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RU, Biochemistry, 07 januari 2005, Promotores : Jong, W.W.W. de, Zoelen, E.J.J. van Co-promotor : Lubsen, N.H., Item does not contain fulltext, The DNA sequences of many genomes have been elucidated in the last years. The challenge now is to predict first the genotype and then the phenotype from the genomic sequences. To predict expression patterns of the genes located on the genome, not only regulation at the level of transcription, but also at the level of translation is important. To obtain more information about regulation of transcription, the promoter and its regulatory elements need to be mapped. In this thesis a conventional search for known and active regulatory elements in the B2-crystallin promoter was performed. For the intergenic region between aB-crystallin and HspB2, an alternative approach was taken to determine which elements are likely to be important for gene regulation, and the evolutionary conservation of sequence elements in this region was studied. Alignments of this region of many species lead to the confirmation of importance of known regulatory elements and the finding of new sites. A good example of regulation of gene expression mediated by regulation of translation, is translation after stress. During and after heat shock, cap-dependent translation is inhibited, while cap-independent translation can still take place. After heat shock translation of (s)Hsps is increased, these (s)Hsps are thought to protect cells from stress. We show that two of the sHsps, aB-crystallin and Hsp27 protect cap-dependent, but not cap-independent translation after heat shock. Inhibition of translation is thought to be mediated by a decrease in activity of the translation factors. However, the protection of translation mediated by sHsps does not seem to affect translation factors directly, but overexpression of aB-crystallin seems to increase the dispersal of stress granules that contain the stalled translation initiation complexes. We propose that aB-crystallin, probably together with other Hsps, resolubilizes the stress granule components and that translational thermotolerance is a cooperative effect

RU Radboud Universiteit Nijmegen, 26 oktober 2005, Promotores : Olijve, W., Zoelen, E.J.J. van, Contains fulltext : 32614.pdf (publisher's version ) (Open Access)

Contains fulltext : 32682.pdf (publisher's version ) (Closed access), EGF activates the ErbB1 receptor, but there appears only a limited correlation between its receptor binding affinity and mitogenic activity. This is indicated by our present observation that in cells with high ErbB1 expression, including SUM102 breast tumor cells, low affinity EGF/Notch chimeras have similarly high mitogenic activity as EGF, in spite of the fact that EGF is superior in inducing receptor tyrosine phosphorylation and p42/p44 MAP-kinase activity. However, as a result of receptor-mediated internalisation high-affinity ligands such as EGF are depleted much more rapidly from the extracellular medium than low-affinity EGF/Notch chimeras. As a consequence, the mitogenic activity of EGF on ErbB1 overexpressing cells is limited by substantial degradation of internalised ligand in the period before cells enter S-phase, a phenomenon that is not observed for low affinity mutant ligands. The mitogenic activity of EGF on ErbB1 overexpressing cells does therefore not only depend on the applied concentration but also on the total amount of ligand added, and is strongly underestimated when tested in a limited assay volume. No such dependence on the incubation volume was observed for EGF activity on cells with low ErbB1 expression levels and on cells for which EGF is growth inhibitory.

Item does not contain fulltext, Wnt signaling has been implicated in regulating bone formation by controlling osteoblast proliferation and function. Although stabilization of beta-catenin by Wnt has been shown to increase alkaline phosphatase expression and osteoblast differentiation, the precise role of Wnt signaling during the process of osteoblast differentiation is largely unknown. In this study, we used microarray technology to investigate expression regulation of Wnt signaling components during in vitro osteoblast differentiation. Expression was analyzed during bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation of murine C2C12 and MC3T3 cells and data were compared with expression in BMP2-treated NIH3T3 fibroblasts. During osteoblast differentiation, particularly strong expression regulation of the Wnt antagonists Sfrp2 (secreted frizzled related protein 2) and Wif1 (Wnt inhibitory factor 1) was observed in the late phase of differentiation. In situ expression analysis in murine tail vertebrae supported Wif1 expression during late phase bone cell differentiation, since Wif1 was found to be expressed in vivo in trabecular, but not in cortical bone. We further analyzed the effects of continuous activation of Wnt signaling by lithium chloride and observed that osteoblast differentiation was reduced, as measured by expression of osteoblast marker genes encoding alkaline phosphatase, osteocalcin, and osterix, as well as by the amount of calcium release. Taken together, our data indicate that endogenous expression of Wnt antagonists by osteoblasts provides a negative Wnt feedback loop which is essential in controlling osteoblast maturation.

Item does not contain fulltext, We have previously shown that polymorphisms in the promoter of the human platelet-derived growth factor alpha-receptor (PDGFRA) gene can be grouped into five distinct haplotypes, designated H1, H 2 alpha, H 2 beta, H 2 gamma and H 2 delta, and that specific combinations of these promoter haplotypes predispose to neural tube defects (NTDs). These promoter haplotypes differ strongly in their ability to drive reporter gene expression in various human cell lines, with highest activity for H 2 alpha and H 2 beta. Here, we show that the haplotype-linked PDGFRA promoter region extends to 3.6 kb upstream from the transcription start site, and contains a total of ten polymorphic sites. For two of these polymorphic sites, i.e. -909 C/A and +68 GAins/del, we observed differential binding of nuclear proteins from human osteosarcoma (HOS) cells. The protein complex binding specifically to -909 C, which is present in all haplotypes except the low activity haplotype H 2 gamma, contained members of the upstream stimulatory factor (USF) family of transcription factors. Furthermore, we identified a protein complex of 125 kDa which bound specifically to the low activity haplotype H1 at position +68 GAdel and may represent an H1-specific PDGFRA transcriptional repressor. The current identification of cis-acting elements in the PDGFRA promoter and the transcription factors that bind them, provides a new strategy for the identification of genes that are potentially involved in neural tube defects.

Item does not contain fulltext, We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor-beta (TGF-beta). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around -70 to -20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca2+ levels and thereby activates Ca2+-dependent Cl- channels. This compound was identified as prostaglandin F2alpha (PGF2alpha) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 +/- 2.5 nM (n = 6), compared with 1.5 +/- 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF2alpha was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF2alpha) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (approximately 25%) suppressed by AL-8810. Our results demonstrate that PGF2alpha acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general.

Item does not contain fulltext, Many mouse models exist for neural tube defects (NTDs), but only few of them are relevant for human patients that are born alive with spina bifida aperta. NTDs in humans show a complex inheritance, which most likely result from the involvement of a variety of predisposing genetic and environmental factors. Hints toward the identity of predisposing genetic factors for human NTDs could come from mouse studies on the development of the neural tube and spinal cord, as well as from studies on associated features of this type of diseases. Among such features is the observation that pregnancies affected by a neural tube defect frequently show changes in thymus morphology, and in both neonatal and maternal T-cell repertoire. The genes for E2a and Pax1 have both been implicated in not only paraxial mesodermal development, but also in that of the immune system. Moreover, Pax1 mutant mice have been shown to display NTDs in digenic mouse models. In the present study we have investigated the phenotype of E2a null mutant mice that are also heterozygous for the so-called undulated mutation in Pax1. Here we report that such double-mutant mice develop a non-lethal NTD that strongly resembles the classic human NTD: spina bifida aperta, associated with defects of the axial skeleton, immune system and urinary tract.

Item does not contain fulltext, Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation. INTRODUCTION: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation. MATERIALS AND METHODS: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation. RESULTS AND CONCLUSIONS: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represente

Item does not contain fulltext, Clubbed digits resemble the human embryonic fingers and toes, which took like the digits of a claw. Clubbed digits, thus, may represent the return of the embryonic claw and may even represent the claws man has lost during evolution, if ontogenesis realty recapitulates phylogenesis. We put forward the hypothesis that secondary clubbing, Like gynecomastia, is caused by a pathologic condition, which alters hormone levels in the blood, leading to the activation of 'dormant' genes, resulting in the development of an organ. However, the nature of the diseases that cause clubbing suggests that these hormones may actually be cytokines, acting as hormones. The nature of these cytokines is not known. They may be identified by comparing their blood levels or the combination of their blood levels to the presence or absence of clubbing, but also to the degree of clubbing and its disappearance after treatment of the primary disease. (C) 2003 Elsevier Ltd. All rights reserved.

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Item does not contain fulltext, The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type and cell differentiation status. In C2C12 mesenchymal cells, BMP-induced osteoblast differentiation can be blocked by TGF-beta. In a search for key regulators of osteoblast differentiation we have used microarray analysis to identify genes which are differentially regulated by BMP2 and TGF-beta. Within the first 24 h following the onset of differentiation, 61 BMP2-regulated genes were identified of which the BMP2 effect was counteracted by TGF-beta. The majority of these differentially expressed transcripts are related to signal transduction. Notably, our data show that three Notch signal transduction pathway genes, Lfng, Hey1, and Hes1, are differentially regulated by BMP2 and TGF-beta. This suggests that these genes might function as the focal point for interaction of Smad and Notch signaling during osteoblast differentiation.

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De aanpak van en ervaringen met kwaliteitszorg, zowel van product als proces, bij de Stichting Bodemsanering NS. In de inventarisatiefase (historisch en oriënterend onderzoek van spoorwegemplacementen) ontstond een kwaliteitszorgsysteem op maat met diverse uitvoeringsrichtlijnen en werkafspraken voor bodemonderzoeken en analyses. In de uitvoeringsfase (van bodemsaneringsprojecten) is de organisatie sterk veranderd, en is er een meer gestandaardiseerde vorm van kwaliteitszorg ontstaan met een specifieke projectorganisatie, managementsystemen, beoordelingsrichtlijnen, en certificering. Een en ander heeft geleid tot een transparante en controleerbare bedrijfsvoering

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RIVM rapport:In 1988 the National Poisons Control Centre (NVIC) of the National Institute of Public Health and the Environment (RIVM) analysed reported expositions to xenobiotics to establish the frequency of accidental intoxication in adolescents, adults and older persons. Adults were involved in 72% of the approximately 20,000 information requests on exposition to xenobiotics. In most (63%) of the intoxication cases, medicines were ingested. Alcohol, drugs and tobacco, and household and industrial products were involved in 21% of the cases. Exposure to pesticides, plants, mushrooms and cosmetics was limited. Some new medicines were highly frequent in intoxication cases after their introduction. Look-alike packages of some "non-food" products, of which the appearance resembles a "food product", increasingly lead to erroneous use and unintended exposure. New illicit drugs can suddenly become available and the revival of old products, like gammahydroxybutyrate, used in a different manner caused severe intoxications. The poisons control centre has an important role in the signalling of unexpected possible dangerous circumstances or exposure to xenobiotics, especially after the introduction of new consumer products and medicines. Informing health authorities enables measures to be found and initiated to diminish the number of specific exposures., Het Nationaal Vergiftigingen Informatie Centrum (NVIC) van het RIVM heeft de in 1998 geregistreerde vergiftigingsgevallen geanalyseeerd om een gedetailleerd beeld te krijgen van de vergiftigingen bij de leeftijdsgroepen "pubers, volwassenen en ouderen". Bij de circa 20.000 onderzochte (vermoede)geintoxiceerden behoort 72% van de personen tot de leeftijdscategorie volwassenen, het aandeel van pubers en bejaarden was relatief klein. Geneesmiddelen werden het meest (63%) ingenomen. In mindere mate (21%) ging het om expositie aan producten uit de categorieen "alcoholica, drugs en tabak", "huishoudmiddelen en doe het zelf producten" en "industrieproducten". Blootstelling aan "bestrijdingsmiddelen", "planten, paddestoelen en dieren" en "cosmetica" was beperkt. Een aantal nieuwe geneesmiddelen blijkt al kort na introductie veelvuldig betrokken te zijn bij bij overdoseringen. Verpakkingen van sommige "non-food" producten, waarvan het uiterlijk lijkt op een "food" product, kunnen aanleiding geven tot verwisseling, en dientengevolge tot een ongewenste blootstelling. Nieuwe drugs kunnen plotseling populair worden. Oude geneesmiddelen die herontdekt worden voor ander gebruik, zoals het gammahydroxyboterzuur (GHB), kunnen bij misbruik ernstige intoxicaties veroorzaken. Het NVIC vervult een belangrijke functie bij het onderkennen van onvoorziene gezondheidsrisico's na de introductie van nieuwe consumenten producten en geneesmiddelen. Door informering van de overheid en beroepsgroepen kan de aanzet gegeven worden tot onderbouwing van specifieke maatregelen ter reductie van blootstelling aan xenobiotica.

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RIVM rapport:Research of the Consumer and Safety Foundation (SCV) has revealed that approximately 1850 children, aged 0 to 5 years needed to be hospitalised each year following accidental intoxication. The National Poisons Control Centre (NVIC) of the National Institute of Public Health and the Environment (RIVM) receives approximately 10,000 information requests per year concerning children aged 0 to 6 years who have been exposed to xenobiotics. In most of these intoxications medicines, household products or cosmetics were involved. Despite the introduction of guidelines on childproof packaging of medicines and household chemicals, intoxications with childproof products frequently occur. The SCV and NVIC (initiated by the Ministry of Health, Welfare and Sport) therefore started a study to establish how the frequency of accidental intoxications in children could be diminished and what specific measures would be needed to reach this goal. The first phase of the study consisted of: a literature search on risk factors of accidental intoxications in children; interviews with parents of intoxicated children to acquire information on the circumstances of exposure, and an analysis of the NVIC database on poisonings to discover which products are most frequently involved in causing exposure in children. On the basis of the data obtained, an estimate was made of the compounds and product groups considered the most hazardous and therefore requiring further analysis into the circumstances of exposure. In the second phase of the study, a large number of telephone inquiries will be held on selected medicines, household products, cosmetics and pesticides., Uit een onderzoek van de Stichting Consument en Veiligheid (SCV) blijkt dat jaarlijks circa 1.850 kinderen van 0 tot en met 4 jaar worden opgenomen in het ziekenhuis na een accidentele vergiftiging. Jaarlijks wordt het Nationaal Vergiftigingen Informatie Centrum (NVIC) circa 10.000 maal gevraagd om informatie te verstrekken over de blootstelling van kinderen van 0 tot en met 5 jaar aan lichaamsvreemde stoffen. Voor een belangrijk deel blijkt het te gaan om geneesmiddelen, huishoudmiddelen en cosmetica. Ondanks besluiten over kinderveilige verpakkingen van huishoud- chemicalikn geneesmiddelen blijken regelmatig intoxicaties op te treden met producten met een kinderveilige verpakking. Het SCV en NVIC zijn daarom, op initiatief van het ministerie van Volksgezondheid Welzijn en Sport, een onderzoek gestart met het doel na te gaan hoe de frequentie van accidentele vergiftigingen bij kinderen verminderd kan worden en welke specifieke maatregelen aan preventie kunnen bijdragen. In deze tussenrapportage wordt de eerste fase van het onderzoek beschreven. Hierin is een literatuurstudie verricht naar risicofactoren voor accidentele vergiftigingen bij kinderen, zijn een beperkt aantal interviews gehouden met ouders/verzorgers van geintoxiceerde kinderen en is een analyse uitgevoerd op het vergiftigingen-informatiebestand van het NVIC. Op grond van de verkregen gegevens is een inschatting gemaakt welke verbindingen en/of productgroepen aanleiding geven tot medisch gezien (potentieel) bedreigende blootstellingen, alsmede voor welke producten een nadere analyse naar de omstandigheden van blootstelling gewenst is. In de tweede fase van dit onderzoek worden een groot aantal telefonische enquetes onder ouders van aan specifieke producten blootgestelde kinderen gehouden. Het onderzoek richt zich met name op een aantal specifieke geneesmiddelen, huishoudmiddelen, cosmetica en bestrijdingsmiddelen.

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Heeft u er ooit bij stilgestaan dat er bij veel soorten dieren en planten evenveel mannetjes als vrouwtjes voorkomen? U gaat er waarschijnlijk van uit dat zoiets normaal is. De verhouding van het aantal mannen tot het aantal vrouwen, de zogenoemde sex-ratio, is bij de mens ongeveer 1:1. Er worden ongeveer evenveel jongens als meisjes geboren. Niemand zal daar wakker van liggen. Het past in ons denken dat een 'samenlevingsverband', een gezin, gemiddeld bestaat uit een man en een vrouw, en hun kinderen. We staan er dus ook niet bij stil dat er bij veel andere dieren die niet in gezinsverband leven ook een sex-ratio van ongeveer 1:1 voorkomt. Planten hebben al helemaal niets dat op een gezinsverband lijkt.

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