Molecular evolution of epididymal lipocalin genes localized on mouse chromosome 2

Producción CientíficaWe previously identified two murine secretory proteins, mE-RABP(Lcn5) and mEP17(Lcn8), belonging to the lipocalin family and specifically expressed in the epididymis. The genes are contiguous and localized on mouse chromosome 2. We now show that five other related lipocalin genes, Lcn9, Lcn10, Lcn11, Lcn12, and Lcn13, that evolved by in situ tandem duplication are present on the same locus. Lcn9, Lcn10, Lcn12, and Lcn13 genes, like Lcn5 and Lcn8 genes, are specifically expressed in the mouse epididymis. However, each gene has a distinct spatial expression within the epididymis and different regulation. Analysis of the human genome sequence shows the presence of genes encoding lipocalins with genomic organization, chromosomal arrangement, and orientation similar to that of the corresponding murine genes, indicating that the epididymal cluster is evolutionary conserved. A phylogenetic analysis of the new epididymal proteins reveals their spread position in the lipocalin protein family tree. This suggests the preservation of the regulatory sequences, while protein sequences have greatly diverged, reflecting functional diversity and possibly multifunctionality. In terms of the cluster ancestry, epididymal expression possibly appeared in a PGDS-like lipocalin in amniotes, and the duplications generating the cluster occurred at least in the common ancestor of rodents and primates. The presence and conservation of a cluster of five genes encoding epididymal lipocalins, differently regulated and regionalized in the epididymis, strongly suggests that these proteins may play an important role for male fertility

Human homolog of Drosophila Hairy and enhancer of split 1, Hes1, negatively regulates δ-catenin (CTNND2) expression in cooperation with E2F1 in prostate cancer

<p>Abstract</p> <p>Background</p> <p>Neuronal synaptic junction protein δ-catenin (<it>CTNND2</it>) is often overexpressed in prostatic adenocarcinomas but the mechanisms of its activation are unknown. To address this question, we studied the hypothesis that Hes1, human homolog of <it>Drosophila </it>Hairy and enhancer of split (Hes) 1, is a transcriptional repressor of δ-catenin expression and plays an important role in molecular carcinogenesis.</p> <p>Results</p> <p>We identified that, using a <it>δ-catenin </it>promoter reporter assay, Hes1, but not its inactive mutant, significantly repressed the upregulation of δ-catenin-luciferase activities induced by E2F1. Hes1 binds directly to the E-boxes on <it>δ-catenin </it>promoter and can reduce the expression of δ-catenin in prostate cancer cells. In prostate cancer CWR22-Rv1 and PC3 cell lines, which showed distinct δ-catenin overexpression, E2F1 and Hes1 expression pattern was altered. The suppression of Hes1 expression, either by γ-secretase inhibitors or by siRNA against Hes1, increased δ-catenin expression. γ-Secretase inhibition delayed S/G2-phase transition during cell cycle progression and induced cell shape changes to extend cellular processes in prostate cancer cells. In neuroendocrine prostate cancer mouse model derived allograft NE-10 tumors, δ-catenin showed an increased expression while Hes1 expression was diminished. Furthermore, <it>E2F1 </it>transcription was very high in subgroup of NE-10 tumors in which <it>Hes1 </it>still displayed residual expression, while its expression was only moderately increased in NE-10 tumors where Hes1 expression was completely suppressed.</p> <p>Conclusion</p> <p>These studies support coordinated regulation of δ-catenin expression by both the activating transcription factor E2F1 and repressive transcription factor Hes1 in prostate cancer progression.</p

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three- dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region- specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE- RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related

KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer

Measurement and comparison of individual external doses of high-school students living in Japan, France, Poland and Belarus -- the "D-shuttle" project --

Twelve high schools in Japan (of which six are in Fukushima Prefecture), four in France, eight in Poland and two in Belarus cooperated in the measurement and comparison of individual external doses in 2014. In total 216 high-school students and teachers participated in the study. Each participant wore an electronic personal dosimeter "D-shuttle" for two weeks, and kept a journal of his/her whereabouts and activities. The distributions of annual external doses estimated for each region overlap with each other, demonstrating that the personal external individual doses in locations where residence is currently allowed in Fukushima Prefecture and in Belarus are well within the range of estimated annual doses due to the background radiation level of other regions/countries

ROCK Inhibitor Y-27632 Suppresses Dissociation-Induced Apoptosis of Murine Prostate Stem/Progenitor Cells and Increases Their Cloning Efficiency

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin−Sca-1+CD49fhigh (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells

Loss of the Urothelial Differentiation Marker FOXA1 Is Associated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation

Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC

Sport facilities as a part of tourism product of Cracow

The subject of my Bachelor’s degree (BA) thesis are sport facilities in Cracow as a part of city tourism product. The main purpose of my thesis is to show the value of sport facilities for tourism in Cracow. The goal of first chapter is to present theory about sport facilities in the structure of urban tourism product. From this chapter we learn what is tourism product and how to shape it. There is also explained the term of sport tourism and there are shown sport fans as tourists. In the end there is shown the place of sport facilities in the structure of city tourism product and mass events and their's protection. Second chapter presents sport establishments and investments in Cracow. There is also placed the calendar of sport events in Cracow in 2011 and there are agendas and agreements concerning sport in Cracow. Third chapter shows sport as a city tourism product. There are presented agendas and agreements concerning sport in Cracow. There are placed graphs showing the results of the survey involving sport facilities in Cracow. In the end there are brought conclusions and recommendations which result from the survey.Tematem mojej pracy licencjackiej są obiekty sportowe w Krakowie jako element miejskiego produktu turystycznego. Głównym celem mojej pracy jest pokazanie znaczenia obiektów sportowych dla Krakowskiej turystyki. Celem pierwszego rozdziału jest zaprezentowanie teorii na temat obiektów sportowych i ich miejsca w strukturze miejskiego produktu turystycznego. Z tego rozdziału dowiadujemy się co to jest produkt turystyczny i jak go kształtować. Jest też wyjaśnione pojęcie turystyki sportowej (usportowionej) i ujęcie fanów sportu jako turystów. Na końcu pokazane jest miejsce obiektów sportowych w strukturze miejskiego produktu turystycznego oraz imprezy masowe i ich zabezpieczanie. Drugi rozdział przedstawia obiekty oraz inwestycje sportowe w Krakowie oraz kalendarz imprez sportowych w Krakowie na rok 2011. Trzeci rozdział przedstawia sport jako miejski produkt turystyczny. Zawiera on wykresy prezentujące wyniki ankiety dotyczącej obiektów sportowych w Krakowie. Na końcu umieszczone zostały wnioski i rekomendacje wynikające z ankiety

DEPARTEMENT OF TOURISM MANAGEMENT

Tematem pracy magisterskiej są cykliczne imprezy sportowe jako narzędzie promocji miasta Krakowa. Głównym celem mojej pracy jest pokazanie znaczenia cyklicznych imprez sportowych w promocji Krakowa. Celem pierwszego rozdziału jest zaprezentowanie teorii dotyczącej produktu turystycznego i marketingu terytorialnego. Z tego rozdziałudowiadujemy się co to jest produkt turystyczny i jak go kształtować, oraz na czym polega marketing terytorialny. W tym rozdziale zaprezentowane zostały również sposoby promocji miasta. Drugi rozdział przedstawia sposoby tworzenia wizerunku miasta jako organizatoraimprez sportowych. Trzeci rozdział przedstawia wpływ cyklicznych imprez sportowych na wizerunek miasta. Rozdział ten zawiera rysunki przedstawiające wyniki ankiety, natomiast na końcu zostały zamieszczone wnioski i rekomendacje wynikające z ankiety.The subject of Master degree thesis are periodic sport events as a way of promoting the city of Cracow. The main purpose of my thesis is to present the meaning of periodic sport events and its influence on tourism popularity of the city. First chapter presents theory concerningthe term of city product and territorial marketing. Second part of my thesis considers the ways of creating the image of the city. It also concerns the meaning of organizing sport events in Cracow.The last chapter is to present the results of the survey considering the whole image of the city.Third chapter also includes conclusions and recommendations based on the survey