Porcine Polymorphonuclear Leukocytes Research Articles

Biosynthesis of eoxin C4 by porcine leukocytes

Human 15-lipoxygenase-1 (LO) possesses mainly 15-lipoxygenase activity whereas the animal ortholog 12/15-LO possesses mainly 12-lipoxygenase activity. These findings have raised the question if studies on animals can predict the function of 15-LO-1 in human. In this study we have characterized the arachidonic acid metabolites formed by porcine 12/15-LO. Mini pigs were infected with a parasite to increase the number of blood eosinophils, which highly express 12/15-LO. Isolated porcine polymorphonuclear leukocytes (PMNL) were incubated with arachidonic acid and the produced metabolites were analysed with HPLC and mass spectrometry (MS). The cells were found to produce 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE at a ratio of 1:5. Furthermore 8,15-dihydroxyeicosatetraenoic acids (DiHETEs) and 14,15-DiHETE were formed. Based on HPLC, UV-spectroscopy and MS analysis it was found that porcine PMNL also produced eoxin (EX) C4. These results demonstrate that although porcine 12/15-LO possesses primarily 12-lipoxygenase activity, the enzyme can catalyse the formation of EXC4.

Pervanadate-induced reverse translocation and tyrosine phosphorylation of phorbol ester-stimulated protein kinase C βII are mediated by Src-family tyrosine kinases in porcine neutrophils

Protein kinase C (PKC), upon activation, translocates from the cytosol to the plasma membrane. Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, is known to induce irreversible translocation of PKC to the plasma membrane, in contrast to the reversible translocation resulting from physiological stimuli and subsequent rapid return to the cytosol (reverse translocation). However, we have previously shown that tyrosine phosphatase (PTPase) inhibitors induce reverse translocation of PMA-stimulated PKCβII in porcine polymorphonuclear leukocytes (PMNs). In the present study, we showed that pervanadate, a potent PTPase inhibitor, also induces tyrosine phosphorylation of PMA-stimulated PKCβII in porcine PMNs. Furthermore, PP2, a specific inhibitor of Src-family tyrosine kinases (PTKs), was found to inhibit both pervanadate-induced reverse translocation and tyrosine phosphorylation of PMA-stimulated PKCβII, suggesting that these two pervanadate-induced responses are mediated by Src-family PTKs. Our findings provide novel insight into the relationship between the subcellular localization and tyrosine phosphorylation of PKC.

Identification of a novel Fc gamma RIIIa alpha-associated molecule that contains significant homology to porcine cathelin.

The following studies are the first to demonstrate the association of porcine FcgammaRIIIaalpha with a molecule that contains significant homology to the cathelin family of antimicrobial proteins. We performed immunoprecipitation of the porcine FcgammaRIIIaalpha multisubunit complex from Brij 96 lysates of polymorphonuclear leukocytes using the G7 mAb, which binds to FcgammaRIIIaalpha on the surface of porcine NK cells and phagocytes. Previous results indicate that the transmembrane alpha subunit of the FcgammaRIIIa complex is associated with the gamma subunit on the surface of porcine polymorphonuclear leukocytes and with several other unique proteins that surface iodinate and migrate at approximately 15, 20, and 25 kDa when analyzed by reducing SDS-PAGE. Through characterization of the porcine FcgammaRIIIa complex, we identified the 15-kDa molecule as a unique FcgammaR-associated protein that has not been described in other systems. We now report an association between FcgammaRIIIaalpha and a 15-kDa molecule that shares homology to cathelin, a protein of undetermined function initially identified in porcine leukocytes. A domain with a high degree of homology to cathelin is found in the proregions of a family of antibiotic proteins referred to as cathelicidins. The results of our studies indicate the presence of a novel FcgammaRIIIa complex in the porcine system, and may provide new insights into the function of this antimicrobial protein homologue in relation to the variety of responses mediated through FcgammaRs.

F1651 fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils

The F1651 fimbrial system has been associated with the resistance of Escherichia coli O115:K “V165” to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F1651 fimbriae. In order to confirm whether or not the F1651 fimbriae are involved in this inhibition, we evaluated the effect of F1651-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F1651-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2−) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F1651-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F1651 fimbriae to the pNs had no effect on the oxidative response.

F165(1) fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils.

The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae. In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.

Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins.

Antimicrobial peptides of several structural classes have been found in phagocytes and epithelial cells of many animals. The broadly microbicidal protegrins (PG1, -2, and -3) were originally isolated as 16 to 18-amino-acid peptides from pig neutrophil lysates, but the corresponding cDNA sequences encoded much larger precursors that belonged to the cathelicidin family of antimicrobial peptides. We explored the storage, secretion, and microbicidal activation of protegrins in porcine neutrophils and in a model system consisting of recombinant proprotegrin 3 (pPG3) and various serine proteases and their inhibitors. Protegrins were stored in neutrophils as inactive proforms that were cleaved by neutrophil elastase to mature protegrins during the preparation of granule lysate and during phorbol myristate acetate-stimulated granule secretion from intact neutrophils. Recombinant pPG3 was efficiently cleaved by trace amounts of human neutrophil elastase or equivalent amounts of elastase activity from porcine neutrophils, but pPG3 was relatively resistant to porcine pancreatic elastase or human neutrophil cathepsin G. The recombinant pPG3 and neutrophil proprotegrins lacked microbicidal activity, but the mature protegrins generated in the elastase-mediated cleavage reaction were as active against Listeria monocytogenes as the chemically synthesized protegrin. The secretion and elastase-mediated activation of proprotegrins accounted for much of the stable microbicidal activity of porcine neutrophil secretions against L. monocytogenes. Secreted proprotegrins and trace amounts of elastase constitute a binary microbicidal system that is likely to contribute to the antimicrobial activity of porcine inflammatory fluids.

Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases

Four different stefin-type cysteine proteinase inhibitors have been isolated from porcine thymus and skin. Amino acid sequence determination revealed the presence of stefin A and stefin B type inhibitors and two new inhibitors, designated as porcine stefin D1 and stefin D2. Stefin D1 was identified as PLCPI, an inhibitor recently characterized from porcine polymorphonuclear leukocytes [Lenarčič et al. (1993) FEBS Lett. 336, 289–292]. Stefin A is composed of 101 amino acids and has an M r of 11391 while stefin B contains 98 amino acids, has an M r of 11174 and is N-terminally blocked. All inhibitors were found to be fast-acting inhibitors of papain, cathepsin L and cathepsin S ( K i = 0.009–0.161 nM). Stefins A and B also bind tightly and rapidly to cathepsin H ( K i = 0.027 and 0.069 nM, respectively), while stefins D1 and D2 have been shown to be very poor inhibitors of cathepsin H ( K i = 102–150 nM). he decreased affinity of these inhibitors toward cathepsin B ( K i = 2–1700 nM) was shown to be mainly due to the low second order association rate constants. The presence of a highly negatively charged N-terminus on stefin D1 constitutes a likely structural determinant of inhibitor specificity.

Dephosphorylation of Cofilin in Polymorphonuclear Leukocytes Derived from Peripheral Blood

We show that human and porcine polymorphonuclear leukocytes express significant amounts of cofilin, a low-molecular-weight actin regulatory protein, as well as profilin. Fifty percent of the cofilin in the resting state was phosphorylated and dephosphorylation occurred after activation by fMLP or TPA. The time course of the dephosphorylation induced by fMLP was very rapid, ending within 1 min, while TPA induced relatively gradual dephosphorylation over a period of 10 min. Surprisingly, okadaic acid and calyculin A, potent inhibitors specific for phosphatase, both induced dephosphorylation of cofilin. This suggests that type 1 alone or both type 1 and 2A phosphatases are involved in the maintenance of the level of phosphorylation of cofilin in resting cells. The dephosphorylation of cofilin was barely detected byin vitrophosphatase assays, which can distinguish activities of types 1, 2B, and 2C. This indicates that cofilin is not dephosphorylated by conventional phosphatases. Although the dephosphorylation of cofilin was observed in cells treated with the calcium ionophore A23187, the phosphorylation level of cofilin was restored when the cells were further incubated in the presence of EGTA. Reactivation of these cells with TPA resulted in the dephosphorylation of cofilin; fMLP activation did not lead to dephosphorylation. Furthermore, a submicromolar concentration of wortmannin, which is an inhibitor specific for phosphatidylinositol 3-kinase, completely inhibited dephosphorylation of cofilin induced by fMLP, but did not suppress TPA-induced dephosphorylation. Thus, we conclude that the dephosphorylation of cofilin is differently regulated depending on either fMLP or TPA activation.

Dose dependent reversal effects of plumbagin on metabolism of arachidonic acid in porcine polymorphonuclear leukocytes

The cytostatic immunostimulating naphthoquinone plumbagin was used as a model to explain the multiple potential of immunostimulants and their interferences with the arachidonic acid metabolism. Plumbagin was found to suppress totally the activity of lipoxygenases and the endogenous production of proinflammatory immunostimulants LTB(4), 5-HETE and 12-HETE in ionophore A-23187 stimulated porcine granulocytes at a concentration of 10(-4) M, whereas it stimulates the AA-metabolism and increases e. g. the 5-lipoxygenase products in a concentration range of 10(-6)-10(-7)M. Surprisingly this stimulating effect is interrupted at a concentration of 10(-8)-10(-9) M resulting in a strong suppression of the biosynthesis of 5-lipoxygenase products. This inhibition might be associated with an increased production of "antiinflammatory" 15-HETE which is known to inhibit 5-lipoxygenase and LTB(4) production. The effects observed for plumbagin support the close linkages of AA metabolism with the unspecific immune system, and might explain the dose dependent antiinflammatory or proinflammatory effects respectively.

Septicemia-inducing Escherichia coli O115:K"V165"F165(1) resists killing by porcine polymorphonuclear leukocytes in vitro: role of F165(1) fimbriae and K"V165" O-antigen capsule.

Escherichia coli O115:K"V165":F165(1) wild-type strain 5131 survives in the bloodstream of experimentally inoculated gnotobiotic pigs and induces septicemia, whereas its afimbriate (F165(1)-negative) TnphoA mutant M48 and its acapsular (K"V165"-negative) spontaneous mutant 5131a are both nonpathogenic. We evaluated the role of the mannose-resistant F165(1) fimbrial system and of the O-antigen K"V165" capsule in resistance to phagocytosis by porcine polymorphonuclear leucocytes (PMNLs) in vitro. F165(1)-positive strains (5131 and 5131a) attached to and were ingested by PMNLs at a significantly higher level than afimbrial mutant M48 (P < 0.001) after 1 h of incubation. During incubation of these strains with PMNLs for up to 6 h, parental strain 5131 resisted killing whereas afimbriate mutant M48 and acapsular mutant 5131a were gradually killed and were found at significantly lower numbers than the parental strain 5131 at 2 (P < 0.05) and 6 (P < 0.001) h. When bacteria were opsonized with normal pig serum, the afimbriate and acapsular mutants survived less well than when the bacteria were nonopsonized. Upon examination by electron microscopy of PMNLs after 2 h of incubation with bacteria, structurally normal bacteria were observed more often within phagosomes of PMNLs incubated with the parental strain than within phagosomes of PMNLs incubated with the afimbriate or the acapsular mutant. The extracellular oxidative response (as tested by release of hydrogen peroxide) of PMNLs stimulated by phorbol myristate acetate was completely inhibited by F165(1)-positive strains but only partially inhibited by the afimbriate mutant. These results suggest that the F165(1) fimbrial system may mediate adherence of E. coli O115 to PMNLs. Survival of the parental strain in the presence of PMNLs, which may be intracellular, is at least partially due to the presence of the F165(1) fimbrial system and of the O-antigen capsule K"V165". Furthermore, the presence of the F165(1) fimbrial system may contribute to the bacterial inhibition of the oxidative response of porcine PMNLs.

Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid to 12-oxo intermediates.

Eicosanoids containing a 12-hydroxyl group preceded by at least two conjugated double bonds are metabolized to 10,11-dihydro and 10,11-dihydro-12-oxo products by porcine polymorphonuclear leukocytes (PMNL) (Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990) Biochemistry 29, 10126-10135). These 10,11-dihydro metabolites could either have been formed by the direct reduction of the 10,11-double bond of the substrate, as previous evidence suggested, or via an initially formed 12-oxo intermediate. To gain some insight into the mechanism for the formation of dihydro products by this pathway, we investigated the metabolism of leukotriene B4 (LTB4), 12(S)-hydroxy-5,8,10,14-eicosatetraenoicacid(12(S)-HETE), and 12(R)-HETE by subcellular fractions from porcine PMNL. In the presence of NAD+ and a microsomal fraction from PMNL, each of the above 12-hydroxyeicosanoids was converted to a single product with a lambda max approximately 40 nm higher than that of the substrate, indicating that the conjugated diene or triene chromophore had been extended by one double bond, presumably by oxidation of the 12-hydroxyl group to an oxo group. In the case of LTB4, this was confirmed by mass spectrometry, which indicated that the product was identical to 12-oxo-LTB4. LTB4 was not converted to any products by a cytosolic fraction from PMNL, but was converted to both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 by the 1500 x g supernatant in the presence of NAD+. Negligible amounts of dihydro products were formed in the presence of NADH or NADPH, suggesting that initial oxidation of the 12-hydroxyl group is a requirement for reduction of the 10,11-double bond. Consistent with this hypothesis, 12-oxo-LTB4 was rapidly metabolized to 10,11-dihydro-12-oxo-LTB4 by the cytosolic fraction in the presence of NADH. Only small amounts of this product, along with some LTB4, were formed by the microsomal fraction. These results indicate that the initial step in the formation of 10,11-dihydro products from 12-hydroxyeicosanoids is oxidation of the 12-hydroxyl group by a microsomal 12-hydroxyeicosanoid dehydrogenase in the presence of NAD+, which is followed by reduction of the olefinic double bond by a cytosolic delta 10-reductase in the presence of NADH.

Immunogenicity of Actinobacillus pleuropneumoniae outer membrane proteins and enhancement of phagocytosis by antibodies to the proteins

To determine the opsonic effect of antibodies to Actinobacillus (Haemophilus) pleuropneumoniae outer membrane proteins on phagocytosis by porcine polymorphonuclear leukocytes (PMN), we separated the integral outer membrane proteins (IOMPs) by Triton X-114 extraction. Four major IOMPs with molecular masses of 76, 50, 39, and 29 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These IOMPs were found to be essentially free of endotoxin in the Limulus amebocyte lysate assay. The 76-kDa protein exhibited a more intensely stained electrophoresis band when isolated from iron-restricted cultures, and a new band at 105 kDa was present in the whole-membrane fraction but not in the integral fraction, indicating that the 105-kDa iron-repressible protein is a peripheral membrane protein. The 76-, 50-, and 39-kDa proteins were shown to be surface exposed, since antibodies to these IOMPs could be absorbed out of convalescent-phase sera by whole cells. Percentages of phagocytosis by porcine PMN of A. pleuropneumoniae opsonized with convalescent-phase sera, convalescent-phase sera absorbed with IOMPs, or convalescent-phase sera absorbed with whole cells were 46.75, 21.81, and 7.96%, respectively. These results demonstrate that antibodies to IOMPs of A. pleuropneumoniae serve as important opsonins in phagocytosis by porcine PMN.

Metabolism of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid and other hydroxylated fatty acids by the reductase pathway in porcine polymorphonuclear leukocytes

We have previously shown that porcine polymorphonuclear leukocytes (PMNL) reduce leukotriene B4 (LTB4) to 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, and 10,11-dihydro-12-oxo-LTB4 [Wainwright et al. (1990) Biochemistry 29, 1180-1185]. We have now demonstrated that 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] is metabolized by a similar pathway in porcine PMNL. 12(S)-HETE was metabolized to two products that were identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy as 12-hydroxy-5,8,14-eicosatrienoic acid (10,11-dihydro-12-HETE) and 12-oxo-5,8,14-eicosatrienoic acid (10,11-dihydro-12-oxo-ETE). Derivatization of 12-hydroxy-5,8,14-eicosatrienoic acid with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid, followed by chromatography on a silicic acid column, enabled the resolution of 12R and 12S stereoisomers, which were identified by cochromatography with synthetic standards. Incubation of 12(S)-HETE with PMNL for various times revealed that the stereochemistry of the 12-hydroxyl group of 12-hydroxy-5,8,14-eicosatrienoic acid was initially the same as that of 12(S)-HETE. However, after 40 min, 30% of the 12-hydroxy-5,8,14-eicosatrienoic acid had the opposite configuration at C12. 13-Hydroxy-9,11-octadecadienoic acid (13-HODE) was metabolized in a similar fashion by porcine PMNL to 13-hydroxy-9-octadecenoic acid (11,12-dihydro-13-HODE) and 13-oxooctadecenoic acid (11,12-dihydro-13-oxo-ODE). The apparent Km values for the reduction of 12-HETE, LTB4, and 13-HODE were 0.21, 0.28, and 2.22 microM, respectively. All three substrates had the same apparent Vmax [0.029 pmol min-1 (10(6) cells)-1]. Competition experiments between LTB4 and 12-HETE indicated that they were metabolized by the same pathway. Various structurally related compounds were metabolized by porcine PMNL in the order LTB4 = 6-trans-LTB4 greater than 12-epi-6-trans,8-cis-LTB4 greater than 12-epi-6-trans-LTB4 greater than 12-HETE greater than LTB5 greater than 15-HETE = 13-HODE much greater than 5-HETE greater than 9-HODE greater than 20-hydroxy-LTB4 greater than 12-hydroxy-5,8,10-heptadecatrienoic acid. Prostaglandins E2 and F2 alpha were not metabolized to any detectable products by porcine PMNL.

Stereochemistry of leukotriene B4 metabolites formed by the reductase pathway in porcine polymorphonuclear leukocytes: inversion of stereochemistry of the 12-hydroxyl group

Leukotriene B4 (LTB4), a potent proinflammatory agent, is a major metabolite of arachidonic acid in polymorphonuclear leukocytes (PMNL). When porcine PMNL were incubated with LTB4 and the products purified by reversed-phase high-pressure liquid chromatography (HPLC), we previously identified two metabolites: 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 [Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369]. Further analysis of the reaction products by normal-phase HPLC has now revealed the presence of a third major metabolite of LTB4. This product is not formed in detectable amounts in the first 5 min of the reaction but accounts for about 20-30% of the reaction products after 60 min, when LTB4 has been completely metabolized. The mass spectrum and gas chromatographic properties of the new metabolite are identical with those of 10,11-dihydro-LTB4, suggesting that it is a stereoisomer of this compound. This product was identified as 10,11-dihydro-12-epi-LTB4 [i.e., 5(S),12(R)-dihydroxy-6,8,14-eicosatrienoic acid] by comparison of its chromatographic properties with those of the authentic chemically synthesized compound. Both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 were enzymatically converted to 10,11-dihydro-12-epi-LTB4 by porcine PMNL, the former compound being the better substrate. The reaction was reversible, since both 10,11-dihydro-12-epi-LTB4 and 10,11-dihydro-12-oxo-LTB4 could be converted to 10,11-dihydro-LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine neutrophil function in the presence of virulent and avirulent Salmonella choleraesuis

Porcine polymorphonuclear leukocytes (PMNLs) may be activated by bacteria to begin phagocytosis followed by oxidative and non-oxidative mechanisms of killing. The purpose of this study was to identify differences between virulent and avirulent Salmonella choleraesuis ( S. choleraesuis) strains, 38 and 9 respectively, in their interactions with porcine PMNLs using five different assays. (1) Staphylococcus aureus ( S. aureus) ingestion was determined by exposure of porcine PMNLs to a mixture of S. choleraesuis and 125I labeled S. aureus. There was a 2.98% and 22.20% decrease in S. aureus ingestion by mouse-avirulent S. choleraesuis 9 and mouse-virulent S. choleraesuis 38 respectively. (2) Iodination of proteins was done by exposing zymosan stimulated porcine PMNLs to S. choleraesuis in the presence of 125I and measuring its incorporation into porcine PMNL proteins. This assay indicated a 73.7% and 74.7% decrease in iodination by S. choleraesuis 9 and S. choleraesuis 38, respectively. (3) Cytochrome c reduction was performed by using porcine PMNLs, zymosan, and S. choleraesuis to determine the bacterial effect on superoxide anion production. S. choleraesuis 9 and S. choleraesuis 38 inhibited superoxide anion production by 78.0% and 92.6%, respectively. (4) Lactoferrin release from porcine PMNLs was measured by an ELISA using the supernatant from the cytochrome c assay. Results indicate a 52.0% and 61.0% increase in lactoferrin release by S. choleraesuis 9 and 38 respectively. (5) The bactericidal assay was performed by counting cfus of S. choleraesuis after preliminary incubation with porcine PMNLs, followed by killing of extracellular S. choleraesuis and lysis of porcine PMNLs. Survival of S. choleraesuis 9 and E. coli (control) were 7.50% and 1.37%, respectively, in contrast to 52.62% survival of the virulent S. choleraesuis 38. These results indicate that both strains inhibited protein iodination and caused a slight increase in lactoferrin release, but the virulent S. choleraesuis 38 inhibited S. aureus ingestion, cytochrome c reduction, and survived porcine PMNL killing more effectively than the avirulent S. choleraesuis 9.

Porcine polymorphonuclear leukocyte NADPH-cytochrome c reductase generates superoxide in the presence of cytochrome b559 and phospholipid

NADPH-cytochrome c reductase and cytochrome b559 were purified from the membrane fraction of phorbol myristate acetate-stimulated porcine polymorphonuclear leukocytes. The highly purified reductase oxidized NADPH and generated superoxide when combined with partially purified cytochrome b559 in the presence of phosphatidylcholine. In the same system, but under the anaerobic condition, the reductase was found to reduce cytochrome b559.

Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

A rapid and sensitive reversed-phase HPLC procedure is reported which allows the simultaneous separation and quantitation of LTC 4, 11 t-LTC 4, LTD 4, LTB 4, 12 epi,6 t,8 c-LTB 4, 6 t-LTB 4 + 12 epi,6 t-LTB 4, two trihydroxy-eicosatetraenoic acids tentatively identified as 20-OH-LTB 4 and 20-OH,12 epi,6 t,8 c-LTB 4 and several not yet identified 15-series leukotrienes produced by the cytosol of porcine polymorphonuclear leukocytes.

Arachidonate 12-lipoxygenase of porcine leukocyte with activity for 5-hydroxyeicosatetraenoic acid

When arachidonic acid was incubated with porcine polymorphonuclear leukocytes, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was produced as a major product. The production of this compound was not accounted for by contaminating platelets known to contain arachidonate 12-lipoxygenase. The enzyme was found predominantly in a cytosol fraction of leukocytes, and the cytosolic enzyme was purified about 30-fold by ammonium sulfate fractionation and DEAE-Sephadex column chromatography. The partially purified enzyme transformed arachidonic acid to a compound which was identified as 12-hy droperoxy-5,8,10,14-eicosatetraenoic acid. 5-Hydroxy-6,8,11,14-eicosatetraenoic acid as substrate was about 40% as active as arachidonic acid, and the reaction was inhibited by the addition of arachidonic acid. The reaction product which was further reduced by sodium borohydride was indistinguishable from authentic 5 S, 12 S-dihydroxy-( E, Z, E, Z)-6,8,10,14-eicosatetraenoic acid.