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Prof John Harwood  -  PhD, DSc


Metabolism and Function of Acyl Lipids

Environmental stress and lipid metabolism

Sensitivity of different plants to graminicides. Front (L to R): Blackgrass (acquired resistance before and 4 days after spraying; Red fescue (inherently resistant) before and 4 days after spraying. Back row (L to R): Barley (sensitive) before, 4 days after (note necrotic patches on leaves, stunted growth and stem collapse and 14 days after spraying; pea (inherently resistant after spraying. Graminicides such as fluazifop or sethoxydim kill grasses by inhibiting their chloroplast acetyl-CoA carboxylase. From Harwood (1999) Pesticide Outlook 10, 154-158.

Sensitivity of different plants to graminicides. Front (L to R): Blackgrass (acquired resistance before and 4 days after spraying; Red fescue (inherently resistant) before and 4 days after spraying. Back row (L to R): Barley (sensitive) before, 4 days after (note necrotic patches on leaves, stunted growth and stem collapse and 14 days after spraying; pea (inherently resistant after spraying. Graminicides such as fluazifop or sethoxydim kill grasses by inhibiting their chloroplast acetyl-CoA carboxylase. From Harwood (1999) Pesticide Outlook 10, 154-158.

Research includes a wide range of environmental factors and target organisms ranging from microbes to higher plants.

The Greenhouse Effect (raised atmospheric CO2 and temperature) has been found to change lipid metabolism, in keeping with its action in stimulating membrane biogenesis and growth. The alterations in metabolism can be explained by changed expression of two key enzymes which is now being tested directly. Linked to this work are experiments on temperature adaptation in the important soil protozoon, Acathamoeba castellanii. Low temperature effects have been separated from oxygen effects in inducing expression of n-6 fatty acid desaturase which renders the membrane lipids more ‘fluid’ and permits phagocytosis, which would otherwise stop at low temperatures.

Elevated CO2 (The Greenhouse Effect) changes cell morphology in wheat plants. The arrows indicate mitochondria revealed by immunofluoresence. From Williams et al. (1998) Plant, Cell and Environment 21, 927-936.

Elevated CO2 (The Greenhouse Effect) changes cell morphology in wheat plants. The arrows indicate mitochondria revealed by immunofluoresence. From Williams et al. (1998) Plant, Cell and Environment 21, 927-936.

Plant growth regulators, such as auxins, stimulate membrane lipid biosynthesis in peas and we have studied the control of phosphatidylcholine formation. An important control point is at the level of cytidylyltransferase and we have studied its regulation as well as isolating its gene. More recently, we have isolated the gene for choline kinase and purified and studied the enzyme. Another important plant membrane constituent is the sulpholipid. We have elucidated its biosynthetic pathway and are now working on its degradation and sulphur re-cycling which has to take place (via soil bacteria) each autumn (Fall).

Several classes of pesticides act through their influence on lipid metabolism. Recently we have studied several fungicides which kill plant fungi (potato blight, mildew) and which appear to alter lipid metabolism. Long-standing studies are also involved with thiocarbamates, a herbicide class that is effective through inhibition of fatty acid elongation and, hence, surface layer formation.

There is increasing interest in the toxic effects of heavy metals. We have shown that such metals interfere with lipid metabolism at various levels in marine algae, bryophytes and lichens. Some bryophytes are rather resistant to lead or copper challenge and may be developed for bioremediation. From brown algae, we have been able to isolate a metallothionein which is important for normal metal homeostasis.

The way in which metabolism is controlled is rather poorly understood. In order to re-dress this deficiency, we have applied the technique of control analysis to the study of lipid accumulation in oil crops. In general, control is shared by both the fatty acid synthesis and the lipid assembly blocks of reactions. In some species, diacylglycerol acyltransferase exhibits strong flux control.

Subcellular localisation of an acyl transferase important in endotoxic shock is revealed by immunoflouresence studies. In (a) and (b) lymphocytes are unchallenged and the enzyme is in the cytosol and nucleus. After interferon-g, challenge, the enzyme is concentrated in the nuclear and plasma membranes. From Neville (2000) PhD Thesis.

Subcellular localisation of an acyl transferase important in endotoxic shock is revealed by immunoflouresence studies. In (a) and (b) lymphocytes are unchallenged and the enzyme is in the cytosol and nucleus. After interferon-γ, challenge, the enzyme is concentrated in the nuclear and plasma membranes. See Jackson, S.K. et al. (2008) J. Leukocyte Biol. 84, 86-92.

Lipid biochemistry and medical aspects

Several projects in the lab. are examining the role of acyl lipids in medical problems.

We have a long-standing interest in pulmonary surfactant, which prevents lung collapse at end-expiration. Having shown that the metabolism of surfactant can be heavily influenced by dust (e.g. silica) inhalation, we have now found a correlation between phosphatidylglycerol deficiency and viral bronchiolitis.

A lipid mixture similar to pulmonary surfactant is also present in the peritoneal cavity. This phospholipid-rich secretion is markedly reduced when filtration problems develop during peritoneal dialysis, suggesting an important role there. We are also testing the possible connection with inflammatory reactions which may be implicated in surgical adhesions.

mRNA expression of cyclooxygenase-2 in oesteoarthritic tissue from a patient undergoing knee-replacement surgery.

mRNA expression of cyclooxygenase-2 in oesteoarthritic tissue from a patient undergoing knee-replacement surgery. Because the tissue is inflamed, COX-2 is already being expressed. This expression is increased by the inflammatory cytokine interleukin-1 and reduced by the n-3 fatty acid, eicosapentaenoic . The n-6 acid, arachidonic does not reduce expression significantly. See Hurst, S. et al. (2009) Lipids 44, 889-896.

Inflammation also plays a role in endotoxic shock — an acute reaction by some patients to gram negative bacterial infections following surgery. We have found a correlation between the metabolism of polyunsaturated fatty acids in certain membrane lipids and pre-disposition to endotoxic shock. This discovery has been followed by the identification of specific acyltransferases which can mediate the changes in metabolism. One of these enzymes has been studied in detail and its gene identified. Interestingly, it shows changes in cell localisation during cytokine stimulation.

Arthritis is a widespread disease causing much suffering and which is mediated through excessive and chronic inflammation. We have been studying the molecular mechanisms behind the relief provided by fish oil diets rich in n-3 polyunsaturated fatty acids. Such acids (but not other types of unsaturated or saturated acids) are able to reduce expression of inflammatory cytokines and eicosanoids and activity of cartilage - degrading proteinases in bovine chondrocyte cultures. We are now following up these exciting results with experiments on human tissue. In addition, we are probing the signalling pathways which underpin the effects on transcription.

Grants

Recent work in the lab. has been funded by the EPSRC, NERC, ARC, BBSRC, MAFF, The Wellcome Trust, The Alzheimer’s Research Trust and the EC.

It has also been supported by industrial collaborations with AgrEvo, Rhône-Poulenc, Yamanouchi, Britannia Pharmaceuticals, DuPont, Syngenta, Obsidian and Johnson and Johnson.

Current Collaborations (external)

Gary Dobson (Scottish Crop Research Inst., Dundee)

Elizabeth Gout (Centre Nucleaires de Grenoble)

Tony Kinney (DuPont, Wilmington)

Johnathan Napier (Rothamsted Research, Herts)

Enrique Martinez-Force (Instituto de la Grasa, Seville)

Laszlo Vigh (Hungarian Academy of Sciences, Szeged)

Randall Weselake (University of Alberta, Edmonton)

Associated scientists (Cardiff)

Colin Berry, Kelly BéruBé, Paola Borri, Bruce Caterson, Irina Guschina, Hefin Jones, Pete Kille, Dipak Ramji (School of Biosciences)

Valerie O’Donnell (School of Medicine)

Mark Good (School of Psychology)

PhD students 

Laura Middleton, Hayley Gallagher