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Research Interests of the SFRR(A)

Some research group highlights....

Genomic and biochemical responses of plants to environmental stress

Dr Barry Pogson (Ph. D)

School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT, Australia 0200

Functional Genomics of Environmental Stress: Stresses such as excess light and drought reduce plant yield and can be lethal. We wish to identify the mechanisms by which plants perceive and respond to environmental or abiotic stress.

The approach utilizes cutting-edge genomic technologies, such as DNA chip arrays and high throughput screening of promoter-reporter gene fusions to identify novel genes and signalling pathways in the model plant, Arabidopsis (a member of the mustard family).

Also, we have identified a class of genes in the alga, Chlamydomonas that enable it to survive and grow under extreme light. Ultimately, an understanding of these mechanisms will be incorporated into traditional plant breeding programs towards improving crop yield.

Photosynthesis and photoprotection: Antixoidants, such as carotenoids and vitamin C (ascorbate) are essential for the survival of plants. We are determining their roles as safety valves against oxidative damage induced during photosynthesis. This program is a combination of biochemical genetics and photophysics.

Chloroplast Development and Function: Our work is focused on the characterization of plant pigments, particularly carotenoids and their role in photosynthesis and chloroplast development. Carotenoids are important pigments in photosystems, many fruits, flowers, and seafood. Nutritionally they are the source of vitamin A and offer some protection against certain cancers, heart and eye diseases. Thus, understanding their biosynthesis may lead to improved nutritional value of foods.

The goals are to elucidate the mechanisms that control carotenoid accumulation and chloroplast biogenesis using biochemical genetics. Of particular interest are mutations that alter seedling and chloroplast development.

Ataxia telangiectasia

Dr. Dianne Watters.

Current address: Dept. of Surgery, University of Queensland. Royal Brisbane Hospital.

Ataxia telangiectasia (A-T) is a rare human autosomal recessive disease affecting 1 in 100,000 live births. Approximately 1 in 400 people are heterozygous for the defective gene. A-T is a multisystem disorder characterised by increased sensitivity to ionising radiation, immunodeficiency and susceptibility to cancer. The major hallmarks of the disease are ataxia (abnormal gait resulting in confinement to a wheel chair by the early teenage years) and occulocutaneous telangiectasia (spidery veins on the whites of the eyes and facial skin often seen in people after radiotherapy). Other symptoms include occulomotor disturbances, premature aging, clinical and cellular sensitivity to ionising radiation and radiomimetic drugs, chromosomal instability, and growth and developmental abnormalities. The major debilitating aspects of the disease, however, are the progressive ataxia with onset in infancy (due to degeneration of Purkinje cells in the cerebellum), and susceptibility to infection. The two major causes of death, usually in the second decade of life, are lymphomas and respiratory infections.

Heterozygotes are intermediate in radiation sensitivity between normal and A-T homozygotes, and there is increasing evidence that heterozygosity for this gene may predispose to cancer, particularly breast cancer. The gene mutated in A-T was recently cloned and shown to code for a large protein (ATM, 350 kDa) with a domain homologous to the kinase domain of phosphatidylinositol (PI) 3-kinase. The mutations in over half of A-T patients are truncating mutations rendering the protein unstable. The other mutations result in loss of kinase activity. ATM is most closely related to proteins, which are involved in maintaining the integrity of DNA and/or cell cycle control in response to DNA damage. It is a protein kinase rather than a lipid kinase and some of its known substrates are p53, and BRCA-1 (the product of the breast cancer susceptibility gene) both of which are involved in DNA damage signaling. ATM is also involved in homologous recombination and Atm knockout mice are sterile due to aberrant recombination during meiosis. We have localised the ATM protein to the nucleus, as expected for a protein with these functions, and also unexpectedly, to peroxisomes and cytoplasmic vesicles (endosomes). While a role for nuclear ATM in DNA damage signalling in proliferating cells is well defined, it does not explain the etiology of the neurodegeneration. In terminally differentiated cells such as the Purkinje cells, ATM is almost exclusively outside the nucleus.

A-T cells appear to be in a state of oxidative stress as evidenced by indirect measures, which show that these cells behave as if they had already been irradiated. In addition genes which are up-regulated after irradiation in normal cells are already up-regulated in A-T cells in the absence of irradiation. We have found decreased activity of the peroxisomal enzyme catalase in cultured A-T cells and in the cerebellum but not the liver of ATM mutant mice. Consistent with these observations, we have found increased lipid hydroperoxides in A-T cells and others have found evidence of oxidative damage in tissues from Atm knockout mice. Interestingly this damage is present in the cerebellum but not in the liver. A-T cells display defective signal transduction in response to a number of different stimuli and there are also reports of altered lipid metabolism in A-T. Our current research is aimed at understanding the role of the ATM protein in peroxisomes and endosomes, in the maintenance of redox homeostasis, and the possibility that the signal transduction defects are related to oxidative damage/alterations of membrane lipids which are crucial for efficient functioning of cell surface receptors. Defining the role of ATM outside the nucleus will help explain aspects of A-T, such as ataxia and defective cell signalling, which have been difficult to explain by aberrant cell cycle checkpoint control after ionising radiation or defective recombination events.

Cell surface respiration by mammalian cells

Mike Berridge

Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand

Mammalian cell respiration occurs primarily in mitochondria, which are thought to be the only cell organelles that consume oxygen for the purpose of ATP production. A small amount of ATP is also produced during glycolysis but this is thought to be supported anaerobically by lactate production. Using mitochondrial gene knockout (r0) cell lines, we have obtained evidence for non-mitochondrial respiration in mammalian cells in which electron transport across the plasma membrane, from cytosolic NADH to molecular oxygen supports glycolytic ATP production. In this process, oxygen is consumed at the cell surface and NADH, produced during glycolysis and during the TCA cycle, is oxidised to NAD+ to facilitate continued glycolytic energy production.

This controversial finding challenges the simplistic textbook view that mitochondria are the sole organelles in mammalian cells involved in aerobic respiration. Two unexpected findings led us to hypothesise cell surface respiration in mammalian cells. The first was the realization that a new sulfonated tetrazolium salt, WST-1, which was claimed to be reduced to its soluble formazan by a mitochondrial enzyme, was actually reduced extracellularly as demonstrated by almost complete inhibition of reduction by superoxide dismutase (SOD), a protein that cannot get into the cell. The second key observation was that mitochondrial gene knockout (ro) cells that are incapable of mitochondrial electron transport and mitochondrial oxygen consumption, not only showed enhanced reduction of the tetrazolium dye, but also grew in an oxygen-dependent manner.

The most obvious approach to addressing the question of whether ro cells "breathed" oxygen was to measure their oxygen consumption. Because no oxygen electrode was available to us in Wellington at the time, I spent a year collaborating with Mike Murphy in Dunedin whose intimate knowledge of the temperamental behaviour of small Clark-type oxygen electrodes allowed me to collect enough data to support the model: ro cells do indeed consume significant amounts of oxygen, a process that is insensitive to mitochondrial electron transport inhibitors and uncouplers of oxidative phosphorylation.

Subsequent work by An Tan, Debbie Scarlett and Patries Herst, a PhD student, has now provided substantial evidence to support the view that oxygen consumption in ro cells occurs at the cell surface. Again, an unexpected result provided key information about involvement of the plasma membrane in oxygen consumption. Thus, an experiment designed to investigate a possible role for a cell surface NADH oxidase in oxygen consumption by ro cells was predicted to show increased oxygen consumption in the presence of NADH. Instead, NADH extensively inhibited oxygen consumption at micromolar concentrations. Because extracellular NADH does not cross the plasma membrane, oxygen consumption at the cell surface is implicated.

Immunochemical Detection of Aldehyde-Modified Proteins in Hepatocytes

Dr. Phil Burcham

Molecular Toxicology Research Group, Dept. Clinical and Experimental Pharmacology, Medical School, Adelaide University, North Terrace Campus, Adelaide, 5005.

Our present interests predominately revolve around the protein-damaging properties of two short-chain 2-trans-alkenals, acrolein (2-t-propenal) and crotonaldehyde (2-t-butenal). These reactive compounds possess diverse toxicological properties and have long concerned toxicologists due to their presence as environmental contaminants in various settings. Somewhat surprisingly, comparatively little is known concerning their mechanism of toxic action at the molecular level. Our interest in these two toxic compounds is partly due to the fact that acrolein and crotonaldehyde have long been identified in peroxidised lipid mixtures, raising the possibility that they contribute to cell damage during oxidative stress. The discoveries in other labs during the mid-1990s that acrolein- and crotonaldehyde-derived DNA adducts are present in the human genome, presumably as a result of endogenous production, suggests these substances might also participate in damaging the proteome. However while the contribution of toxic aldehydes such as malondialdehyde and 4-hydroxy-2-nonenal to protein damage during oxidative stress is receiving attention in a number of laboratories, less is known concerning the significance of acrolein or crotonaldehyde production during oxidative cell injury. Using an immunohistochemical approach, Uchida and associates have established that acrolein-modified proteins are present in the affected tissues of patients with a number of degenerative conditions that are known to involve oxidative stress (eg. diabetic nephropathy, AlzheimerÍs, etc). At present, which cell proteins are involved in the formation of these adducts is not known. Thus one goal of work in our lab is to identify critical proteins that sustain damage during acrolein- and crotonaldehyde-mediated cell injury, in the expectation that such knowledge will illuminate the critical biochemical perturbations underlying the toxicity of these substances.

The main experimental approach we are using to detect damaged proteins during aldehyde-mediated toxicity involves the use of antibodies in Western blot procedures. In the past we have exploited the fact that Michael addition reactions (to form carbonyl-containing adducts) feature strongly during the reaction of 2-trans-alkenals with proteins. Thus we have used an immunochemical assay based on derivatisation of carbonylated proteins with 2,4-dinitrophenylhydrazine followed by immunodetection with anti-dinitrophenyl sera to detect modification of a wide range of proteins in the early stages of aldehyde-mediated toxicity in mouse hepatocyte monolayers.

To selectively study protein damage by acrolein and crotonaldehyde in isolated cells, we have explored the use of unsaturated alcohol precursors as a means to generate these aldehydes directly within the intracellular environment. This avoids the problem of side reactions between these reactive aldehydes and cell culture media components that confound experiments where reactive aldehydes are directly added to cells. Allyl alcohol, which undergoes rapid oxidation to acrolein via an alcohol dehydrogenase-dependent pathway, has enabled us to establish that extensive protein carbonylation precedes acrolein-mediated cell-killing in isolated liver cells. Furthermore, novel work conducted by Honours student Ms. Rachael Dunlop (recently commenced candidature as a PhD student at the HRI in Sydney) as well as Dr Frank Fontaine established that 2-trans-crotyl alcohol is useful for studying crotonaldehyde-mediated protein damage and toxicity in isolated cells. As with allyl alcohol, the toxicity of crotyl alcohol in isolated hepatocytes was completely abolished by 4-methyl pyrazole, while it was enhanced by the aldehyde dehydrogenase inhibitor cyanamide. This confirms that an aldehydic oxidation product (crotonaldehyde) was responsible for the acute toxicity of crotyl alcohol. This ability to control the generation of acrolein and crotonaldehyde directly within the intracellular environment of hepatocytes provides a powerful approach to comparing patterns of protein damage by these substances in a model where such damage can be carefully related to cytotoxicity and other biochemical end-points.

Recognising that protein carbonylation is a blunt tool for studying protein damage by unsaturated aldehydes, we have recently commenced production of polyclonal antibodies that recognise protein adducts formed by individual 2-trans-alkenals, including acrolein and crotonaldehyde. This work is conducted primarily by Dr Fontaine as part of a NH&MRC-funded project. The first antibody produced during this effort recognises acrolein-modified lysine residues in proteins with high sensitivity. More importantly, cross-reactivity of this antibody with adducts formed by other endogenous aldehydes (such as malondialdehyde, methyl glyoxal or even crotonaldehyde) is essentially negligible, at least under conditions of an ELISA-based assay. Dr Burcham is currently optimising the use of the anti-acrolein sera in Western blot procedures during a sabbatical in the laboratory of Prof. Dennis Petersen at the University of Colorado Health Science Center. The preliminary data from this effort suggests that this approach provides more toxicologically-significant insights into protein damage during allyl alcohol toxicity than is provided by protein carbonylation assays.

Cardiovascular Disease

Kevin Croft and Trevor Mori

Department of Medicine, Royal Perth Hospital University of Western Australia

The major research interest of the Department is CD with a particular emphasis in human intervention studies investigating the role of diet and other life-style factors on hypertension and atherosclerosis. Supporting this applied area is a strong basic research program focusing on the role of lipid peroxidation in atherosclerosis with a particular interest on markers of in vivo free radical lipid damage (e.g., isoprostanes), bioactive lipid oxidation products (such as isoeicosanoids) and dietary phenolic antioxidants. The Department also houses the Biomedical Mass Spectromer Unit. The Mass Spectrometry Unit is largely concerned with gas chromatography combined with mass spectrometry (GC-MS). Currently we operate 2 GC-MS instruments using both electron impact and chemical ionisation methods to generate mass ions. Negotiations are currently underway to obtain funding for a third independent instrument. The MS facility is also further supported by a variety of HPLC and GC equipment. The Department is happy to collaborate with external groups wishing to make use of the facility and potential collaborators can contact senior staff at the address shown above.

Apoptosis

Dr. Jiri Neuzil

Apoptosis Research Group Heart Foundation Research Centre

School of Medical Science, Griffith University Gold Coast Campus, Southport, Qld, Australia

Anti-cancer activities of vitamin E analogues: The major thrust of our interest focuses on vitamin E (VE) analogues, epitomized by α-tocopheryl succinate (α-TOS), selective inducers of apoptosis and anti-cancer agents. We have shown that VE analogues induce apoptosis primarily by activating the intrinsic pathway and suppress cancer in pre-clinical models of colon and breast carcinomas as well as mesotheliomas, a fatal type of cancer. We study the molecular mechanism by which α-TOS induces apoptosis and by which it sensitizes resistant cancer cells to other apoptogens, such as the TNF family members. We are synthesizing novel analogues of α-TOS with higher apoptogenic activity and specificity for cancer cells, including specific adducts of the agents targeting malignant cells. We also investigate effects of VE analogues on signaling pathways that promote proliferation and/or survival of cancer cells, such as the FGF autocrine look and the Akt pathway, including effects on transcriptional regulation of expression of key members. Further, we are interested in suppression of angiogenesis by VE analogues, since we found that α-TOS causes apoptosis of proliferating (angiogenic) endothelial cells but not normal, arrested endothelial cells. Another focus of our studies is to understand the reasons for selectivity of VE analogues for malignant cells. We believe that in near future, we will be in a position to commence testing of selected VE analogues in human patients. This project is supported by grants provided by ARC, QCF and NBCF.

Role of mitochondria in apoptosis of heart muscle cells during myocardial infarction: During myocardial infarction, muscle heart cells die by apoptosis. This leads to major complications associated with the heart muscle insufficiency. We are interested in furthering our understanding of the processes that underlie this process. Current data strongly suggest that mitochondria may be the major organelle modulating cardiomyocyte cell death, but the precise mechanism is not well understood. We are interested in the molecular mechanism of apoptosis of cardiomyocytes in situation like ischemia/reperfusion (I/R). To study this, we use several models of I/R, including cultured cells, isolated mouse hearts and whole animal models. Out major interest here is to extend the knowledge gained from cell culture studies to the in situ heart model and to the whole animal. We are studying not only the mitochondrial pathways of cell death in cardiomyocytes but also the alternative/parallel signaling pathways that may play a significant role, including the Daxx pathway. At present, we are establishing a method that allows studying apoptosis on the beating heart in a mouse model of myocardial infarction on the level of single cells, which will make it possible to study intervention that may protect the heart muscle from infarction-induced death. We hope that results of these studies may be used in the future for human patients. This project is supported by grants provided by NHMRC and NHF. PDF with bibliography and contacts

Redox Reactions and Vascular Disease

Dr Steven Gieseg

Free Radical Biochemistry Laboratory School of Biological Science, University of Canterbury, Christchurch, New Zealand

Laboratory Research Interests:

• Heart disease and the role of cellular antioxidants
• Neopterin in inflammatory diseases
• Inhibition of oxLDL mediated apoptosis
• Protein oxidation as an initiator of apoptosis
• Redox stress in fish - Effect on vascular control

Our research is focused on three major areas, free radical damage to cellular proteins (protein hydroperoxides and protein bound DOPA), the role of the antioxidant 7,8-dihydroneopterin in inflammation, and cellular disfunction by oxidant during vascular disease (heart disease and complications of diabetes). We are primarily a free radical biochemistry laboratory specialising in tissue culture based research with HPLC and GC analysis. Our principal cell models are human monocytes like U937 and THP-1 cells and macrophages purified from human blood. PDF with bibliography and contacts

Redox Reactions in Vascular Homeostasis

Dr Shane Thomas

Centre for Vascular Research, University of New South Wales, Australia

Role of redox reactions in endothelial cell signaling and function: The endothelium is critical for maintenance of vascular homeostasis. Central to this is endothelial derived nitric oxide (EDNO), synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Vascular diseases including atherosclerosis are characterized by endothelial dysfunction that is manifested as impaired EDNO bioactivity that may contribute to clinical events. Considerable evidence indicates that endothelial dysfunction is due, in part, to vascular oxidative stress and there is great interest in defining the oxidative processes involved. Diseased blood vessels produce increased amounts of reactive oxygen species, derived primarily from endothelial and smooth muscle cells and detected principally as superoxide anion radical (O₂·) and its dismutation product hydrogen peroxide (H₂O₂). It is established that O₂· rapidly reacts with nitric oxide (NO) to limit EDNO bioactivity. Increasing evidence indicate that H2O2 also represent an important signaling molecule governing vascular cell phenotype and vascular tone. Our research focuses on defining to what extent and how H₂O₂ impacts on endothelial function and phenotype and EDNO bioactivity during vascular disease. In collaboration with John Keaney and Kai Chen (Whitaker Cardiovascular Institute, Boston University, USA) to date we have discovered that H₂O₂ activates eNOS by altering the enzyme's phosphorylation status. We have also identified mitochondria as a novel target that mediates the proximal cell signaling events induced by H₂O₂ in endothelial cells. Currently we are investigating the effects of H₂O₂ on EDNO bioactivity. Our recent data indicates that despite activating eNOS the oxidant can limit EDNO bioactivity by promoting oxidative inactivation of NO. We are in the process of characterizing the nature of the oxidative reactions limiting NO and the extent to which these processes are important for endothelial dysfunction during vascular disease states. Our research also focuses on determining the role of myeloperoxidase for endothelial dysfunction and redox control of cell signaling in endothelial cells stimulated with physiological agonists, in particular vascular endothelial growth factor.

Roles and regulation of indoleamine 2,3-dioxygenase: Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme protein that catalyses the oxidative metabolism of L-Trp via the kynurenine pathway. IDO is induced at sites of inflammation by interferon-_γ (IFN_γ) and is traditionally thought to function as an anti-microbial and anti-tumour effector of the cytokine. Recent groundbreaking studies have established that IDO also represents an important immune regulatory enzyme that inhibits T lymphocyte activation by reducing the local concentrations of L-Trp, the least abundant of all essential amino acids. Considerable evidence supports an IDO-based mechanism for immune suppression. For example, induction of IDO and inhibition of T cell activation protects against inflammatory disorders including colitis and collagen-induced arthritis in mice. We have discovered increased IDO expression in atherosclerotic lesions and have initiated a NHMRC funded project examining the role of IDO in this disease in which inflammation of the vascular wall represents an important pathogenic event. In light of the important immune regulatory role of IDO our research also focuses on determining the molecular mechanisms by which the enzyme is controlled. Together with Roland Stocker, we have previously described that IDO is inhibited by nitric oxide (NO). Currently we are investigating how NO inhibits IDO by characterizing the nature of the inactive NO-IDO heme adduct using Resonance Raman Spectroscopy with Andrew Terentis (Florida Atlantic University, USA). We have also identified that IDO is subject to post-translational control and that this form of control may be subject to redox control. We are in the process of extending these studies to determine the mechanisms by which IDO is subject to post-translational control and if these processes are important in the regulation of the enzyme's immune regulatory actions in innate immune cells. PDF with bibliography and contacts

For more information, contact the Society.