University General - Research Publications
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ItemNo Preview AvailableMof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture(AMER SOC MICROBIOLOGY, 2008-08)Acetylation of histone tails is a hallmark of transcriptionally active chromatin. Mof (males absent on the first; also called MYST1 or KAT8) is a member of the MYST family of histone acetyltransferases and was originally discovered as an essential component of the X chromosome dosage compensation system in Drosophila. In order to examine the role of Mof in mammals in vivo, we generated mice carrying a null mutation of the Mof gene. All Mof-deficient embryos fail to develop beyond the expanded blastocyst stage and die at implantation in vivo. Mof-deficient cell lines cannot be derived from Mof(-/-) embryos in vitro. Mof(-/-) embryos fail to acetylate histone 4 lysine 16 (H4K16) but have normal acetylation of other N-terminal histone lysine residues. Mof(-/-) cell nuclei exhibit abnormal chromatin aggregation preceding activation of caspase 3 and DNA fragmentation. We conclude that Mof is functionally nonredundant with the closely related MYST histone acetyltransferase Tip60. Our results show that Mof performs a different role in mammals from that in flies at the organism level, although the molecular function is conserved. We demonstrate that Mof is required specifically for the maintenance of H4K16 acetylation and normal chromatin architecture of all cells of early male and female embryos.
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ItemOsteoclast Inhibitory Lectin, an Immune Cell Product That Is Required for Normal Bone Physiology in Vivo(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2008-11-07)Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil(-/-) mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil(-/-) mice compared with wild type mice. Furthermore, ocil(-/-) mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil(-/-) mice. Examination of bone resorption markers in the long bones of ocil(-/-) mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil(-/-) mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil(-/-) mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.
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ItemNo Preview AvailableFaces of integration.(Ubiquity Press, Ltd., 2009-12-22)THEME: Two central themes permeate this paper-the interplay between structure and agency in integration processes and the extent to which this is mediated through sensemaking by individual actors. CASE STUDY: The empirical base for the paper is provided by case study research from Wales which draws on examples of different types of integration in health and social care. The individual case studies highlight different interpretations of integration set against a background of the resources involved, processes employed and outcomes achieved. DISCUSSION: A wide ranging discussion exposes the complex interplay and dynamics between structural factors and the manner in which they enable or constrain integration, and individual actors realising their potential agency through leadership, professionalism and boundary spanning to influence outcomes. The importance of structure and agency complementing each other to determine effective integration is emphasised, together with the scope that is available for interpretation and meaning by individual actors within the contested discourse of integration.
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ItemNo Preview AvailableMembrane-bound Fas ligand only is essential for Fas-induced apoptosis(NATURE PUBLISHING GROUP, 2009-10-01)Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.
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ItemIn vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs(NATURE PUBLISHING GROUP, 2008-07-15)TRAIL/Apo-2L has shown promise as an anti-glioma drug, based on investigations of TRAIL sensitivity in established glioma cell lines, but it is not known how accurately TRAIL signalling pathways of glioma cells in vivo are reproduced in these cell lines in vitro. To replicate as closely as possible the in vivo behaviour of malignant glioma cells, 17 early passage glioma cell lines and 5 freshly resected gliomas were exposed to TRAIL-based agents and/or chemotherapeutic drugs. Normal human hepatocytes and astrocytes and established glioma cell lines were also tested. Cross-linked TRAIL, but not soluble TRAIL, killed both normal cell types and cells from three tumours. Cells from only one glioma were killed by soluble TRAIL, although only inefficiently. High concentrations of cisplatin were lethal to glioma cells, hepatocytes and astrocytes. Isolated combinations of TRAIL and chemotherapy drugs were more toxic to particular gliomas than normal cells, but no combination was generally selective for glioma cells. This study highlights the widespread resistance of glioma cells to TRAIL-based agents, but suggests that a minority of high-grade glioma patients may benefit from particular combinations of TRAIL and chemotherapy drugs. In vitro sensitivity assays may help identify effective drug combinations for individual glioma patients.
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ItemUnderstanding the Relationship between Activity and Neighbourhoods (URBAN) Study: research design and methodology(BMC, 2009-07-10)BACKGROUND: Built environment attributes are recognized as being important contributors to physical activity (PA) engagement and body size in adults and children. However, much of the existing research in this emergent public health field is hindered by methodological limitations, including: population and site homogeneity, reliance on self-report measures, aggregated measures of PA, and inadequate statistical modeling. As an integral component of multi-country collaborative research, the Understanding the Relationship between Activity and Neighbourhoods (URBAN) Study seeks to overcome these limitations by determining the strengths of association between detailed measures of the neighborhood built environment with PA levels across multiple domains and body size measures in adults and children. This article outlines the research protocol developed for the URBAN Study. METHODS AND DESIGN: The URBAN Study is a multi-centered, stratified, cross-sectional research design, collecting data across four New Zealand cities. Within each city, 12 neighborhoods were identified and selected for investigation based on higher or lower walkability and Māori demographic attributes. Neighborhoods were selected to ensure equal representation of these characteristics. Within each selected neighborhood, 42 households are being randomly selected and an adult and child (where possible) recruited into the study. Data collection includes: objective and self-reported PA engagement, neighborhood perceptions, demographics, and body size measures. The study was designed to recruit approximately 2,000 adults and 250 children into the project. Other aspects of the study include photovoice, which is a qualitative assessment of built environment features associated with PA engagement, an audit of the neighborhood streetscape environment, and an individualized neighborhood walkability profile centered on each participant's residential address. Multilevel modeling will be used to examine the individual-level and neighborhood-level relationships with PA engagement and body size. DISCUSSION: The URBAN Study is applying a novel scientifically robust research design to provide urgently needed epidemiological information regarding the associations between the built environment and health outcomes. The findings will contribute to a larger, international initiative in which similar neighborhood selection and PA measurement procedures are utilized across eight countries. Accordingly, this study directly addresses the international priority issues of increasing PA engagement and decreasing obesity levels.
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ItemA novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation(ROCKEFELLER UNIV PRESS, 2008-01-28)Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bim(S)2A, which is highly selective for Mcl-1. Unlike Noxa, Bim(S)2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bim(S)2A promotes cell killing only when Bcl-x(L) is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.
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ItemTWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFα(ROCKEFELLER UNIV PRESS, 2008-07-14)Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.
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ItemTake the sex out of STI screening! Views of young women on implementing chlamydia screening in General Practice(BMC, 2008-05-09)BACKGROUND: Australia is developing a chlamydia screening program. This study aimed to determine the attitudes of young women to the introduction of chlamydia screening in Australian General Practice. METHODS: In-depth face-to-face interviews with 24 young women from across Victoria, Australia, attending a randomly selected sample of general practices. RESULTS: Young women reported that they would accept age-based screening for chlamydia in general practice, during both sexual-health and non-sexual-health related consultations. Trust in their general practitioner (GP) was reported to be a major factor in the acceptability of chlamydia screening. The women felt chlamydia screening should be offered to all young women rather than targeted at "high risk" women based on sexual history and they particularly emphasised the importance of normalising chlamydia screening. The women reported that they did not want to be asked to provide a sexual history as part of being asked to have a chlamydia test. Some reported that they would lie if asked how many partners they had had CONCLUSION: Women do not want a sexual history taken when being asked to have a chlamydia test while attending a general practitioner. They prefer the offer of chlamydia screening to be based on age rather than assessment of sexual risk. Chlamydia screening needs to be normalised and destigmatised.
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ItemA Continuum of Cell States Spans Pluripotency and Lineage Commitment in Human Embryonic Stem Cells(PUBLIC LIBRARY SCIENCE, 2009-11-05)BACKGROUND: Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment. METHODOLOGY/PRINCIPAL FINDINGS: We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3. SIGNIFICANCE: These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes) characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency.