Showing papers by "John L. Harwood published in 2002"

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids.

Abstract: Objective To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. Methods The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). Results Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1–induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase–activating protein, tumor necrosis factor α, IL-1α, and IL-1β) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. Conclusion These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.

Biosynthesis of triacylglycerols and volatiles in olives

Abstract: For many reasons (history, myth, oil quality, etc.) olive is unique among the commercially important oil crops. The biochemistry of the olive tree is also singular. From the photosynthetic point of view olive is one of the few species capable of synthesising both polyols (mannitol) and oligosaccharides (raffinose and stachyose) as the final products of the photosynthetic CO2 fixation in the leaf cell. These carbohydrates, together with sucrose, can be exported from the leaves to the fruits to fulfil the metabolic requirements for oil synthesis. On the other hand, contrary to oilseeds, which are absolutely dependent on the leaves to supply photoassimilates for the synthesis of storage oil, developing olives contain active chloroplasts capable of fixing CO2. Thus, the olive contributes to its own carbon economy. In fact, detached olives have been demonstrated to be capable of fixing radiolabelled CO2 in the light and using the reduced photosynthetic products to form storage oil. Soluble fractions from olive pulp have been demonstrated to catalyse the synthesis of fatty acids from malonyl-CoA. The properties of this subcellular fraction indicate that fatty acids are formed by the same type of fatty acid synthase complex established for other plant systems. By the same token, glycerolipids, including storage triacylglycerols, are formed from glycerophosphate and acyl-CoAs according to the Kennedy pathway, as it has been demonstrated in particulate fractions from olive pulp and tissue culture. Also unique to virgin olive oil is its characteristic aroma. The most abundant volatile compounds in the aroma of olive oil are aldehydes and alcohols of six carbon atoms. Such compounds are formed from linoleic and α-linolenic acids through a sequence of enzymatic reactions known as the lipoxygenase pathway, which is stimulated when olives are crushed during the process of oil extraction. The properties of the different reactions involved in the metabolic pathway leading to the formation of such volatile compounds are described in this paper.

Nutritional and health aspects of olive oil

Abstract: This review describes the types of lipids in human diets and their possible implications for health and disease. The high oleate and significant linoleate contents of olive oil are important for nutrition; moreover, when olive oil is used for deep-fat frying, as in a typ- ical “Mediterranean Diet”, then many of its desirable properties are retained and there is much less deterioration than for some other cooking oils. The health-promoting ef- fects of olive oil have been cited anecdotally for many years. However, as highlighted in this review, the scientific basis for these effects is still unclear. In this review, partic- ular attention is paid to obesity, cardiovascular disease, cancer and inflammatory dis- eases. There is good evidence that olive oil is protective in cardiovascular diseases. Its mechanism of action may involve effects on blood lipids, but other mechanisms, in- cluding effects on immune function, endothelial function and the coagulation pathways remain possible and are discussed. The effects of olive oil in obesity and cancer are less clear. Finally, many questions still remain about the potential health effects of the many non-lipid components of olive oil.

Control analysis of lipid biosynthesis in tissue cultures from oil crops shows that flux control is shared between fatty acid synthesis and lipid assembly

Abstract: Top-Down (Metabolic) Control Analysis (TDCA) was used to examine, quantitatively, lipid biosynthesis in tissue cultures from two commercially important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). A conceptually simplified system was defined comprising two blocks of reactions: fatty acid synthesis (Block A) and lipid assembly (Block B), which produced and consumed, respectively, a common and unique system intermediate, cytosolic acyl-CoA. We manipulated the steady-state levels of the system intermediate by adding exogenous oleic acid and, using two independent assays, measured the effect of the addition on the system fluxes (JA and JB). These were the rate of incorporation of radioactivity: (i) through Block A from [1-14C]acetate into fatty acids and (ii) via Block B from [U-14C]glycerol into complex lipids respectively. The data showed that fatty acid formation (Block A) exerted higher control than lipid assembly (Block B) in both tissues with the following group flux control coefficients (C):(i) Oil palm: *CJTL/BikA=0.64

Effects of n-3 fatty acids on cartilage metabolism.

Abstract: Although the clinical benefits of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) has been recognised for a number of years, the molecular mechanisms by which particular PUFA affect metabolism of cells within the synovial joint tissues are not understood. This study set out to investigate how n-3 PUFA and other classes of fatty acids affect both degradative and inflammatory aspects of metabolism of articular cartilage chondrocytes using an in vitro model of cartilage degradation. Using well-established culture models, cartilage explants from normal bovine and human osteoarthritic cartilage were supplemented with either n-3 or n-6 PUFA, and cultures were subsequently treated with interleukin 1 to initiate catabolic processes that mimic cartilage degradation in arthritis. Results show that supplementation specifically with n-3 PUFA, but not n-6 PUFA, causes a decrease in both degradative and inflammatory aspects of chondrocyte metabolism, whilst having no effect on the normal tissue homeostasis. Collectively, our data provide evidence supporting dietary supplementation of n-3 PUFA, which in turn may have a beneficial effect of slowing and reducing inflammation in the pathogenesis of degenerative joint diseases in man.

Abscisic acid modifies the changes in lipids brought about by water stress in the moss Atrichum androgynum

Abstract: Summary • Mosses are particularly able to withstand drought stress. Moreover, abscisic acid (ABA), which is intimately involved during stress in higher plants, has also been implicated in bryophytes. Because membrane damage is a common feature of drought stress, we have studied changes in lipid composition during desiccation and rehydration of the moss Atrichum androgynum and the effect of exogenous ABA on these processes. • In order to correlate any membrane changes with drought stress, we analysed different lipid classes by thin-layer chromatography, fatty acid composition by gas–liquid chromatography and lipid peroxidation. • Water stress caused changes in phosphoglyceride composition consistent with an activation of phospholipase D and of phosphatidylinositol metabolism. Recovery of phosphoglyceride composition towards original levels occurred during rehydration and ABA treatment reduced the overall extent of changes. Reduction in thylakoid lipids and chlorophyll coincided with loss of photosynthesis. • The data show that mosses respond to drought stress similarly to higher plants and that ABA may reduce membrane damage by diminishing the lipid changes.

Control mechanisms operating for lipid biosynthesis differ in oil-palm (Elaeis guineensis Jacq.) and olive (Olea europaea L.) callus cultures.

Abstract: As a prelude to detailed flux control analysis of lipid synthesis in plants, we have examined the latter in tissue cultures from two important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). Temperature was used to manipulate the overall rate of lipid formation in order to characterize and validate the system to be used for analysis. With [1-14C]acetate as a precursor, an increase in temperature from 20 to 30 degrees C produced nearly a doubling of total lipid labelling. This increase in total lipids did not change the radioactivity in the intermediate acyl-(acyl carrier protein) or acyl-CoA pools, indicating that metabolism of these pools did not exert any significant constraint for overall synthesis. In contrast, there were some differences in the proportional labelling of fatty acids and of lipid classes at the two temperatures. The higher temperature caused a decrease in polyunsaturated fatty acid labelling and an increase in the proportion of triacylglycerol labelling in both calli. The intermediate diacylglycerol was increased in olive, but not in oil palm. Overall the data indicate the suitability of olive and oil-palm cultures for the study of lipid synthesis and indicate that de novo fatty acid synthesis may exert more flux control than complex lipid assembly. In olive, diacylglycerol acyltransferase may exert significant flux control when lipid synthesis is rapid.

Lipid metabolism in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. from lead-contaminated and non-contaminated populations.

Abstract: Lipid metabolism and the effect of Pb 2q and Cu 2q on this process was studied in the moss Rhytidiadelphus squarrosus collected from both a lead-contaminated and a non-contaminated site. Total radiolabelling of lipids from [1- 14 C]acetate was similar in both populations and Cu or Pb (1 mM, 10 mM) did not cause much alteration in acute exposure experiments. However, there were significant qualitative changes. Of the major labelled neutral lipid classes, samples from the lead-polluted site showed a decrease in labelling of triacylglycerols and an increase in wax esters. Acute lead exposure caused similar effects. Cu caused a decrease in the labelling of wax esters and an increase in diacylglycerols. These data suggest that heavy metals cause a change in carbon flux through the acylation reactions associated with the Kennedy pathway. R. squarrosus obtained from the Pb-contaminated site also showed changes in polar lipid labelling compared to the uncontaminated site. The labelling of phosphatidylcholine was more than halved and replaced by increased labelling of other zwitterionic lipids. The chloroplast glycerolipids were also increasingly labelled. Acute exposure to Pb, however, caused little alteration of labelling patterns within 24 h. R. squarrosus contains high levels of polyunsaturated fatty acids (PUFAs), but moss obtained from the Pb-polluted site had significantly less PUFAs containing three or more double bonds. Such samples, when incubated with [1- 14 C]acetate also showed decreased PUFA labelling. By contrast, acute exposure to Pb produced different results. These data provide a foundation for examining lipid metabolism in bryophytes and the effects of pollution in this important class of organism. The results also emphasize that acute and chronic exposure to heavy metals may produce different effects and that caution must be exercised in extrapolating data from one system to another.

Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoon, Acanthamoeba castellanii.

Abstract: Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoeba castellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with [Delta]10-nonadecenoate showed that the enzyme is an n-6 desaturase rather than a [Delta]12-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b5 as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b5) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n-6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n-6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms.

Lipid metabolism in the moss Dicranum scoparium: effect of light conditions and heavy metals on the accumulation of acetylenic triacylglycerols

Abstract: Lipid metabolism in the moss Dicranum scoparium Hedw. was studied using radiolabelling from [1- 1 4 C]acetate. The effect of two environmental parameters, light and heavy metals, on such metabolism was examined. Radiolabelling was approximately linear for 48 h after which the radioactivity in the total and polar lipid fractions remained constant in the light, whereas it declined in the dark. Pulse-chase experiments confirmed that lipid labelling was influenced by light exposure. Light exposure altered the pattern of polar lipid labelling, especially of those lipids associated with chloroplast membranes. Within the neutral lipids, diacylglycerols and triacylglycerols (TAGs) were the major classes labelled. D. scoparium contained up to 45% of total acyl moieties as 9,12,15-octadecatrien-6-ynoic (18:3A) acid and this was found in a TAG subfraction which could be separated by TLC. Although TAGs always represented 65-75% of total neutral lipid labelling, the proportion of TAGs containing 18:3A was increased with time and by light but reduced by exposure to environmentally relevant levels of Cu 2 + and Pb 2 + . The data suggest that 18:3A is produced in D. scoparium by the action of a bi-functional Δ6-desaturase on α-linolenate. Furthermore, important environmental factors (such as light and heavy metals) significantly change the metabolism of TAGs containing 18:3A.