új miR-126 célgént a kissejtes tüdőrákban, az SLC7A5 proteint. Az SCLC sejtekben, más tumorokhoz hasonlóan, az SLC7A5 expressziójának gátlása anti-proliferatív hatással bírt. Mind az SLC7A5 expressziójának gátlása, mind a miR-126 expressziójának növelése késleltette az SCLC sejtek kilépését a sejtciklus G1 fázisából, azt mutatva, hogy a miR-126 sejtciklusra kifejtett hatása részben az SLC7A5 fehérje expressziójának gátlásán keresztül megy végbe. Az SLC7A5 aminosav transzporter gondoskodik az esszenciális aminosavak felvételéről, melyek az mTOR szignál útvonal aktiválásán keresztül szignál molekulaként szolgálnak a tumor sejtek proliferációjához. A miR-126 több fehérjén keresztül vesz részt a PI3K/Akt útvonal szabályozásában, mely az SCLC tumorok nagy részében aberránsan aktív. A miR-126 az SCLC sejtek növekedésének egy fontos negatív regulátora, részben a PI3K/Akt/mTOR szignál útvonalra gyakorolt hatása révén, melyet olyan target géneken keresztül fejthet ki, mint az SLC7A5

The Trinity with Saints formerly Attributed to Jacopo Palma the Younger

Durand l'année académique 2015/2016 la conservation d'une peinture sur toile du 16e siècle, autrefois attribuée à Jacopo Palama a été réalisé au département de l'Université hongroise des Beaux-Arts. La restauration a été rendue nécessaire à cause de l'assombrissement du vernis et la détérioration due à des dégâts des eaux. Une série d'examens techniques a été effectuée. L'examen photographique (réflectographie IR, radiographie sous rayon X, fluorescence sous UV, lumière rasante) et l'examen de coupe stratigraphique d'échantillons de la couche picturale (microscope optique, fluorescent, polarisant et test microchimique) ont permis de décrire les matériaux et le processus de peinture.In the 2015/16 academic year the conservation of a 16th century canvas painting formerly attributed to Jacopo Palma took place at the Conservation Department of the Hungarian University of Fine Arts. The darkened varnish layers and the severe deterioration caused by water damage made the conservation of the painting necessary. A series of technical examinations were carried out. Photographic examination (IR-reflectography, X-radiography, UV-fluorescence, raking light images) and examination of the cross sections of the samples taken from the painted surface (normal, fluorescent and polarized light microscopy and microchemical tests) made it possible to describe the materials and the painting process

VALUE IN GRASS – MATTER OF FIBRE AND CARBS

Climate adaptation is a major challenge. Chasing the sufficient amount of hay is getting in higher priority. Distant mass hay producers give favourable offers despite long distances. Quality is also gaining position and indicators like RFQ (Relative Forage Quality) is highlighting the marketing language. Hay market as we knew no longer exists in Hungary. Most farmers produce their own hay and do not spend extra cents to buy bales. Climate change however, force them to adapt and store more bales for the future. Horse owners and dairy farmers are the main driver to convince hay producers to provide high quality forage. We gathered Hungarian regional hay-price information and evaluated the trends in this sector. The demand-driven hay-price is in contradiction with premium quality timothy grass hay

VALUE IN GRASS – MATTER OF FIBRE AND CARBS

Climate adaptation is a major challenge. Chasing the sufficient amount of hay is getting in higher priority. Distant mass hay producers give favourable offers despite long distances. Quality is also gaining position and indicators like RFQ (Relative Forage Quality) is highlighting the marketing language. Hay market as we knew no longer exists in Hungary. Most farmers produce their own hay and do not spend extra cents to buy bales. Climate change however, force them to adapt and store more bales for the future. Horse owners and dairy farmers are the main driver to convince hay producers to provide high quality forage. We gathered Hungarian regional hay-price information and evaluated the trends in this sector. The demand-driven hay-price is in contradiction with premium quality timothy grass hay

Summarized fold change values by microarray results of the selected 9 CPD-dependent genes.

<p>active PL: samples containing active CPD-photolyase, inactive PL: samples containing inactive CPD-photolyase.</p><p>Summarized fold change values by microarray results of the selected 9 CPD-dependent genes.</p

Accelerated photorepair of CPDs in HaCaT cells transfected with CPD-PL Ψ-mRNA.

<p>HaCaT cells were transfected with lipofectamine-complexed Ψ-mRNA encoding CPD-photolyase. Twelve hours later, cells were subjected to 20 mJ/cm² UVB and immediately exposed to photoreactivating light (photoreactivated) or left in the dark (non-photoreactivated) for 1 h and then maintained at 37°C for 5 and 23 hs. After incubation at 5 and 23 h, genomic DNA was isolated at the indicated times after UVB irradiation and the amount of CPDs was measured by ELISA. The values were calculated relative to those obtained with cells that were not UVB-irradiated. Significance was assessed by unpaired, two-sample <i>t</i>-test, p<0.05. Error bars represent the standard error of the mean from three experiments performed independently.</p

Induction of cyclin E1 and p15INK4b protein expression upon UVB exposure is regulated through the JNK signalling pathway in HaCaT cells.

<p>Keratinocytes transfected with CPD-PL Ψ-mRNA were incubated in serum-free medium supplemented with JNK inhibitor (SP600125) for 1 h. Immediately thereafter, cells were irradiated with a physiological dose of UVB or left untreated followed by exposure to photoreactivating light (or not) for 1 h. The cells were cultured further in serum-free medium supplemented with the inhibitor. Cells were harvested for western blot assay at the indicated time. Protein levels of cyclin E1, p15INK4b, and β-actin are noted. The figure shows representative results from three independent experiments.</p

Experimental verification of microarray results for 9 selected genes.

<p>HaCaT cells were exposed to 20 mJ/cm² UVB at 12 h after delivery of CPD-PL Ψ-mRNA. Immediately thereafter, cells were either subjected to photoreactivating light (active CPD-photolyase) or left in the dark (inactive CPD-photolyase) for 1 h. Following incubation total RNA was extracted at 5 and 23 h, then real-time RT-qPCR was performed to validate the CPD-dependent expression of ATF3, CCNE1, CDKN2B, EGR1, ID2, PTGS2, RUNX1, SNAI1 and SNAI2. Values measured in UVB irradiated cells with or without photoreactivation were related to those measured in non-UVB irradiated cells that were transfected control Ψ-mRNA, (pecked lines). Asterisks indicate significant differences (two-tailed, unpaired <i>t</i>-test; p<0.05) between photoreactivated (active CPD-photolyase) and non-photoreactivated (inactive CPD-photolyase) samples. The results of RT-qPCR are means ± SEM from three independent experiments in triplicate.</p

Photorepair of CPDs prevents altered expression of cyclin E1 and p15INK4b protein in UVB irradiated HaCaT cells.

<p>Cells were transfected with lipofectamine-complexed CPD-PL Ψ-mRNA, 12 hs later irradiated with 20 mJ/cm² UVB and immediately exposed to photoreactivating light (active CPD-photolyase) or kept in the dark (inactive CPD-photolyase) for 1 h. Subsequently, cells were cultured at 37°C until harvested at the indicated time after UVB irradiation. (A) The expression of cyclin E1 and p15INK4b were analyzed by Western blot. (B) Quantitation of western blots displays relative changes in protein expression normalized to β-actin. Pixel densities were calculated relative to those obtained with cells that were not UVB irradiated (pecked lines). Significance was assessed by two-tailed, unpaired <i>t</i>-test (asterisk, p<0.05) showing differences between photoreactivated and non-photoreactivated samples. Error bars represent the standard error of the mean. The results are means of three independent experiments.</p