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Biomedical subjects

J L Harwood

Publications and source records attributed to J L Harwood.

At least 19 recordsLinked to original sources
PubMed2005

Triclosan inhibition of fatty acid synthesis and its effect on growth of Escherichia coli and Pseudomonas aeruginosa.

OBJECTIVES: To assess the effect of triclosan on fatty acid synthesis and to relate the inhibition of enoyl reductase to bacterial viability. METHODS: The effect of triclosan on fatty acid synthesis in a triclosan-resistant Escherichia coli and its sensitive counterpart and in Pseudomonas aeruginosa was investigated by measuring acetate incorporation into total lipid followed by analysis of fatty acid methyl esters by gas chromatography. Concurrently, the bactericidal effect of triclosan against these bacterial strains was assessed. RESULTS: Triclosan inhibited fatty acid biosynthesis in all the strains tested. However, for triclosan-resistant E. coli (MIC > 1000 mg/L) the concentration required to achieve inhibition was higher than that required for the susceptible counterpart. These concentrations did not significantly affect cell survival in any of the strains tested. CONCLUSIONS: This study shows that the inhibition of fatty acid biosynthesis by the bisphenol might be involved in its growth-inhibitory action and that other mechanisms are involved in its lethal effect. In addition, although microorganisms with a high triclosan MIC were still susceptible to the inhibitory effect of the bisphenol on fatty acid biosynthesis, a higher concentration of the compound was required. This suggested that triclosan bioavailability was different in these strains.

Escherichia coli↗
PubMed2005

Daphnia magna can tolerate short-term starvation without major changes in lipid metabolism.

Daphnia magna is a common crustacean that is adapted to brief spells of fasting. Lipids are naturally a major component of their diet and are stored as energy reserves. However, there has been some controversy in the literature on the extent to which dietary lipids are used directly for complex lipid formation in Daphnia. We examined lipid metabolism in D. magna by labeling the animals using [1-14C]acetate and then followed the turnover of radiolabeled lipids during a pulse chase. Daphnia were either fed or maintained without food during the chase period. The decrease in radioactivity during the chase was relatively unaffected by feeding, although there were some differences in the distribution of radioactivity between lipid classes or individual FA. The polar lipids, which were four times better labeled than nonpolar lipids, contained the most radioactivity in the zwitterionic phosphoglycerides, PE and PC. Under the experimental conditions, the turnover of the polar membrane lipids was unaffected by feeding. Within nonpolar lipids, TAG accounted for up to about 80% of the label, followed by DAG. Overall, our data show that D. magna is capable of high rates of lipid radiolabeling de novo and, in addition, is able to use--and indeed may be dependent on--some dietary components such as the PUFA linoleate and alpha-linolenate. The results also clearly show that Daphnia is able to tolerate brief spells of fasting (24 h) with very little change to its lipid metabolism.

Animals↗
PubMed2002

Use of control analysis to study the regulation of plant lipid biosynthesis.

Control analysis is a powerful method to quantify the regulation of metabolic pathways. We have applied it to lipid biosynthesis for the first time by using model tissue culture systems from the important oil crops, olive ( Olea europaea L.) and oil palm ( Elaeis guineensis Jacq.). By the use of top-down control analysis, fatty acid biosynthesis has been shown to exert more control than lipid assembly under different experimental conditions. However, both parts of the lipid biosynthetic pathway are important, so that attempts to alter oil yield by manipulating the activity of a single enzyme step are very unlikely to produce significant increases.

Biochemistry↗
PubMed2000

Novel inhibitors of the condensing enzymes of the type II fatty acid synthase of pea (Pisum sativum).

The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called beta-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-(14)C]acetate substrate and chloroplast preparations from pea leaves, and [1-(14)C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2000

n-3 fatty acids specifically modulate catabolic factors involved in articular cartilage degradation.

This study describes specific molecular mechanisms by which supplementation with n-3 fatty acids (i.e. those present in fish oils) can modulate the expression and activity of degradative and inflammatory factors that cause cartilage destruction during arthritis. Our data show that incorporation of n-3 fatty acids (but not other polyunsaturated or saturated fatty acids) into articular cartilage chondrocyte membranes results in a dose-dependent reduction in: (i) the expression and activity of proteoglycan degrading enzymes (aggrecanases) and (ii) the expression of inflammation-inducible cytokines (interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha) and cyclooxygenase (COX-2), but not the constitutively expressed cyclooxygenase COX-1. These findings provide evidence that n-3 fatty acid supplementation can specifically affect regulatory mechanisms involved in chondrocyte gene transcription and thus further advocate a beneficial role for dietary fish oil supplementation in alleviation of several of the physiological parameters that cause and propogate arthritic disease.

Animals↗
PubMed2000

Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues.

Glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.15) catalyses the first step of the Kennedy pathway for acyl lipid formation. This enzyme was studied using high-speed particulate fractions from oil palm (Elaeis guineensis Jacq.) tissue cultures and mesocarp acetone powders. The fractions were incubated with [(14)C]glycerol 3-phosphate and incorporation of radioactivity into Kennedy pathway intermediates studied. Optimal conditions were broadly similar between the two preparations but those from fruit mesocarp clearly contained more active enzymes for the subsequent stages of the Kennedy pathway - as exemplified by the appreciable accumulation of radioactivity in triacylglycerol. Experiments with different acyl-CoA substrates showed that the GPAT in both high-speed particulate preparations had a significant preference for palmitate. Glycerol 3-phosphate acyltransferase was solubilised from both preparations with optimal solubilisation being achieved at 0.5% (w/v) CHAPS concentrations. Solubilised GPATs were purified further using DE52 ion-exchange chromatography and Sephadex G-100 molecular exclusion chromatography. Purifications of up to about 70-fold were achieved. The purified GPATs showed a strong preference for palmitoyl-CoA compared to other acyl-CoA donors, in keeping with the importance of palmitate in palm oil.

Acyl Coenzyme A↗
PubMed2000

Lipoxygenase pathway in olive callus cultures (Olea europaea).

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.

Aldehyde-Lyases↗
PubMed2000

The inhibition of fatty acid elongation by pebulate can be effectively counteracted by the safener dichlormid.

The thiocarbamate herbicide pebulate inhibits fatty acid elongation, which is necessary for surface lipid biosynthesis. As both barley and wild oats are susceptible to pebulate, the safener dichlormid was used to study the reversal of its herbicidal effect. Fatty acid elongation was restored by a dichlormid pretreatment in barley, but not in pebulate-expressed oats.

Acetamides↗
PubMed2000

Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme.

Arabidopsis↗
PubMed2000

Pea choline kinase: purification, properties and isolation of a cDNA.

Choline kinase has been partially purified from pea seedlings and its properties studied. Using sequence information from soya bean and other choline kinases, we have also isolated a cDNA encoding the enzyme. It encodes a protein of 343 amino acids (calculated molecular mass of 39785 Da), which shows 82% homology with the soya bean choline kinase. The protein has been expressed in Escherichia coli with very good activity and high expression levels.

Animals↗
PubMed2000

Characterization of lipoxygenase isoforms in olive callus cultures.

Lipoxygenase activity is critical for the development of flavours and aromas in olive oils. We have partly purified isoforms of molecular mass 95 kDa that have activity against linoleic or alpha-linolenic acids by a simple procedure from olive callus cultures.

Cell Fractionation↗
PubMed2000

Effect of copper and lead on lipid metabolism in bryophytes and lichens.

Bryophytes and lichens have a widespread occurrence and can survive under extreme environmental conditions, such as drought, low temperatures, continuous light or prolonged darkness. It has been shown that lipid metabolism is sensitive to both metal response and metal resistance mechanisms in many organisms, including yeast, Silene cucubalus, and in the marine brown algae Fucus spp. and Ascophyllum nodosum. In the present study, the effects of lead and copper on lipid metabolism have been studied in two moss species, Rhytidiadelphus squarrosus and Dicranum scoparium, and also in the lichen Peltigera horizontalis with a cyanobacterial Nostoc photobiont.

Acetates↗
PubMed1999

Re-evaluation of plant sulpholipid labelling from UDP-[14C]glucose in pea chloroplasts.

The sulphoquinovosyldiacylglycerol (sulpholipid) zone obtained by TLC of pea chloroplast lipids yields, on hydrolysis, not only sulphoquinovose but also galactose and glucose. Following incorporation from UDP-[(14)C]glucose, the percentages of the total radioactivity in these three sugars were typically 1, 5 and 85%, respectively. The occurrence of the glucolipid causes difficulties in elucidating further the biosynthetic pathway for sulphoquinovose.

Carbon Radioisotopes↗
PubMed1999

Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Amino Acid Sequence↗
PubMed1999

Abnormal surfactant composition and activity in severe bronchiolitis.

A prospective study of infants under 1 y of age, ventilated for severe viral bronchiolitis, was carried out in four paediatric intensive care units in order to study surfactant activity and composition in this condition. Lung lavage fluid from 24 infants with bronchiolitis, 19 with bronchiolitis and sepsis or cardiac failure and 12 controls were analysed by the "click test" for surfactant activity and for phospholipids. Surfactant activity was present in all controls, but in only 2 of the 24 infants with bronchiolitis alone. The presence of phosphatidylglycerol correlated perfectly with the click test, suggesting that reduced activity is due to changes in surfactant lipid composition. In those with bronchiolitis plus coexisting disease, surfactant activity and phosphatidylglycerol were absent in only half. Surfactant activity and phosphatidylglycerol re-appeared by extubation. Severe viral bronchiolitis is associated with an absence of surfactant activity and PG, which resolves by clinical recovery. Infants with coexisting conditions are not always surfactant deficient. Surfactant administration is likely to be beneficial, but requires a selective approach.

Bronchiolitis↗