Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2. (1/97)

The lipopolysaccharide (LPS) of Chlamydia trachomatis L2 was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. From a total of 5 x 10(4) cm2 of infected monolayers, 22.3 mg of LPS were obtained. Compositional analysis indicated the presence of 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, phosphorus, and fatty acids in a molar ratio of 2.8:2:2.1:4.5. Matrix-assisted laser-desorption ionization mass spectrometry performed on the de-O-acylated LPS gave a major molecular ion peak at m/z 1781.1 corresponding to a molecule of 3 Kdo, 2 GlcN, 2 phosphates, and two 3-hydroxyeicosanoic acid residues. The structure of deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide was determined by 600 MHz NMR spectroscopy as Kdoalpha2-->8Kdoalpha2-->4Kdoalpha2-->6D-GlcpNbeta1 -->6D-GlcpNalpha 1,4'-bisphosphate. These data, together with those published recently on the acylation pattern of chlamydial lipid A (Qureshi, N., Kaltashov, I., Walker, K., Doroshenko, V., Cotter, R. J., Takayama, K, Sievert, T. R., Rice, P. A., Lin, J.-S. L., and Golenbock, D. T. (1997) J. Biol. Chem. 272, 10594-10600) allow us to present for the first time the complete structure of a major molecular species of a chlamydial LPS.  (+info)

Measurement of in vivo rectal mucosal cytokine and eicosanoid production in ulcerative colitis using filter paper. (2/97)

BACKGROUND: Excessive mucosal generation of cytokines and eicosanoids has been reported in vitro in ulcerative colitis (UC) using traumatising biopsy techniques, and in vivo using time consuming rectal dialysis. AIMS: To validate a simple filter paper technique to profile rectal mucosal production of cytokines and eicosanoids in vivo in patients with UC compared with controls. PATIENTS: Forty one patients with UC (21 with active disease) and 16 controls were studied. METHODS: In vitro, recovery of known concentrations of cytokine or mediator applied to filter papers was measured by ELISA following incubation in buffer. In vivo, patients and controls had filter papers apposed to the rectal mucosa briefly through a rigid sigmoidoscope. Filter papers were then incubated prior to assay by ELISA. RESULTS: In vitro validation studies showed that the filter paper technique could be used to measure mucosal release of interleukin-1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), thromboxane B(2) (TXB(2)), and prostaglandin E(2) (PGE(2)), but not interferon gamma (IFN-gamma). Mucosal release of IL-1beta, TNF-alpha, TXB(2) and PGE(2) were significantly increased in active UC (p=0.001) and correlated directly with disease activity (p=0.02). CONCLUSIONS: The filter paper technique confirmed increased rectal mucosal release of cytokines and eicosanoids in UC, in proportion to disease activity. The simplicity, safety and speed of the technique make it a practicable option for use in the outpatient clinic to study the pathogenesis of inflammatory bowel disease, and potentially its response to treatment.  (+info)

Requirements for unsaturated fatty acids for the induction on respiration in Saccharomyces cerevisiae. (3/97)

Unsaturated fatty acids provided during the release from glucose repression were shown to be essential for derepression of respiration in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Cells derepressed in the presence of oleic acid contained three to six times as much cytochrome per cell as those derepressed in the absence of unsaturated fatty acid or those derepressed with eicosaenoic acid. The delta9 isomer was the most efficient of the cis-octadecenoic acid isomers in supporting that increase, and eicosaenoic acid supported an increase at only 15% the rate observed with oleic acid. Derepression, even in the presence of oleic acid, proceeded only after a lag of 3 hours. When glucose was removed prior to the addition of oleate, the lag was reduced by the time of the preincubation with glycerol. This result suggests that some processes necessary for increased respiration can proceed in the absence of an added unsaturated fatty acid, but these processes apparently require certain levels of unsaturated acids in the pre-existing lipids, since they occurred in cells whose membranes contained 50 mol % oleate, but not in cells containing only 20 mol %. These processes leading to eventual increased respiration were inhibited by cycloheximide but not chloramphenicol, suggesting that protein synthesis on cytoplasmic ribosomes but not mitochondrial ribosomes were required. Derepression in the absence of oleate for 3 hours lessened the inhibition or respiration induction by ethidium bromide. This result indicates that the transcription of mitochondrial DNA necessary for the induction of respiration may have occurred in the absence of added unsaturated fatty acid, but that some subsequent event required added esterified unsaturated fatty acid.  (+info)

Stable ordering in Langmuir-Blodgett films. (4/97)

Defects in the layering of Langmuir-Blodgett (LB) films can be eliminated by depositing from the appropriate monolayer phase at the air-water interface. LB films deposited from the hexagonal phase of cadmium arachidate (CdA2) at pH 7 spontaneously transform into the bulk soap structure, a centrosymmetric bilayer with an orthorhombic herringbone packing. A large wavelength folding mechanism accelerates the conversion between the two structures, leading to a disruption of the desired layering. At pH > 8.5, though it is more difficult to draw LB films, almost perfect layering is obtained due to the inability to convert from the as-deposited structure to the equilibrium one.  (+info)

Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon:nitrogen availability. (5/97)

The objective of the current work was to establish the degree to which the effects of carbon and nitrogen availability on Arabidopsis seedling growth and development are due to these nutrients acting independently or together. Growth of seedlings on low (0.1 mM) nitrogen results in a significant reduction of seedling and cotyledon size, fresh weight, chlorophyll, and anthocyanin content but a slight increase in endogenous sugars. The addition of 100 mM sucrose (Suc) to the nitrogen-depleted growth media results in a further reduction in cotyledon size and chlorophyll content and an overall increase in anthocyanins and endogenous sugars. Storage lipid breakdown is almost completely blocked in seedlings grown on low nitrogen and 100 mM Suc and is significantly inhibited when seedlings are grown on either low nitrogen or high Suc. Carbohydrate repression of photosynthetic gene expression can only be observed under low nitrogen conditions. Low (0.1 mM) nitrogen in the absence of exogenous carbohydrate results in a significant decrease in chlorophyll a/b-binding protein and ribulose bisphosphate carboxylase small subunit gene transcript levels. Thus, carbon to nitrogen ratio rather than carbohydrate status alone appears to play the predominant role in regulating various aspects of seedling growth including storage reserve mobilization and photosynthetic gene expression.  (+info)

Phospholipase A2 activity triggers the wound-activated chemical defense in the diatom Thalassiosira rotula. (6/97)

The activation of oxylipin-based chemical defense in the diatom Thalassiosira rotula is initiated by phospholipases that act immediately after cell damage. This lipase activity is responsible for the preferential release of free mono- and polyunsaturated fatty acids. Among these, eicosatetraenoic- and eicosapentaenoic acid are further converted by lipoxygenases to reactive defensive metabolites such as the antiproliferative alpha,beta,gamma,delta-unsaturated aldehydes 2,4-decadienal and 2,4,7-decatrienal. We show that mainly saturated free fatty acids are present in the intact diatom T. rotula, whereas the amount of free polyunsaturated eicosanoids is drastically increased in the first minutes after wounding. Using fluorescent probes, the main enzyme activity responsible for initiation of the aldehyde-generating lipase/lipoxygenase/hydroperoxide lyase cascade was characterized as a phospholipase A2. All enzymes involved in this specific defensive reaction are active in seawater over several minutes. Thus, the mechanism allows the unicellular algae to overcome restrictions arising out of potential dilution of defensive metabolites. Only upon predation are high local concentrations of aldehydes formed in the vicinity of the herbivores, whereas in times of low stress, cellular resources can be invested in the formation of eicosanoid-rich phospholipids. In contrast to higher plants, which use lipases acting on galactolipids to release C18 fatty acids for production of leaf-volatile aldehydes, diatoms rely on phospholipids and the transformation of C20 fatty acids to form 2,4-decadienal and 2,4,7-decatrienal as an activated defense.  (+info)

Effect of arachidonic and eicosapentaenoic acids on acute lung injury induced by hypochlorous acid. (7/97)

BACKGROUND: Hypochlorous acid (HOCl) is the main oxidant of activated polymorphonuclear neutrophil granulocytes (PMN) and generated by myeloperoxidase during respiratory burst. This study investigates the effects of HOCl on pulmonary artery pressure (PAP) and vascular permeability and characterises the influence of arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the observed effects. METHODS: HOCl (500, 1,000, 2,000 nmol/min) was continuously infused into the perfusate (Krebs-Henseleit buffer solution, KHB). AA or EPA in subthreshold doses (both 2 nmol/min) or buffer were simultaneously infused using a separate port. PAP, pulmonary venous pressure (PVP), ventilation pressure, and lung weight gain were continuously recorded. The capillary filtration coefficient (Kf,c) was calculated before and 30, 60, and 90 minutes after starting the HOCl infusion. RESULTS: HOCl application resulted in a dose dependent increase in PAP and Kf,c. The onset of these changes was inversely related to the HOCl dose used. The combined infusion of AA with HOCl resulted in a significant additional rise in pressure and oedema formation which forced premature termination of all experiments. The combination of EPA with HOCl did not result in an enhancement of the HOCl induced rise in pressure and oedema formation. CONCLUSIONS: Changes in the pulmonary microvasculature caused by HOCl are differently influenced by omega-6 and omega-3 polyunsaturated free fatty acids, suggesting a link between neutrophil derived oxidative stress and pulmonary eicosanoid metabolism.  (+info)

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells. (8/97)

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega. cm(2). When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 omega x cm(2.) These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.  (+info)

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