Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.

Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.

A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

Endemic infection reduces transmission potential of an epidemic parasite during co-infection.

Endemic, low-virulence parasitic infections are common in nature. Such infections may deplete host resources, which in turn could affect the reproduction of other parasites during co-infection. We aimed to determine whether the reproduction, and therefore transmission potential, of an epidemic parasite was limited by energy costs imposed on the host by an endemic infection. Total lipids, triacylglycerols (TAG) and polar lipids were measured in cockroaches (Blattella germanica) that were fed ad libitum, starved or infected with an endemic parasite, Gregarina blattarum. Reproductive output of an epidemic parasite, Steinernema carpocapsae, was then assessed by counting the number of infective stages emerging from these three host groups. We found both starvation and gregarine infection reduced cockroach lipids, mainly through depletion of TAG. Further, both starvation and G. blattarum infection resulted in reduced emergence of nematode transmission stages. This is, to our knowledge, the first study to demonstrate directly that host resource depletion caused by endemic infection could affect epidemic disease transmission. In view of the ubiquity of endemic infections in nature, future studies of epidemic transmission should take greater account of endemic co-infections.

Dietary fatty acids and arthritis.

Musculoskeletal complaints are the second most frequent reason for medical treatments. Within these diseases rheumatoid arthritis (RA) and, especially, osteoarthritis (OA) are common. Although the causes of arthritis are multifactorial and not fully understood, clinical trials have generally shown benefit from dietary n-3 polyunsaturated fatty acids. This has usually been attributed to their anti-inflammatory properties. Recently we have used in vitro model systems to study the molecular mechanism(s) by which n-3 PUFAs may act to alleviate the symptoms of arthritis. These experiments showed that n-3 PUFAs reduce expression of cartilage-degrading proteinases, cyclooxygenase-2 and inflammatory cytokines. Eicosapentaenoic acid (EPA) was more effective than docosahexaenoic acid (DHA) or alpha-linolenic acid. The data provide a scientific rationale for the consumption of n-3 fatty acids as part of a healthy diet and perhaps in treating arthritis.

Relative efficacies of omega-3 polyunsaturated fatty acids in reducing expression of key proteins in a model system for studying osteoarthritis.

OBJECTIVE: To assess the relative efficacy of three different omega-3 (n-3) polyunsaturated fatty acids (PUFAs) in suppressing the mRNA levels for important proteins involved in the etiology of osteoarthritis (OA). METHODS: A model cell culture system (bovine chondrocytes) was used. Inflammatory factors and enzymes involved in OA were induced by exposure of the chondrocyte cultures to interleukin-1alpha (IL-1alpha). The effect of pre-incubating cultures with various amounts of exogenous fatty acids on subsequent levels of mRNAs was assessed by reverse transcription-polymerase chain reactions (RT-PCR). RESULTS: Exposure of cultures to IL-1alpha induced expression of the cartilage proteinases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)-4 and ADAMTS-5, cyclooxygenase (COX)-2, the matrix metalloproteinase (MMP)-3 and the inflammatory cytokines IL-1alpha, interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). n-3 PUFAs were able to reduce the levels of mRNA for ADAMTS-4, ADAMTS-5, MMP-3, MMP-13, COX-2 (but not COX-1), IL-1alpha, IL-1beta and TNF-alpha. Eicosapentaenoic acid (EPA) was the most effective, followed by docosahexaenoic (DHA) and then alpha-linolenic (ALA) acid. The n-6 PUFA, arachidonic acid (AA) had no effect. CONCLUSION: These results show that omega-3 (n-3) PUFAs cause a reduction in the mRNA levels for various proteins known to be important in the pathology of OA. They provide a molecular explanation, at least in part, for beneficial effects of dietary omega-3 PUFAs for the amelioration of symptoms of the disease. The relative efficacy of EPA suggests that this omega-3 PUFA may be especially useful for dietary supplementation in patients with OA.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Microbial degradation of the plant sulpholipid.

Five bacterial strains capable of using sulphoquinovose (6-deoxy-6-sulpho-D-glucopyranose) as a sole source of carbon have been isolated and partially characterized.

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.

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.