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John M Murray

Publications and source records attributed to John M Murray.

17 recordsLinked to original sources
PubMed2006

Cytoskeletal components of an invasion machine--the apical complex of Toxoplasma gondii.

The apical complex of Toxoplasma gondii is widely believed to serve essential functions in both invasion of its host cells (including human cells), and in replication of the parasite. The understanding of apical complex function, the basis for its novel structure, and the mechanism for its motility are greatly impeded by lack of knowledge of its molecular composition. We have partially purified the conoid/apical complex, identified approximately 200 proteins that represent 70% of its cytoskeletal protein components, characterized seven novel proteins, and determined the sequence of recruitment of five of these proteins into the cytoskeleton during cell division. Our results provide new markers for the different subcompartments within the apical complex, and revealed previously unknown cellular compartments, which facilitate our understanding of how the invasion machinery is built. Surprisingly, the extreme apical and extreme basal structures of this highly polarized cell originate in the same location and at the same time very early during parasite replication.

Animals↗
PubMed2006

The half-life of hepatitis B virions.

The virion half-life of hepatitis B virus (HBV) is currently estimated at approximately 1 day. This estimate has been obtained from drug perturbation experiments with reverse transcriptase inhibitors. However, the analyses of those experiments have not considered the export of virions produced from preformed mature DNA-containing HBV capsids in infected cells. Data from 3 acutely infected chimpanzees indicates that there is approximately 10-fold more total intracellular HBV DNA than HBV DNA in blood, and therefore the half-life of virions for chimpanzees during acute infection is 10-fold shorter at 3.8 hours than the half-life associated with export of total intracellular HBV DNA. Mathematical model simulations duplicating the viral dynamics observed in drug perturbation experiments suggest a half-life of at most 4.4 hours for HBV virions in chronically infected humans, significantly shorter than current estimates, but consistent with the half-lives of virions for hepatitis C virus and HIV. This faster turnover of HBV in blood indicates a correspondingly higher replication rate and risk of mutation against hepatitis B antiviral therapy. In conclusion, we find the half-life of HBV virions is approximately 4 hours, significantly shorter than current estimates of 1 day. This new value is consistent with virion half-life estimates for HIV and hepatitis C virus.

Animals↗
PubMed2006

Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[(125)I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [(125)I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

Amino Acid Sequence↗
PubMed2005

Dynamics of hepatitis B virus clearance in chimpanzees.

Mathematical modeling was performed to test the extent to which cytopathic and noncytopathic T cell effector functions contribute to resolution of hepatitis B virus (HBV) infection in three acutely infected chimpanzees. Simulations based exclusively on cytopathic functions show a poor fit to the data and would require the destruction and regeneration of approximately 11 livers for clearance to occur. In contrast, a simulation based on a combination of cytopathic and noncytopathic functions provided a significantly better fit to the data (P < 0.001) and required as much as 5-fold less destruction to clear the virus from the liver. The best fit simulation supports the notion that during the early phase of HBV clearance, noncytopathic T cell effector mechanisms inhibit viral replication and greatly shorten the half-life of the long lived covalently closed circular viral DNA transcriptional template, thereby limiting the extent to which cytopathic T cell effector functions and tissue destruction are required to terminate acute HBV infection.

Acute Disease↗
PubMed2005

Plastid segregation and cell division in the apicomplexan parasite Sarcocystis neurona.

Apicomplexan parasites harbor a secondary plastid that is essential to their survival. Several metabolic pathways confined to this organelle have emerged as promising parasite-specific drug targets. The maintenance of the organelle and its genome is an equally valuable target. We have studied the replication and segregation of this important organelle using the parasite Sarcocystis neurona as a cell biological model. This model system makes it possible to differentiate and dissect organellar growth, fission and segregation over time, because of the parasite's peculiar mode of cell division. S. neurona undergoes five cycles of chromosomal replication without nuclear division, thus yielding a cell with a 32N nucleus. This nucleus undergoes a sixth replication cycle concurrent with nuclear division and cell budding to give rise to 64 haploid daughter cells. Interestingly, intranuclear spindles persist throughout the cell cycle, thereby providing a potential mechanism to organize chromosomes and organelles in an organism that undergoes dramatic changes in ploidy. The development of the plastid mirrors that of the nucleus, a continuous organelle, which grows throughout the parasite's development and shows association with all centrosomes. Pharmacological ablation of the parasite's multiple spindles demonstrates their essential role in the organization and faithful segregation of the plastid. By using several molecular markers we have timed organelle fission to the last replication cycle and tied it to daughter cell budding. Finally, plastids were labeled by fluorescent protein expression using a newly developed S. neurona transfection system. With these transgenic parasites we have tested our model in living cells employing laser bleaching experiments.

Animals↗
PubMed2004

ADAR1 RNA deaminase limits short interfering RNA efficacy in mammalian cells.

Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.

Adenosine Deaminase↗
PubMed2004

Variability and heritability of cell division pathways in Toxoplasma gondii.

A histone 2b-YFP fusion protein stably expressed in Toxoplasma gondii has several advantages: it reveals previously hidden details of nuclear morphology; it makes it possible to observe cell-cycle events; it provides a basis for quantitative measurements of DNA content in living cells; and it enables sorting of live cells according to cell-cycle phase or ploidy. With this cell line it was possible to recognize and directly clone individual progeny arising from different patterns of cell division that produce two, three or four daughter cells. These experiments established that the progeny produced by all cell division pathways are viable and infective. Furthermore, the number of progeny produced by a mature parasite during cell division is not correlated with the number of its siblings. The complete repertoire of cell division pathways is therefore inherited by a single cell produced through any one of the individual paths. The results expand the range of what must be considered normal in T. gondii cell division and provide a useful tool for further study of nuclear structure and proliferation in this important human pathogen.

Animals↗
PubMed2004

Altered RNA editing of serotonin 5-HT2C receptor induced by interferon: implications for depression associated with cytokine therapy.

Members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in one type of RNA editing that converts adenosine residues to inosine. The A-to-I editing of serotonin receptor subtype 2C (5-HT(2C)R) mRNA leads to replacement of three amino acid residues located within the intracellular loop II domain, resulting in dramatic alterations in G-protein coupling functions of the receptor. It has been speculated that RNA editing may play a role in several pharmacological and behavioral processes where the serotonergic plasticity is mediated through 5-HT(2C)R. Interferon-alpha (IFN-alpha) often causes severe depression in patients treated for chronic viral hepatitis and certain malignancies. In this study, we examined the effects of IFN-alpha on RNA editing in human glioblastoma cell lines, which express 5-HT(2C)R mRNAs. ADAR1 expression and the pattern of the 5-HT(2C)R mRNA editing rapidly changed in response to IFN-alpha, leading to the dominant expression of the 5-HT(2C)R-VSI isoform predicted to have reduced G-protein coupling functions. Our results support the hypothesis that 5-HT(2C)R mRNA editing has causative relevance in the pathophysiology of depression associated with cytokine therapy.

Adenosine Deaminase↗
PubMed2003

Requirement of dimerization for RNA editing activity of adenosine deaminases acting on RNA.

Adenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA. Three vertebrate ADAR gene family members, ADAR1, ADAR2, and ADAR3, have been identified. The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of those cytidine deaminases. In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form. No heterodimer complex formation among different ADAR gene family members was detected. Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex. Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization. Homodimer formation may be necessary for ADAR to act as active deaminases. Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing.

Adenosine Deaminase↗
PubMed2003

Naive T cells are maintained by thymic output in early ages but by proliferation without phenotypic change after age twenty.

Analysing T-cell receptor excision circle numbers in healthy individuals we find a marked change in the source of naive T cells before and after 20 years of age. The bulk of the naive T cell pool is sustained primarily from thymic output for individuals younger than 20 years of age whereas proliferation within the naive phenotype is dominant for older individuals. Over 90% of phenotypically naive T cells in middle age are not of direct thymic origin. Moreover, this change in source of naive T cells is accompanied either by an increased death rate of T cells from the thymus or reduced thymic export. Modelling of these processes shows that new naive T cells of a thymic origin have a half-life of approximately 50 days before this change occurs, and that either the life-span of recent thymic emigrants (but not necessarily of all naive cells) decreases approximately threefold in middle age, or thymic production drops by this same amount. The decay rate of T-cell receptor excision circle levels for individuals over 20 years of age is consistent with the decay rate of the productive thymus. Our modelling suggests that at age 25, thymic export is responsible for 20% of naive T-cell production and that this percentage decreases with the 15.7 year half-life of the productive thymus so that by age 55 only 5% of naive production arises from thymic export.

Adolescent↗
PubMed2003

The impact of behavioural changes on the prevalence of human immunodeficiency virus and hepatitis C among injecting drug users.

BACKGROUND: Prevalence of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in the injecting drug user (IDU) community can differ considerably. In Australia in 1997, HIV prevalence among attendees at Needle Exchange Programs was 1% while HCV prevalence was 50%. The impact that different needle-sharing behaviour and drug injecting use may have on the future levels of these viruses is uncertain. METHOD: We develop a mathematical model of the number of people who inject drugs with each of these infections to determine their changes under different scenarios. The impact of transmission probabilities and needle sharing on the incidence and prevalence of HIV and HCV infections are assessed. RESULTS: Critical levels of needle sharing, below which total infections would fall to minimal levels, were estimated to be 17 IDU partners per year for HIV compared with 3 IDU partners per year for HCV. Current average levels of needle sharing in Australia are estimated to be six IDU partners per year. CONCLUSIONS: This analysis suggests that under current drug injecting behaviour, HIV prevalence in IDU in Australia should remain below 1% but that HCV prevalence will stay elevated.

Australia↗
PubMed2002

A novel polymer of tubulin forms the conoid of Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasite, an important human pathogen, and a convenient laboratory model for many other human and veterinary pathogens in the phylum Apicomplexa, such as Plasmodium, Eimeria, and Cryptosporidia. 22 subpellicular microtubules form a scaffold that defines the cell shape of T. gondii. Its cytoskeleton also includes an intricate apical structure consisting of the conoid, two intraconoid microtubules, and two polar rings. The conoid is a 380-nm diameter motile organelle, consisting of fibers wound into a spiral like a compressed spring. FRAP analysis of transgenic T. gondii expressing YFP-alpha-tubulin reveals that the conoid fibers are assembled by rapid incorporation of tubulin subunits during early, but not late, stages of cell division. Electron microscopic analysis shows that in the mature conoid, tubulin is arranged into a novel polymer form that is quite different from typical microtubules.

Actin Cytoskeleton↗
PubMed2002

Interferon regulatory factor-3 is an in vivo target of DNA-PK.

Eukaryotic cells have evolved complex signaling networks to sense environmental stress and to repair stress-induced damage. IFN regulatory factor-3 (IRF-3) is a transcription factor that plays a central role in the host response to viral infection. Although the main activity of IRF-3 characterized to date has been its role in the induction of IFN-alpha and -beta after virus infection, recent evidence indicates additional roles for IRF-3 in the response to DNA damage and in virus-induced apoptosis. Here we identify IRF-3 as the first in vivo target for DNA-dependent protein kinase (DNA-PK). Phosphorylation of IRF-3 by DNA-PK after virus infection results in its nuclear retention and delayed proteolysis. These results expand the known roles of DNA-PK and provide a functional link between the cellular machineries that regulate the innate immune response and that sense and respond to DNA damage. As such this study contributes to a more integrated view of the cellular responses to various cellular stress signals.

3T3 Cells↗
PubMed2002

Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

Algorithms↗
PubMed2002

Daughter cell assembly in the protozoan parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii. Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.

Animals↗