Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens.

Adhesion of meningitis-associated Escherichia coli O18acK1H7 to collagens was characterized. The E. coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass. Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of alpha-methyl-D-mannoside. A cat cassette was introduced into the filmA gene of IHE 3034, and the resulting mutant strain IHE 3034-2 failed to adhere to collagens. In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S-fimbrillin, had no significant effect on the adhesiveness. The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034-2. The complemented strain IHE 3034-2(pRPO-1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion. We have previously shown that the type 1 fimbria from E. coli K-12 strain PC31 does not confer bacterial adhesiveness to collagens. The fimH genes from E. coli IHE 3034 as well as from PC31 were expressed in the fimH-null strain MS4. The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive. Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino-acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201. Each of these residues in the IHE 3034-FimH were individually substituted to the corresponding amino acid in the PC31-FimH. The substitution S62-->A completely abolished collagen adhesiveness. The reverse substitution A62-->S in the PC31-FimH as well as in the FimH from another E. coli strain induced collagen adhesiveness to the level seen with IHE 3034-FimH. Out of nine fimH genes analysed from isolates of E. coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1. Our results demonstrate that the amino-acid residue Ala-62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E. coli.

Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles.

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.

Effects of repeated oral doses of fenflumizole on platelet aggregation and thromboxane formation in man ex vivo.

Anti-platelet effects of fenflumizole, a new cyclo-oxygenase inhibitor, were studied in man ex vivo. Fenflumizole was given to male volunteers at the oral doses of 25, 50 or 100 mg per day, each dose for a period of seven days. The formation of thromboxane B2 (TXB2) during whole blood clotting, platelet aggregation induced by arachidonic acid and ADP, the formation of TXB2 during aggregation as well as serum concentration of fenflumizole were measured repeatedly during drug administration and for a fortnight after drug discontinuation. TXB2 formation during whole blood clotting was decreased dose-dependently by fenflumizole. The degree of inhibition of TXB2 formation was proportional to fenflumizole concentration in serum within each individual. The lag phase of platelet aggregation induced by arachidonic acid was prolonged and the formation of TXB2 during aggregation decreased by fenflumizole. No total inhibition of either TXB2 synthesis or platelet aggregation was caused by the fenflumizole doses used. The results show that the degree of inhibition of platelet thromboxane forming capacity by repeated doses of fenflumizole is closely related to the concentration of the drug in blood. Platelet aggregation however is less sensitive to changes in fenflumizole levels and cannot be assessed solely on the basis of cyclo-oxygenase activity.

The antipsoriatic drug metabolite etretin (Ro 10-1670) alters the metabolism of fatty acids in human keratinocytes in culture.

We have studied the effect of etretin (Ro 10-1670), the active metabolite of the widely used antipsoriatic drug etretinate (Ro 10-9359), on the incorporation and release of arachidonic acid in human skin keratinocytes. During 24-h culture, radioactive 14C-arachidonic acid was avidly incorporated into the cellular lipids of the keratinocytes. When the cells were cultured for another 48 h in fresh medium, 8.8% +/- 0.3% of the incorporated radioactivity was released from the cells. The presence of etretin (10(-8) M to 10(-5) M) in the medium stimulated the release of radiolabel. With 10(-5) M etretin in the culture medium, 13.0% +/- 0.4% of the incorporated radioactivity was released, and this was accompanied by decreased labelling of phosphatidylethanolamine. This suggests that phosphatidylethanolamine may be an important source of the released arachidonic acid. Etretin pretreatment reduced the incorporation of 14C-arachidonic acid into diacylglycerols, triacylglycerols, and cholesteryl esters. Pretreatment for 48 h with 10(-5) M etretin reduced subsequent 14C-arachidonic acid incorporation into nonphosphorus lipids from a mean total of 8.2% +/- 0.2% to 3.2% +/- 0.1% (p less than 0.001). These findings suggest that etretin interferes with the esterification of arachidonic acid into nonphosphorus lipids. Etretin was also found to cause changes in the fatty acid composition of keratinocytes. Following 48 h culture with etretin, the percentage amount of the fatty acids belonging to the n3 series was increased whereas that of palmitic acid (16:0) and palmitoleic acid (16:1n7) was decreased. In conclusion, our study suggests that etretin in therapeutical concentrations affects fatty acid metabolism in human keratinocytes in culture.

Incorporation and distribution of eicosanoid precursor fatty acids in murine fibroblasts in culture.

Murine fibroblasts in culture were labelled with either [14C]arachidonic acid, [14C]dihomo-gamma-linolenic acid or [14C]eicosapentaenoic acid. All these [14C]fatty acids were effectively incorporated into the fibroblasts and the bulk of the radioactivity was recovered in various phospholipids. The major radiolabelled phospholipids were phosphatidylethanolamine, phosphatidylcholine and the phosphatidylinositol + phosphatidylserine fraction. Significant amounts of radiolabel were found also in the triacylglycerols: even as much as 30% of the total of the incorporated dihomo-gamma-linolenic acid was recovered in the triacylglycerols. The present study suggests that arachidonic acid, dihomo-gamma-linolenic acid and eicosapentaenoic acid are effectively taken up and esterified into different lipid fractions of murine fibroblasts and that also the triacylglycerols are significantly involved in the incorporation, storage, and release of the eicosanoid precursor fatty acids.

Interference with the distribution and release of arachidonic acid in human keratinocytes by bradykinin, histamine and phosphatidic acid.

The effects of bradykinin, histamine, phosphatidic acid and leukotrienes B4 and C4 on the distribution and release of 14C-arachidonic acid in human keratinocytes in culture were investigated. Bradykinin, histamine, and phosphatidic acid were found to liberate 14C-arachidonic acid from membrane phospholipids, whereas leukotrienes B4 and C4 were ineffective in this respect. The decrease in the labeling of phospholipids was accompanied by increased labeling of the non-phosphorus lipids. The present study suggests that bradykinin, histamine, and phosphatidic acid may interfere with the distribution and release of arachidonic acid in human keratinocytes in culture.

Endogenously generated 5-hydroperoxyeicosatetraenoic acid is the preferred substrate for human leukocyte leukotriene A4 synthase activity.

A single protein from human leukocytes possesses both 5-lipoxygenase and leukotriene A4 (LTA4) synthase activities. It has been reported that LTA4 production is more efficient when the enzyme utilizes arachidonic acid, than when 5-HPETE is exogenously supplied as substrate. In the present study, human leukocyte homogenate 100,000 X g supernatant was incubated with 100 microM octadeuterated arachidonic acid and exogenous 5-HPETE (0-80 microM), and the isotopic composition of LTA4 hydrolysis products was determined by gas chromatography-mass spectrometry. Even though 100 microM deuterated arachidonic acid results in 20-30 microM deuterated 5-HPETE, 80 microM exogenous 5-HPETE in the incubation could reduce the amount of deuterated LTA4 by only approx. 20%. The present study would thus indicate that the arachidonic acid moiety is preferentially converted to LTA4 in a concerted reaction without dissociation of a 5-HPETE intermediate.

Ultraviolet B irradiation induces changes in the distribution and release of arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture.

There is increasing evidence that derivatives of 20-carbon polyunsaturated fatty acids, the eicosanoids, play an important role in the inflammatory responses of the human skin. To better understand the metabolic fate of fatty acids in the skin, the effect of ultraviolet B (UVB) irradiation (280-320 nm) on the distribution and release of 14C-labeled arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture was investigated. Ultraviolet B irradiation induced the release of all three 14C-labeled fatty acids from the phospholipids, especially from phosphatidylethanolamine, and this was accompanied by increased labeling of the nonphosphorus lipids. This finding suggests that UVB induces a significant liberation of eicosanoid precursor fatty acids from cellular phospholipids, but the liberated fatty acids are largely reincorporated into the nonphosphorus lipids. In conclusion, the present study suggests that not only arachidonic acid but also dihomo-gamma-linolenic acid, and eicosapentaenoic acid might be involved in the UVB irradiation-induced inflammatory reactions of human skin.

Time course of incorporation of 20-carbon polyunsaturated fatty acids in a human keratinocyte cell line.

Human keratinocytes (NCTC 2544) in culture were labeled with equal amounts of either 14C-arachidonic acid, 14C-dihomo-gamma-linolenic acid or 14C-eicosapentaenoic acid. At various time points, the incubations were stopped and the distribution of the 14C-fatty acids was analyzed. All these eicosanoid precursor fatty acids were effectively incorporated into the cellular lipids of the keratinocytes, and the major radiolabeled individual lipid fraction was phosphatidylethanolamine. The distributions of arachidonic acid and dihomo-gamma-linolenic acid within cellular lipids were rather the same. However, less eicosapentaenoic acid than either arachidonic acid or dihomo-gamma-linolenic acid was incorporated into the phospholipids and, correspondingly, more eicosapentaenoic acid was incorporated into the nonphosphorus lipids. In the phosphatidylinositol + phosphatidylserine fraction, there was significantly less eicosapentaenoic acid than either arachidonic acid or dihomo-gamma-linolenic acid. The present study suggests that these eicosanoid precursor fatty acids are effectively incorporated into the human keratinocytes and that the pattern of incorporation and distribution of eicosapentaenoic acid appears to differ slightly from that of either arachidonic acid or dihomo-gamma-linolenic acid.

Lipoxin A. Stereochemistry and biosynthesis.

Lipoxin A (LXA) was prepared by incubation of either (15S)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) or (15S)-15-hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic (15-HPETE) with human leukocytes stimulated by either the ionophore A23187 or the chemotactic peptide fMet-Leu-Phe. Comparison with four trihydroxyeicosatetraenoic acids prepared by total synthesis showed that biologically derived LXA is 5S,6R,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid. Three isomers of LXA were also identified in extracts of leukocytes utilizing an improved isolation procedure. These were (5S,6S,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (6S-LXA), (5S,6R,15S)-5,6,15-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid (11-trans-LXA), and (5S,6S,15S)-5,6,15-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid (6S-11-trans-LXA). 18O2-labeling studies indicated that formation of LXA and its isomers occurred with incorporation of 18O at their C-5 but not C-6 positions. These results suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-eicosatetraenoic acid or its equivalent may serve as one intermediate in the biosynthesis of LXA and 6S-LXA. When added to guinea pig lung strips LXA provoked contractions which were slow in onset and long lasting. In addition, dose response studies showed that biologically derived LXA and synthetic LXA were indistinguishable in this bioassay whereas synthetic 6S-LXA and biologically derived 6S-LXA did not share this activity. Taken together, these results suggest that activated leukocytes utilize exogenous 15-HETE to generate lipoxins which in turn can modulate cellular responses.

Evidence for a 5(6)-epoxytetraene intermediate in the biosynthesis of lipoxins in human leukocytes. Conversion into lipoxin A by cytosolic epoxide hydrolase.

The existence of a 15(S)-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid intermediate in the biosynthesis of lipoxins A and B has recently been proposed. In the present study, human leukocytes were exposed to 15-HETE and the divalent cation ionophore A23187 and alcohol trapping studies were performed. The products containing alkyltetraenes were isolated and characterized. HPLC analysis, UV spectroscopy and GC/MS of the products showed that 5,15-dihydroxy-14-O-alkyleicosatetraenoic acids were formed, indicating that 5(6)-epoxytetraenes (precursor of the trapping product) were formed in human leukocytes. To gain further evidence for the role of 5(6)-epoxytetraene intermediate in the biosynthesis of lipoxins, (15)-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid was prepared by total chemical synthesis. When added to purified human liver cytosolic epoxide hydrolase, the epoxide was rapidly and quantitatively converted into LXA. The results provide further evidence for the role of a 5(6)epoxytetraene intermediate in the biosynthesis of lipoxins.

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes.

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

UVB irradiation induces changes in the distribution and release of 14C-arachidonic acid in human keratinocytes in culture.

Following labeling of human keratinocytes in culture for 48 h with 14C-arachidonic acid (800,000 cpm), 86.8 +/- 0.5% (mean +/- SEM) of the radioactivity was incorporated into the cells. Two hours after exposure to UVB irradiation at doses up to 392 mJ/cm2 of erythemally effective (EE) UVB irradiation, only slight changes in the distribution of arachidonic acid could be detected. However, 24 h after irradiation the release of arachidonic acid into the culture medium was significantly increased. The distribution of arachidonic acid was also changed: there was a considerable loss in the amount of radioactivity associated with phosphatidylethanolamine. With doses up to 174 mJ/cm2 (EE) of UVB, the decrease in the labeling of phospholipids was accompanied by an increased arachidonic acid content in the nonphosphorus lipids, especially in the triacylglycerols. Following a high dose of UVB (392 mJ/cm2, EE), a substantial release of label was detected, but the labeling of triacylglycerols was unaltered. The present study suggests that in human keratinocytes UVB irradiation induces the release of arachidonic acid from the cellular lipids and that the major source of the released arachidonic acid is phosphatidylethanolamine.

Effect of prednisolone on the fatty acid composition and the formation of arachidonate metabolites in human keratinocytes in culture.

Human keratinocytes were cultured for eight days in a medium containing 0 - 100 microM of prednisolone. Subsequently, the amounts of prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TXB2) released into the culture medium were measured by radioimmunoassay and the fatty acid composition of the keratinocytes was investigated. Prednisolone significantly inhibited the production of PGF2 alpha and 6-keto-PGF1 alpha by keratinocytes whereas the formation of TXB2 remained unaffected. The total molar formation of the cyclo-oxygenase metabolites of arachidonic acid appeared to be inhibited by prednisolone, suggesting that prednisolone might interfere with the phospholipase activities and the release of arachidonic acid in human keratinocytes. Prednisolone had only a weak effect on the fatty acid composition of keratinocytes as the percentage amount of oleic acid (18:1) was slightly decreased and that of eicosenoic acid (20:1) correspondingly increased while the percentage amounts of the other fatty acids remained unaffected. The present study might suggest that the anti-inflammatory action of glucocorticoids in the human skin would at least in part be due to the decreased production of cyclo-oxygenase metabolites of arachidonic acid in human keratinocytes. The present investigation also suggests that prolonged glucocorticoid treatment of the skin would not cause changes in the fatty acid composition of keratinocytes.

The effect of selenium and vitamin E deficiencies on the fate of arachidonic acid in rat isolated lungs.

The fate of exogenous 14C-arachidonic acid (14C-AA) was investigated in the isolated lungs of rats fed selenium and vitamin E deficient diet or diets supplemented with selenium and/or vitamin E. When 80 nmol of 14C-AA was infused into the pulmonary circulation most of the infused 14C-AA was found in different phospholipid and neutral lipid fractions of the perfused lungs. Only less than ten percent of the infused radioactivity was recovered in the perfusion effluent. The amount of arachidonate metabolites in the perfusion effluent was negligible, and most of the radioactivity in the perfusion effluent consisted of unmetabolized arachidonate. Selenium deficiency had no significant effect on the distribution of 14C-AA in different lung lipid fractions. However, in the lungs of vitamin E deficient rats the amount of radioactivity was slightly increased in the neutral lipid fraction, which was due to the increased amount of 14C-AA in the diacylglycerols. The amount of radioactivity was increased especially in the 1,3-diacylglycerols. The amount of radioactivity was increased especially in the 1,3-diacylglycerols. The amount of 14C-AA in the triacylglycerols and in different phospholipids was not significantly changed. The present study might indicate that selenium deficiency has no significant effect on the fate of exogenous arachidonic acid in isolated rat lungs, and that vitamin E deficiency would slightly increase the amount of arachidonic acid in the diacylglycerols.

Carbon monoxide is not responsible for the cigarette smoke-induced changes in the pulmonary metabolism of arachidonic acid and prostaglandin E2.

Cigarette smoke is known to interfere with the pulmonary metabolism of arachidonic acid and prostaglandin E2 (PGE2). We investigated the possible role of carbon monoxide in these cigarette smoke-induced alterations. 14C-Arachidonic acid (50 nmol) was infused into the pulmonary circulation of isolated perfused hamster lungs, and the radioactive metabolites in the perfusion effluent, as well as the distribution of incorporated radioactive arachidonic acid within the lung lipids, were analysed. Carbon monoxide, added into the ventilatory air, had no effect on the oxidative metabolism of arachidonic acid or on the distribution of radioactive arachidonic acid within the lung. In addition, carbon monoxide had no effect on the metabolism of PGE2 following infusion of 100 nmol of 14C-PGE2 into the rat pulmonary circulation. The present study suggests that carbon monoxide is not responsible for the cigarette smoke-induced changes in the pulmonary metabolism of arachidonic acid and PGE2.

The fatty acid composition of 12 North-European fish species.

Cardiovascular diseases among Greenland Eskimos are rare because their diet is rich in fatty fish and marine mammals. The beneficial effect of the fish diet appears to be mediated, at least in part, by the high amount of eicosapentaenoic acid in fish. We investigated the total lipid amount and fatty acid composition of 12 commonly eaten North-European fish species. Most of the detected fatty acids were unsaturated, and the content of eicosapentaenoic acid varied usually between 6 and 16%. The amount of total lipid varied between 3.5 and 216 mg/g wet tissue. The total amount of lipid in different fish species seems to be more important than the respective fatty acid composition when considering which fish should be especially beneficial in the diet. Herring, salmon, Baltic herring, turbot and trout seem to contain most abundantly eicosapentaenoic acid.