A5032896683- Cell death mechanisms and regulation
- Mitochondrial Function and Pathology
- Immune Cell Function and Interaction
- Autophagy in Disease and Therapy
- Immunotherapy and Immune Responses
- RNA Interference and Gene Delivery
- ATP Synthase and ATPases Research
- Phagocytosis and Immune Regulation
- T-cell and B-cell Immunology
- Cancer Immunotherapy and Biomarkers
- interferon and immune responses
- Cancer-related Molecular Pathways
- Microtubule and mitosis dynamics
- Autoimmune and Inflammatory Disorders Research
- DNA Repair Mechanisms
- Immune Response and Inflammation
- Endoplasmic Reticulum Stress and Disease
- NF-κB Signaling Pathways
- Cell Adhesion Molecules Research
- Ubiquitin and proteasome pathways
- Cancer Cells and Metastasis
- Molecular Biology Techniques and Applications
- Cell Image Analysis Techniques
- Anesthesia and Neurotoxicity Research
- Protein Degradation and Inhibitors
QIMR Berghofer Medical Research Institute
1996-2023
Mater Research
2011-2016
Translational Research Institute
2013-2016
The University of Queensland
1996-2016
Medical Research Institute
2015
Fluorescence Innovations (United States)
2013
Mater Health Services
2011
Peter MacCallum Cancer Centre
2002-2010
The University of Melbourne
2006-2010
Cancer Genetics (United States)
2010
A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochromec, key component electron transport chain, that been reported to translocate from cytosol cells undergoing apoptosis. The mechanism this release is, as yet, unknown. Here we report ectopic expression Bax induces apoptosis with an early cytochrome c preceding many apoptosis-associated morphological alterations well...
The surface of healthy cells is composed lipids that are asymmetrically distributed on the inner and outer leaflet plasma membrane. One these lipids, phosphatidylserine (PS), normally restricted to membrane is, therefore, only exposed cell cytoplasm. However, during apoptosis lipid asymmetry lost PS becomes Annexin V, a 36-kDa calcium-binding protein, binds PS; fluorescently labeled V can be used detect outside apoptotic cells. also stain necrotic because have ruptured membranes permit...
During apoptosis, cytochrome c is released into the cytosol as outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when activity inhibited, permeabilization causes a rapid depolarization transmembrane potential, which recovers original levels over next 30–60 min then maintained. After permeabilization, can use cytoplasmic maintain potential...
Abstract Interferon gamma (IFN γ ) is a key moderator of cell-mediated immunity with diverse, mainly pro-inflammatory actions on immunocytes and target tissue. Recent studies have shown it may enhance anti-tumor antiviral effects CD8 T cells. Here we investigate the mechanisms by which IFN mediates T-cell cytotoxic function. We show that in vivo , antigen-specific cells produce INF are necessary to effect rejection skin grafts expressing OVA as transgene keratinocytes. The ability enhanced...
Propidium iodide (PI) is a small fluorescent molecule that binds to DNA but cannot passively traverse into cells possess an intact plasma membrane. PI uptake versus exclusion can be used discriminate dead cells, in which membranes become permeable regardless of the mechanism death, from live with membranes. excited by wavelengths between 400 and 600 nm emits light 700 nm, therefore compatible lasers photodetectors commonly available flow cytometers. This protocol for staining quantitate cell...
The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic from or necrotic cells. Dyes that bind DNA, such as Hoechst 33342 4′,6-diamidino-2-phenylindole (DAPI), observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light visualized using conventional fluorescent microscopes...
Apoptosis is orchestrated by caspases, a family of cysteine proteases that cleave their substrates on the carboxy-terminal side specific aspartic acid residues. These are generally present in healthy cells as inactive zymogens, but when stimulated they undergo autolytic cleavage to become fully active. They subsequently at one or two sites, which can result activation, inactivation, relocalization, remodeling substrate. Consequently, many cleaved fragments remain intact during apoptosis and...
Adenosine triphosphate (ATP) is the main source of energy for metabolism. Mitochondria provide majority this ATP by a process known as oxidative phosphorylation. This involves active transfer positively charged protons across mitochondrial inner membrane resulting in net internal negative charge, transmembrane potential (ΔΨm). The proton gradient then used synthase to produce fusing adenosine diphosphate and free phosphate. charge healthy mitochondrion maintained at approximately −180 mV,...
The MUC13 transmembrane mucin is highly and constitutively expressed in the small large intestine. Although polymorphisms have been associated with human inflammatory bowel diseases susceptibility to Escherichia coli infection pigs, biological functions of are unknown. This study aimed explore whether modulates intestinal inflammation.Muc13(-/-) mice were generated, phenotyped challenged colitis-inducing agent, dextran sodium sulphate (DSS). Colitis was assessed by clinical symptoms...
Trypan blue is a colorimetric dye that stains dead cells with color easily observed using light microscopy at low resolution. The staining procedure rapid and can be analyzed within minutes. number of live (unstained) (blue) counted hemocytometer on basic upright microscope. therefore convenient assay for rapidly determining the overall viability in culture before commencing scientific experimentation, or quantitating cell death following treatment any cytotoxic stimuli.
Measuring cell death with colorimetric or fluorimetric dyes such as trypan blue and propidium iodide (PI) can provide an accurate measure of the number dead cells in a population at specific time; however, these assays cannot be used to distinguish that are dying marked for future death. In many cases it is essential proliferative capacity treated indirect measurement This achieved using colony-forming assay described here. protocol specifically applies HeLa but most adherent lines limited motility.
Preclinical studies have suggested that purified populations of CD1c (BDCA-1+) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use CD1c+ BDC in patients advanced metastatic hormone refractory cancer. HLA-A*0201-positive were recruited and consented. The vaccine was manufactured by pulsing autologous BDC, prepared magnetic bead...
Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B perforin. Granzyme B, a serine proteinase with substrate specificity similar to caspase family apoptotic cysteine proteinases, is capable cleaving activating number death target cells. Despite ability engage pathway at multiple entry points, preferred mechanism for rapid induction apoptosis by has yet be clearly established. Here we...
Cell cycle checkpoints act to protect cells from external stresses and internal errors that would compromise the integrity of cell. Checkpoints are often defective in cancer cells. Drugs target checkpoint mechanisms should therefore be selective for tumor drug-sensitive checkpoint. Histone deacetylase inhibitors typify this class agents. They trigger a G2-phase response normal but cytotoxic which is defective. In study, we investigated molecular basis tumor-selective cytotoxicity these drugs...
Cell death is mediated by cytotoxic lymphocytes through various granule serine proteases released with perforin. The unique protease activity, restricted expression, and distinct gene locus of granzyme M suggested this enzyme might have a novel biological function or trigger form cell death. Herein, we demonstrate that in the presence perforin, activity rapidly effectively induces target In contrast to B, induced does not feature obvious DNA fragmentation, occurs independently caspases,...
Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It also that granzyme B can caspases directly, caspase substrates, and/or several non-caspase substrates. The relative importance of Bid in B-induced therefore remained unclear. Here we report cells isolated various tissues Bid-deficient mice were resistant death. Consistent with role regulating mitochondrial outer...
Asymmetric cell division is a potential means by which fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric of T cells paramount for determining role this process in generation effector and memory subsets. In other types, regulated conserved polarity protein complexes control localization determinants spindle orientation division. We have developed tractable, vitro model naive CD8(+) undergoing initial while attached to dendritic...