John L. Harwood

Bio: John L. Harwood is an academic researcher from Cardiff University. The author has contributed to research in topics: Lipid metabolism & Fatty acid. The author has an hindex of 60, co-authored 420 publications receiving 16081 citations. Previous affiliations of John L. Harwood include John L. Scott & Spanish National Research Council.

Overexpression of phospholipid: diacylglycerol acyltransferase in Brassica napus results in changes in lipid metabolism and oil accumulation

Abstract: The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.

Effects of n‐3 polyunsaturated fatty acids on COX‐2 and PGE2 protein levels in articular cartilage chondrocytes

Abstract: Introduction Previous studies within our laboratory have shown that supplementation with n-3 polyunsaturated fatty acids (PUFAs), but not other fatty acids, has a beneficial effect on reducing the expression and activity of degradative and inflammatory factors known to cause damage and destruction of cartilage in arthritic diseases (Curtis et al. 2000,2002). Cyclooxygenase (COX), also known as prostaglandin H synthase, catalyses the rate-limiting step in the formation of inflammatory prostaglandins (PGs) (Hla & Neilson 1992). PG synthesis requires conversion of arachidonic acid to PGH2 by either the constitutive COX-1 or by COX-2, which is induced by inflammatory and mitogenic stimuli (Smith et al. 2000). Prostaglandin E2 (PGE2) is produced from PGH2 and has been shown to have a number of functions including both anti- and pro-inflammatory actions (Christman et al. 1991). The aim of this project was to investigate the effects of n-3 PUFAs on cyclooxygenase-2 and prostaglandin E2 protein levels in articular cartilage chondrocytes. Materials and methods Articular cartilage was obtained both from 7-day-old bovine metacarpo-metatarsophalangeal joints and from human patients undergoing total knee replacement surgery for osteoarthritis (Llandough Hospital, S.Wales, UK). Both explant and monolayer cultures were set up in DMEM with or without 10–300 µg/ml n-3[eicosapentaenoic acid (EPA)] or n-6[arachidonic acid (AA)] PUFAs (minimum 8 h, at 37°C, in 5%CO2) and in the absence or presence of IL-1 (10 ng/ml) for a further 4 days. Results Total RNA was extracted and RT-PCR performed using oligonucleotide primers specific to COX-2 (Invitrogen, UK). COX-2 mRNA was found to be absent or only present at very low levels in both bovine and human control cultures. After treatment with IL-1, this expression greatly increased. However, supplementation of IL-1-treated cultures with n-3 PUFA (EPA) resulted in a loss of COX-2 mRNA expression. In contrast, supplementation with n-6 PUFA (AA) had no effect. Western blot analysis, using a polyclonal antibody specific to COX-2 (Santa Cruz Biotechnology Inc., USA), showed that COX-2 protein was absent in all control samples and the IL-1 induction of bovine COX-2 protein could also be reduced when supplemented with n-3 PUFAs but not n-6 PUFAs. Using a commercially available PGE2 ELISA Immunoassay kit, it was possible to analyse the PGE2 levels present in explant media from bovine or human samples. In one example, in a 74-year-old female patient, PGE2 protein was very low in the cartilage cultured without IL-1 treatment or PUFA supplementation (control). When the cartilage was treated with IL-1, there was a huge induction of PGE2 levels by as much as 60 fold. This induction is in turn reduced greatly with the supplementation of n-3 PUFA (EPA) but not with n-6 PUFA (AA). Discussion It has long been accepted that COX-2 plays an important role in inflammation. COX-2 has been found in joints affected by arthritic diseases (Hla & Neilson 1992) and in cultures induced by IL-1. The current study has shown that both the IL-1-induced COX-2 message and protein levels can be reduced with supplementation by n-3 PUFAs but not n-6 PUFAs. This work has also shown that with a decrease in COX-2 protein levels, there is also a corresponding decrease in prostaglandin E2 levels caused by n-3 PUFA supplementation.

Interferon-γ: an overview of signals, mechanisms and functions

Abstract: Interferon-gamma (IFN-gamma) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN-gamma ligand, receptor, signal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that influence macrophage function during infection. The current model for IFN-gamma signal transduction is discussed, as well as signal regulation and factors conferring signal specificity. Cellular effects of IFN-gamma are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. In addition, integration of signaling and response with other cytokines and pathogen-associated molecular patterns, such as tumor necrosis factor-alpha, interleukin-4, type I IFNs, and lipopolysaccharide are discussed.

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances

Abstract: Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.