ORCID Profile
0000-0002-4504-6534
Current Organisation
University of Adelaide
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Publisher: Wiley
Date: 02-1999
DOI: 10.1002/(SICI)1098-2795(199902)52:2<174::AID-MRD8>3.0.CO;2-7
Publisher: Springer Science and Business Media LLC
Date: 02-2000
Abstract: RAIDD, a caspase recruitment domain (CARD) containing molecule, interacts with procaspase-2 in a CARD-dependent manner. This interaction has been suggested to mediate the recruitment of caspase-2 to the tumour necrosis factor receptor 1 (TNFR1). In this paper we have studied the subcellular localization of RAIDD and its interaction with caspase-2. We demonstrate that endogenous RAIDD is mostly localized in the cytoplasm and to some extent in the nucleus. RAIDD localization is not affected by TNF-treatment of HeLa cells, but in cells ectopically expressing caspase-2, a fraction of RAIDD is recruited to the nucleus. In transfected cells, coexpression of RAIDD and caspase-2 leads to CARD-dependent colocalization of the two proteins to discrete subcellular structures. We further show that overexpression of the RAIDD-CARD results in the formation of filamentous structures due to CARD-mediated oligomerization. These structures were similar to death effector filaments (DEFs) formed by FADD and FLICE death effector domains (DEDs), and partially colocalized with DEFs. Our results suggest that similar to the DED, the RAIDD-CARD has the ability to form higher order complexes, believed to be important in apoptotic execution. We also present evidence that RAIDD-CARD oligomerization may be regulated by intramolecular folding of the RAIDD molecule.
Publisher: Oxford University Press (OUP)
Date: 06-1997
Abstract: The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.
Publisher: International Union of Crystallography (IUCr)
Date: 07-2019
DOI: 10.1107/S2059798319009008
Abstract: Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.
Publisher: Springer Science and Business Media LLC
Date: 06-01-2021
DOI: 10.1038/S41586-020-03039-0
Abstract: Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution 1,2 . Here we generate and analyse reference genomes of the platypus ( Ornithorhynchus anatinus ) and echidna ( Tachyglossus aculeatus ), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.
Publisher: Wiley
Date: 2006
DOI: 10.1002/BIES.20422
Abstract: Ubiquitination is essential in mediating erse cellular functions including protein degradation and trafficking. Ubiquitin-protein (E3) ligases determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3 ligases is an evolutionarily conserved family of proteins required for the ubiquitination of a large number of cellular targets. As a result, this family regulates a wide variety of cellular processes including transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. The modular architecture of the proteins, comprising a C2 domain, multiple WW domains and a catalytic domain, enables erse intermolecular interactions and recruitment to various subcellular locations. The WW domains commonly mediate interaction with substrate proteins however, an increasing number of Nedd4 targets do not contain obvious WW domain-interaction motifs suggesting the involvement of accessory proteins. This review discusses recent insights into how accessory and adaptor proteins modulate the activities of Nedd4 family members, including recruitment of novel substrates, alteration of subcellular localisation and effects on ubiquitination.
Publisher: Elsevier BV
Date: 04-2001
Publisher: Informa UK Limited
Date: 12-1993
Abstract: Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.
Publisher: Elsevier BV
Date: 10-1998
Publisher: MDPI AG
Date: 26-08-2021
Abstract: Chromosome segregation at mitosis and meiosis is a highly dynamic and tightly regulated process that involves a large number of components. Due to the fundamental nature of chromosome segregation, many genes involved in this process are evolutionarily highly conserved, but duplications and functional ersification has occurred in various lineages. In order to better understand the evolution of genes involved in chromosome segregation in mammals, we analyzed some of the key components in the basal mammalian lineage of egg-laying mammals. The chromosome passenger complex is a multiprotein complex central to chromosome segregation during both mitosis and meiosis. It consists of survivin, borealin, inner centromere protein, and Aurora kinase B or C. We confirm the absence of Aurora kinase C in marsupials and show its absence in both platypus and echidna, which supports the current model of the evolution of Aurora kinases. High expression of AURKBC, an ancestor of AURKB and AURKC present in monotremes, suggests that this gene is performing all necessary meiotic functions in monotremes. Other genes of the chromosome passenger complex complex are present and conserved in monotremes, suggesting that their function has been preserved in mammals. Cohesins are another family of genes that are of vital importance for chromosome cohesion and segregation at mitosis and meiosis. Previous work has demonstrated an accumulation and differential loading of structural maintenance of chromosomes 3 (SMC3) on the platypus sex chromosome complex at meiotic prophase I. We investigated if a similar accumulation occurs in the echidna during meiosis I. In contrast to platypus, SMC3 was only found on the synaptonemal complex in echidna. This indicates that the specific distribution of SMC3 on the sex chromosome complex may have evolved specifically in platypus.
Publisher: Wiley
Date: 16-05-2021
DOI: 10.1111/ALL.14883
Abstract: Staphylococcus aureus is a pathogen of major concern in both acute infections and chronic conditions such as chronic rhinosinusitis (CRS). Bacteriophage (phage) therapy has recently regained interest for its potential to treat infections caused by antibiotic resistant strains including Methicillin Resistant Staphylococcus aureus (MRSA). However, bacteria can adapt and become resistant to phages. The aim of this study is to determine the potential for antibiotics to overcome phage resistance. The susceptibility of S. aureus clinical isolates (CIs) to phages J‐Sa36, Sa83 and Sa87 alone or in combination with protein synthesis inhibitor (PSI) antibiotics clindamycin, azithromycin and erythromycin was assessed using plaque spot assays, minimum inhibitory concentration (MIC) assays, double layer spot assays and resazurin assays. The safety and efficacy of subinhibitory PSI antibiotics in combination with phage was tested in a Sprague Dawley rat model of sinusitis infected with a phage resistant S. aureus CI. All three antibiotics at subinhibitory concentrations showed synergy when combined with all 3 phages against S. aureus CIs in planktonic and biofilm form and could sensitize phage‐resistant S. aureus to promote phage infection. The combination of topical subinhibitory clindamycin or azithromycin and phage was safe and could eradicate S . aureus sinonasal biofilms in vivo . Subinhibitory concentrations of PSI antibiotics could sensitize phage‐resistant S . aureus and MRSA strains to phages in vitro and in vivo . This data supports the potential use of phage‐PSI antibiotic combination therapies, in particular for difficult‐to‐treat infections with phage‐resistant S . aureus and MRSA strains.
Publisher: American Society of Hematology
Date: 15-11-2008
DOI: 10.1182/BLOOD-2008-04-150953
Abstract: Many ion channels and transporters are regulated by ubiquitination mediated by the Nedd4 family of HECT-type ubiquitin ligases (E3s). These E3s commonly interact with substrates via their WW domains that bind to specific motifs in target proteins. However, not all potential targets of these E3s contain WW-binding motifs. Therefore, accessory proteins may mediate the interaction between Nedd4 family members and their targets. Here we report that the alent metal ion transporter DMT1, the primary nonheme iron transporter in mammals, is regulated by ubiquitination mediated by the Nedd4 family member WWP2. DMT1 interacts with 2 WW domain-interacting proteins, Ndfip1 and Ndfip2, previously proposed to have roles in protein trafficking. This promotes DMT1 ubiquitination and degradation by WWP2. Consistent with these observations, Ndfip1−/− mice show increased DMT1 activity and a concomitant increase in hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. This novel mechanism of regulating iron homeostasis suggests that Ndfips and WWP2 may contribute to diseases involving aberrant iron transport.
Publisher: Elsevier BV
Date: 03-2002
Publisher: Informa UK Limited
Date: 08-1999
Publisher: MDPI AG
Date: 28-08-2021
Abstract: Segregation of chromosomes is a multistep process occurring both at mitosis and meiosis to ensure that daughter cells receive a complete set of genetic information. Critical components in the chromosome segregation include centromeres, kinetochores, components of sister chromatid and homologous chromosomes cohesion, microtubule organizing centres, and spindles. Based on the cytological work in the grasshopper Brachystola, it has been accepted for decades that segregation of homologs at meiosis is fundamentally random. This ensures that alleles on chromosomes have equal chance to be transmitted to progeny. At the same time mechanisms of meiotic drive and an increasing number of other ex les of non-random segregation of autosomes and sex chromosomes provide insights into the underlying mechanisms of chromosome segregation but also question the textbook dogma of random chromosome segregation. Recent advances provide a better understanding of meiotic drive as a prominent force where cellular and chromosomal changes allow autosomes to bias their segregation. Less understood are mechanisms explaining observations that autosomal heteromorphism may cause biased segregation and regulate alternating segregation of multiple sex chromosome systems or translocation heterozygotes as an extreme case of non-random segregation. We speculate that molecular and cytological mechanisms of non-random segregation might be common in these cases and that there might be a continuous transition between random and non-random segregation which may play a role in the evolution of sexually antagonistic genes and sex chromosome evolution.
Publisher: CSIRO Publishing
Date: 1998
DOI: 10.1071/R98041
Abstract: Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.
Publisher: The Company of Biologists
Date: 15-07-2004
DOI: 10.1242/JCS.01212
Abstract: N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.
Publisher: Elsevier BV
Date: 10-2013
Publisher: Elsevier BV
Date: 08-2002
No related grants have been discovered for Linda Shearwin.