Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in ...

Molecular Human Reproduction vol.5 no.7 pp. 668–674, 1999

Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term

N.L.Brown, D.M.Slater, S.A.Alvi, M.G.Elder, M.H.F.Sullivan1 and P.R.Bennett Department of Maternal and Fetal Medicine, Division of Paediatrics, Obstetrics and Gynaecology, Imperial College School of Medicine, Queen Charlottes and Chelsea Hospital, Goldhawk Road, London W6 0XG, UK 1To

whom correspondence should be addressed

Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion–decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion–decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion–decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy. Key words: 5-LOX/chorion–decidua/eicosanoids/FLAP/labour

Introduction There is now substantial evidence for the involvement of arachidonic acid metabolites of the cyclo-oxygenase pathway, particularly prostaglandins (PG) E2 and F2α, in the biochemical mechanisms of human parturition (Ellwood et al., 1980; Okazaki et al., 1981; Challis and Olson, 1988). Arachidonic acid can also be metabolized by the lipoxygenase pathways to generate various products that include hydroperoxyeicosatetraenoic acids (HPETEs) which are rapidly reduced to give hydroxyeicosatetraenoic acids (HETEs) and, in the case of 5-HPETE, to leukotrienes (LTs). In association with labour, there is an increase in arachidonic acid metabolism by the cyclooxygenase pathway compared to the lipoxygenase pathways in amnion cells which increases PGE2 release (Bennett et al., 1993). However, there may also be an increase in the expression of lipoxygenase enzymes, as there is growing evidence suggesting a role for lipoxygenase metabolites in the parturitional process. Lipoxygenase metabolites can be synthesized by human intrauterine tissues (Saeed and Mitchell, 1982), and have been found to be involved in myometrial contractions (Carraher et al., 1983; Ritchie et al., 1984). The concentrations of three products of lipoxygenase metabolism (12-HETE, 15-HETE, and LTB4) in amniotic fluid have been found to be significantly higher in patients at term in active labour than in those at term but not in labour (Romero et al., 1987a). The levels of arachidonate lipoxygenase metabolites in amniotic fluid are also affected differently by the presence of infection and labour in women with premature rupture of membranes 668

(Romero et al., 1987b), such that LTB4 concentrations are increased by both infection and labour, and additively by the combination. 15-HETE concentrations increased only in women with both labour and infection, and 12-HETE was unaffected in all patients, indicating that differential processes control lipoxygenase metabolite production in human pregnancy. The levels of LTC4 have also been found to increase in amniotic fluid in women undergoing labour, compared with women not undergoing labour (Romero et al., 1988). The 5-LOX product 5-HETE has been related to myometrial contractures (weak contractions which precede labour) in the rhesus monkey (Walsh et al., 1986; Walsh 1989). 5-HETE has also been shown to produce a dose-dependent increase in human myometrial contractility whereas the 12-LOX product 12-HETE had no effect (Bennett et al., 1987a). These results suggest there may be a change in the expression of 5-LOX with parturition. There may also be a complex interaction between lipoxygenase and cyclo-oxygenase products during human labour. LTB4 can inhibit the release of PGE2 from term fetal membranes (Ticconi et al., 1995), and may thus facilitate pregnancy. Oxytocin increased PGE2 output, but not LTB4 production (Ticconi et al., 1998), which suggests that a simple release of substrate does not cause a general increase in eicosanoid production. There is a switch from lipoxygenase to cyclooxygenase products in labour (Rose et al., 1990), and bacteria have been shown to have a similar effect on amnion cells in vitro (Bennett et al., 1987b). Finally, progesterone and glucocorticoids can stimulate the production of LTB4 (but not © European Society of Human Reproduction and Embryology

5-LOX and FLAP expression during pregnancy and labour

Figure 1. Reverse transcription–polymerase chain reaction (RT–PCR) analyses investigating the expression of 5-LOX, FLAP and GAPDH mRNA in amnion (A), chorion–decidua (CD) and placenta (P) at term before labour. The left-hand lane represents a 100 bp ladder marker. For the products shown, 32 cycles were used for each reaction.

Figure 3. The mRNA expression of FLAP in (A) amnion and (B) chorion–decidua in samples taken at different gestational ages. The levels of FLAP mRNA are shown as a ratio relative to the level of GAPDH mRNA in each sample. Results are expressed as mean 6 SEM. *Significant difference (P , 0.05) in the level of FLAP mRNA expression in the chorion–decidua, between the first and third trimesters of pregnancy. Sample numbers in each group are shown on the figure. The number of polymerase chain reaction (PCR) cycles used was 34 for the amnion and the chorion–decidua.

Figure 2. The mRNA expression of 5-LOX in (A) amnion and (B) chorion–decidua in samples taken at different gestational ages. The values of 5-LOX mRNA are shown as a ratio relative to the level of GAPDH mRNA in each sample. Results are expressed as mean 6 SEM. *Significant difference (P , 0.05) in the level of 5-LOX expression in the chorion–decidua between different trimesters of pregnancy. Sample numbers in each group are shown on the figure. The number of polymerase chain reaction (PCR) cycles used was 34 for the amnion and 30 for the chorion–decidua.

PGE2) from fetal membrane explants (Zicari et al, 1997). Taken together, these findings indicate that increased release of LTB4 may (before the onset of labour) have certain effects on pregnancy, but it is not clear whether this persists into labour. 5-Lipoxygenase (5-LOX) is a member of a family of lipoxygenases which include 12-LOX and 15-LOX and is part of the primary pathway of LT production. 5-LOX catalyses both the first step in the oxygenation of arachidonic acid to produce 5-HPETE and the second dehydration step to produce LTA4. LTA4 can then be metabolized by other enzymes to generate biologically active compounds, including LTB4, which has been implicated as a potential mediator of inflammation, and LTs C4, D4, and E4 which are smooth muscle contractile agents. The cDNA sequence for 5-LOX has been cloned and its amino acid sequence deduced (Matsumoto et al., 1988). 5-LOX is a 78 kDa protein with a requirement for Ca21 and ATP. The activation of 5-LOX involves translocation from the cytosol to the membrane fraction (Rouzer and Kargman 1988). This activation is also dependent on a 5-lipoxygenase669

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Figure 4. (A) Immunoblot showing the expression of 5-LOX in first trimester amnion and chorion samples collected from two patients after termination of pregnancy. A 5 amnion; C 5 chorion; P/C 5 placenta/chorion. (B) Graphical representation showing the signal intensity for 5-LOX relative to that for β-actin.

activating protein (FLAP) (Dixon et al., 1990). The cDNA sequence of FLAP is known (Dixon et al., 1990) and is an 18 kDa integral membrane protein which activates 5-LOX by specifically binding arachidonic acid and transferring it to the enzyme (Mancini et al., 1993). Immunoreactive 5-LOX has been localized in human myometrium, placental syncytiotrophoblasts, decidua and amniotic membrane from patients after normal labour and was found to be lower in myometrial smooth muscle cells in patients who did not respond to labour induction (Faber et al., 1996). This evidence for a role for lipoxygenase metabolites in parturition suggests that the expression of 5-LOX and FLAP may change during pregnancy and/or at term. Therefore, this study examined the expression of 5-LOX and FLAP in amnion and chorion–decidua samples taken throughout gestation, and at term before and after labour.

Materials and methods Amnion and chorion–decidua samples were collected at various gestational ages from nine patients undergoing termination of pregnancy (8–18 weeks), from nine preterm deliveries by Caesarean section for fetal distress (25–36 weeks), from 11 term deliveries in the absence of labour (elective caesarean section) and from 12 patients after labour (normal vaginal deliveries) (37–41 weeks for both groups). Any tissues showing evidence of chorioamnionitis were not included in this study. Only patients from the last group underwent

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clinically recognizable labour. It was not always possible to collect recognizable paired samples of amnion and chorion–decidua from material following termination of pregnancy. Gestational ages were calculated from the last menstrual period, and are given to the nearest completed week of pregnancy. Ethics committee approval was obtained for tissues which would normally be discarded and all patients undergoing pregnancy termination gave written consent. The tissues were snap frozen in liquid nitrogen and stored at –70°C until use. Total RNA was extracted using a standard guanidinium–isothiocyanate technique (Chomczynski and Sacchi, 1987) and stored at –80°C until use. RNA samples (1 µg) were denatured at 70°C for 5 min and cooled to 37°C. Reverse transcription (RT) was carried using 13 first strand buffer (Gibco; Gibco BRL Life Technologies Ltd, Paisley, UK), 0.25 µg random hexanucleotide primers (Pharmacia; Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK), 10 mM dithiothreitol, 1 mM each deoxynucleotide triphosphate (Gibco), 1 IU RNase inhibitor (Pharmacia) and 40 IU M-MLV (Maloney murine leukaemia virus) reverse transcriptase (Gibco) for 60 min at 37°C in a reaction volume of 20 µl. The reaction was stopped by incubation at 90°C for 4 min, and the resultant cDNA stored at –80°C until polymerase chain reaction (PCR) amplification. The primers for the polymerase reaction were for 5-lipoxygenase: 59-ATC AGG ACG TTC ACG GCC GAG G-39 (sense) and 59-CCA GGA ACA GCT CGT TTT CCT G-39 (antisense); 5-lipoxygenaseactivating protein (FLAP): 59-ATG GAT CAA GAA ACT GTA CGC39 (sense) and 59-ATG AGA AGT AGA GGG GGA GAT G-39 (antisense) (Bonnet et al., 1995). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were as described previously (Tso et al., 1985). PCR was performed in a volume of 25 µl containing 1:20 volume of the cDNA, 13 NH4 buffer (Bioline, London, UK), 1.5 mM MgCl2, 0.2 mM dNTP, 62.5 ng each sense and antisense primer and 0.5 IU Taq polymerase (Bioline). Following an initial denaturation step of 2 min at 94°C, target cDNA was amplified by denaturing at 94°C for 30 s, primer annealing (68°C for 5-LOX; 62°C for FLAP; 58°C for GAPDH) for 30 s and primer extension at 72°C for 30 s. Cycle profiles were performed as described previously to ensure that the exponential phase was used to amplify the products (Slater et al., 1995). The cycle numbers chosen are indicated in the figure legends. RT–PCR products for both FLAP and 5-LOX were subcloned into pGEM-Teasy vector (Promega, Southampton, UK), and verified by double stranded sequencing. These subcloned products were used as probes for dot-blot hybridization as described below. To quantify the PCR, 5 µl of each PCR reaction was dotted onto Hybond nylon filters (Amersham Pharmacia Biotech). The filters were first washed in denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralized (1.5 M NaCl, 0.5 M Tris–HCl pH 7.5) and finally in 33 sodium citrate buffer [203 sodium chloride/sodium citrate (SSC):0.3 M NaCl, 0.3 M sodium citrate pH 7.0] for 5 min each. The DNA was fixed to the filters by UV cross-linking. Filters were first prehybridized for 1–2 h at 65°C followed by hybridization overnight with the appropriate [32P]-dCTP-labelled cDNA probe at 65°C. Excess probe was removed by washing in a series of SSC buffer washes containing 0.1% SDS from 33 to 0.13 SSC. The levels of cDNA were then determined by β-counting of the filter sections. The expression of 5-LOX and FLAP were expressed as a ratio relative to the expression of GAPDH.

Immunoblot analysis for 5-lipoxygenase protein (5-LOX) Tissue samples were allowed to thaw in 0.5ml of homogenization buffer (10 mM Tris–HCl pH 7.4, 1 mM EDTA, and 1 mM sodium orthovanadate) containing the protease inhibitors pepstatin A (1 µM), phenylmethylsulphonyl fluoride (PMSF, 0.1 mM), and E-64 (trans-


Figure 5. (A) Immunoblot showing protein expression of 5-LOX in samples of amnion and chorion–decidua taken from three patients before the onset of labour and three patients after normal labour. A 5 amnion; CD 5 chorion–decidua. (B) Graphical representation showing the signal density for 5-LOX relative to the density of β-actin (n 5 3). Results are expressed as mean 6 SEM. *Significant difference (P , 0.05) in 5-LOX protein expression between groups.

epoxysuccinyl L-leucylamido-(4-guanidino)butane) (10 µM). The tissue was homogenized in short bursts on ice with a tissue homogenizer. The total lysate was then centrifuged for 10 min at 13000 g to sediment the cell debris. Protein concentrations in the supernatant were determined by a Bradford assay using bovine serum albumin (BSA) as a standard and 200 µg of each sample were loaded on a 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel. To improve the resolution of the protein bands, 67 mM imidazole was included in both the stacking gel and the sample buffer (Rittenhouse and Marcus, 1984). A sample of cell lysate from U937 cells was also included on each gel as a positive control for 5-LOX. The proteins were then electrophoretically transferred at 350 mA for 4 h to a nitrocellulose filter in transfer buffer containing 16.5 mM Tris, 191 mM glycine and 20% methanol. The filter was then blocked for 2 h in phosphate-buffered saline (PBS) containing 0.1% Tween and 5% non-fat milk. 5-LOX protein was detected by incubation of the filter overnight at 4°C with a mouse monoclonal antibody (RDILIPOXY5abm; Research Diagnostics Inc, Flanders, NJ, USA) which was diluted to 1 µg/ml in PBS–0.1% Tween containing 1% non-fat milk. This was followed by a 1 h incubation with an anti-mouse IgG antibody labelled with peroxidase (Sigma, Dorset, UK). Immunolocalized proteins were detected using a chemi-luminescent detection system (ECL; Amersham Pharmacia Biotech). The filters were also incubated with an β-actin goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:2000 dilution) and then an anti-goat peroxidase labelled antibody (Santa Cruz) (1:1000 dilution) to verify equal loading of the samples.

Analysis of immunoblots The autoradiographs of each blot were scanned by a UMAX Mirage D-16L scanner and the integrated density of each band determined using the software package Whole Band Analyser (Bioimage Services, Genomic Solutions Inc, Ann Arbor, MI, USA). The integrated density for 5-LOX was then expressed relative to the density of β-actin for each sample. Statistical analysis Results were analysed by two-way analysis of variance, followed by Fisher’s exact test, or by the Mann–Whitney U-test (for non-parametric data). P , 0.05 was considered to be statistically significant.

Results Preliminary studies showed that mRNA for 5-LOX and for FLAP were expressed in amnion, chorion–decidua, and placenta tissues (Figure 1). The expression of both 5-LOX and FLAP mRNA appeared to be higher in the chorion–decidua and placenta compared with the amnion at term. To determine whether there were any changes in the expression of FLAP or 5-LOX mRNA either throughout pregnancy or in association with labour onset at term, semi-quantitative RT–PCR was performed as described in the Materials and methods section. The exponential phase of amplification occurred between 30–34 cycles and linearized at 36–38 cycles. 671

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Figure 6. Expression of 5-LOX mRNA in (A) amnion prior to labour (n 5 11) and post-labour (n 5 11) and (B) chorion–decidua before labour (n 5 11) and after labour (n 5 12). The 5-LOX mRNA levels are shown as a ratio relative to GAPDH mRNA. Results are expressed as mean 6 SEM. *Significant difference between the non-labour and post-labour 5-LOX mRNA expression in chorion–decidua where P 5 0.0096. The number of polymerase chain reaction (PCR) cycles used was 30 cycles for both the amnion and chorion–decidua.

There were no apparent changes in the amnion throughout gestation in the expression of either 5-LOX or FLAP mRNA (Figures 2A and 3A). However, the expression of 5-LOX and FLAP mRNA in the chorion–decidua declined with increasing gestational age; comparison across the three trimesters of pregnancy showed that the 5-LOX mRNA values decreased during the second and third trimesters (Figure 2B), whereas the values of FLAP mRNA (Figure 3B) differed significantly between the first and third trimesters. Expression of 5-LOX protein was found to be very low in both amnion and chorion– decidua collected at 9–10 weeks as an ECL exposure time of 16 h was required in order to detect the immunolocalized proteins (Figure 4). This was different to tissue obtained at term where 5-LOX protein could be detected following an ECL exposure time of 4 min suggesting that expression is much higher at term compared to the first trimester (Figure 5). The expressions of 5-LOX and FLAP mRNA at term before and after labour were also compared. No significant changes were found in the expressions of either transcript in the amnion 672

Figure 7. Expression of FLAP mRNA in (A) amnion prior to labour (n 5 11) and post-labour (n 5 10) and (B) chorion–decidua before labour (n 5 11) and after labour (n 5 12). The FLAP mRNA levels are shown as a ratio relative to GAPDH mRNA. Results are expressed as mean 6 SEM. *Significant difference between the non-labour and post-labour FLAP mRNA expression in chorion–decidua where P 5 0.032. The number of PCR cycles used were 33 for the amnion and 30 for the chorion–decidua.

between the non-labour and post-labour groups (Figures 6A and 7A). Significant increases however were found in both 5-LOX (P 5 0.01) and FLAP (P 5 0.032) mRNA expression in the chorion–decidua following labour with ~3-fold increases in both (Figures 6B and 7B). 5-LOX protein concentrations increased in chorion–decidua in association with labour (Figure 5B); but no such change in the amnion concentrations of 5-LOX protein was observed.

Discussion The present study demonstrates for the first time that human amnion, chorion–decidua and placenta express the transcript for FLAP. The expression of both 5-LOX and FLAP transcripts in the chorion–decidua appear to be significantly higher in the first trimester of pregnancy compared to the rest of pregnancy up to 36 weeks gestation. These two proteins are functionally dependent on one another (Dixon et al., 1990) and the observation that the expression of both changes at the same time suggests that they may play a role early in pregnancy and are then switched off until nearer term. Leukotrienes B4, C4, D4 and E4 have been shown to increase vascular permeability in


other tissues (Sugio et al., 1986; Huang et al., 1993; Yoshimura et al., 1994). A possible role for 5-LOX in early pregnancy may be in regulating vascular permeability in the decidua and the chorion. Leukotrienes may also regulate the development of the placental vascular bed (Parisi and Walsh 1986). There is a decrease in 5-LOX and FLAP production during the second trimester which may be because leukotrienes can promote uterine contractility. There was a marked increase in values of 5-LOX mRNA at term in chorion–decidua, compared with earlier in the third trimester. The same numbers of PCR cycles were used for these samples, enabling a direct comparison to be made. 5-LOX protein was also readily detectable at term. This suggests that there is an induction of mRNA for 5-LOX before labour, and a further induction in association with labour. An increase in mRNA prior to labour, and a further increase associated with labour has been reported for COX-2 (Slater et al., 1999). A recent paper (Brown et al., 1998) has shown that COX-2 expression and activity may be maximal prior to clinical labour, showing that this induction is not caused by the process of labour. The data on 5-LOX and FLAP do not allow us to draw such definite conclusions, although 5-LOX mRNA appeared to increase before labour. Differences in the expression of mRNA of 5-LOX and FLAP were also found at term when comparing tissues collected before and after labour. We found there was a significant increase in the values of 5-LOX and FLAP mRNA in the chorion–decidua after labour compared to before labour. The expression of 5-LOX protein was also increased after labour in the chorion–decidua. 5-LOX and FLAP genes may respond to similar transcription signals as their mRNA levels have been found to increase concurrently (Reid et al., 1990). The FLAP gene promoter region contains possible motifs for AP-2 binding and a glucocorticoid-responsive element (GRE) (Kennedy et al., 1991). The 5-LOX gene promoter contains several response elements which include a Sp1 site, an AP-2site, and a NF-kB-binding sequence (Hoshiko et al., 1990). The changes in mRNA or protein values identified in this study may not necessarily reflect changes in the synthesis of 5-LOX products. Substrate availability, intracellular calcium concentrations, and subcellular distribution may all affect overall enzyme activity (Edwin et al., 1995), and cannot be investigated by the approach taken in this study. However, it has been established that 5-LOX products are increased during human parturition (Romero et al., 1987a, 1988), which is consistent with the marked increase in expression of 5-LOX at term in chorion–decidua. FLAP mRNA expression increased only in association with labour, which may suggest activation of the enzyme during labour. The 5-LOX product, 5-HETE, may have another role in the parturitional process, in addition to the stimulation of myometrial contractions (Walsh et al., 1986; Bennett et al., 1987a). HETEs have chemokinetic and chemotaxic effects on white blood cells (Stenson and Parker 1984). The numbers of mononuclear phagocytes increase in human peripheral blood in association with labour (Buchan et al., 1985), and the cytotoxic activity of human lymphocytes increases significantly at the time of labour (Szekeres-Bartho et al., 1986). It has

been postulated (Walsh, 1989) that the intrauterine production of HETEs may act as a signal for the recruitment of white blood cells to the uterus and then activate them, in order to increase prostaglandin and leukotriene production, and to act as a defence against entry of infection into the uterus during or after delivery.

Acknowledgements The authors would like to thank Action Research (NLB) and WellBeing (SAA, DMS) for supporting this work. We also gratefully acknowledge Dr Steve Dilworth for the use of his image scanner and the Bioimage Whole Band Analyser software.

References Bennett, P.R., Elder, M.G. and Myatt, L. (1987a) The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility. Prostaglandins, 33, 837–844. Bennett, P.R., Rose, M.P., Elder, M.G. et al. (1987b) Preterm labor: stimulation of arachidonic acid metabolism in human amnion cells by bacterial products. Am. J. Obstet. Gynecol., 156, 649–655. Bennett, P.R., Slater, D., Sullivan, M. et al. (1993) Changes in amniotic arachidonic acid metabolism associated with increased cyclo-oxygenase gene expression. Br. J. Obstet. Gynaecol., 100, 1037–1042. Bonnet, C., Bertin, P., Cook-Moreau, J. et al. (1995) Lipoxygenase products and expression of 5-lipoxygenase-activating protein in human cultured synovial cells. Prostaglandins, 50, 127–135. Brown, N.L., Alvi, S.A., Elder, M.G. et al. (1998) A spontaneous induction of fetal membrane prostaglandin production precedes clinical labour. J. Endocrinol., 157, R1–R6. Buchan, G.S., Gibbins, B.L. and Griffin, J.F. (1985) The influence of parturition on peripheral blood mononuclear phagocyte subpopulations in pregnant women. J. Leukoc. Biol., 37, 231–242. Carraher, R., Hahn, D.W., Ritchie, D.M. et al. (1983) Involvement of lipoxygenase products in myometrial contractions. Prostaglandins, 26, 23–32. Challis, J.R.G. and Olson, D.M. (1988) Parturition. In Knobil, E. and Neill, J. (eds), The Physiology of Reproduction. Raven Press, New York, USA, pp. 2177. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by guanidinium-thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156–159. Dixon, R.A.F., Diehl, R.E., Opas, R. et al. (1990) Requirement of a 5lipoxygenase-activating protein for leukotriene synthesis. Nature, 343, 282–284. Edwin, S.S., Thai, D., LaMarche, S. et al. (1995) The regulation of arachidonate lipoxygenase metabolite formation in cells derived from intrauterine tissues. Prostaglandins Leukot. Essent. Fatty Acids, 52, 229–233. Ellwood, D.A., Mitchell, M.D. and Anderson, A.B.M. (1980) The in vitro production of prostanoids by the human cervix during pregnancy: preliminary observations. Br. J. Obstet. Gynaecol., 87 210–214. Faber, B.M., Metz, S.A. and Chegini, N. (1996) Immunolocalization of eicosinoid enzymes and growth factors in human myometrium and fetoplacental tissues in failed labor inductions. Obstet. Gynecol., 88, 174–179. Hoshiko, S., Radmark, O. and Samuelsson, B. (1990) Characterization of the human 5-lipoxygenase gene promoter. Proc. Natl Acad. Sci. USA, 87, 9073–9077. Huang, A.J., Manning, J.E., Bandak, T.M. et al. (1993) Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J. Cell. Biol., 120, 1371–1380. Kennedy, B.P., Diehl, R.E., Boie, Y. et al. (1991) Gene characterisation and promoter analysis of the human 5-lipoxygenase-activating protein (FLAP). J. Biol. Chem., 266, 8511–8516. Mancini, J.A., Abramovitz, M., Cox, M.E. et al. (1993) 5-lipoxygenaseactivating protein is an arachidonate binding protein. FEBS Letts., 318, 277–281. Matsumoto, T., Funk, C.D., Radmark, O. et al. (1988) Molecular cloning and amino acid sequence of human 5-lipoxygenase. Proc. Natl. Acad. Sci. USA, 85, 26–30.

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N.L.Brown et al. Okazaki, T., Casey, M.L., MacDonald, P.C. et al. (1981) Initiation of human parturition. XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua. Am. J. Obstet. Gynecol., 139, 373–381. Parisi, V.M. and Walsh, S.W. (1986) Arachidonic acid metabolites and the regulation of placental and other vascular tone during pregnancy. Semin. Perinatol., 10, 288–298. Reid, G.K., Kargman, S., Vickers, P.J. et al. (1990) Correlation between expression of 5-lipoxygenase-activating protein, 5-lipoxygenase, and cellular leukotriene synthesis. J. Biol. Chem., 265, 19818–19823. Ritchie, D.M., Hahn, D.W. and McGuire, J.L. (1984) Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid. Life Sci., 34, 509–513. Rittenhouse, J. and Marcus, F. (1984) Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl–prolyl peptide bonds in sodium dodecyl sulfate-containing buffers. Anal. Biochem., 138, 442–448. Romero, R., Emamian, M., Wan, M. et al. (1987a) Increased concentrations of arachidonic acid lipoxygenase metabolites in amniotic fluid during parturition. Obstet. Gynaecol., 70, 849–851. Romero, R., Quintero, R., Emamian, M. et al. (1987b) Arachidonate lipoxygenase metabolites in amniotic fluid of women with intra-amniotic infection and preterm labor. Am. J. Obstet. Gynecol., 157, 1454–1460. Romero, R., Wu, Y.K., Mazor, M. et al. (1988) Increased amniotic fluid leukotriene C4 concentration in term human parturition. Am. J. Obstet. Gynecol., 159, 655–657. Rose, M.P., Myatt, L. and Elder, M.G. (1990) Pathways of arachidonic metabolism in human amnion cells at term. Prostaglandins Leukot. Essent. Fatty Acids, 39, 303–309. Rouzer, C.A. and Kargman, S. (1988) Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. J. Biol. Chem., 263, 10980–10988. Saeed, S.A. and Mitchell, M.D. (1982) Formation of arachidonate lipoxygenase metabolites by human fetal membranes, uterine decidua vera, and placenta. Prostaglandins Leukot. Med., 8, 635–640. Slater, D.M., Berger, L.C., Newton, R. et al. (1995) Expression of cyclooxygenase types 1 and 2 in human fetal membranes at term. Am. J. Obstet. Gynecol., 172, 77–82. Slater, D., Dennes, W., Sawdy, R. et al. (1999) Expression of cyclooxygenase types 1 and 2 throughout pregnancy. J. Mol. Endocrinol., 22, 125–130. Stenson, W.F. and Parker, C.W. (1984) Leukotrienes. Adv. Intern. Med., 30, 175–199. Sugio, K., Tsurufuji, S. and Daly, J.W. (1986) Effects of forskolin and prostaglandin E1 on leukotriene C- and D-induced plasma exudation in the rat skin. Life Sci., 39, 229–233. Szekeres-Bartho, J., Varga, P. and Pacsa, A.S. (1986) Immunologic factors contributing to the initiation of labor lymphocyte reactivity in term labor and threatened preterm delivery. Am. J. Obstet. Gynecol., 155, 108–112. Ticconi, C., Zicari, A., Pontieri, G. et al. (1995) Release of arachidonic acid metabolites by human fetal membranes: interrelationship between leukotriene B4 and prostaglandin E2. Prostaglandins, 49, 197–204. Ticconi, C., Mauri, A., Zicari, A. et al. (1998) Interrelationships between oxytocin and eicosanoids in human fetal membranes at term gestation: which role for leukotriene B4? Gynecol. Endocrinol., 12, 129–134. Tso, J.Y., Sun, X.H., Kao, T.H. et al. (1985) Isolation and characterisation of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res., 13, 2485–2502. Walsh, S.W., Young, S.R. and Stockmar, E.J. (1986) Increased 5-lipoxygenase activity precedes labour. [Abstr. no. 31] In 33rd Annual Meeting for the Society for Gynecologic Investigation.. Walsh, S.W. (1989) 5-Hydroxyeicosatetraenoic acid, leukotriene C4, and prostaglandin F2α in amniotic fluid before and during term and preterm labour. Am. J. Obstet. Gynecol., 161, 1352–1360. Yoshimura, K., Nakagawa, S., Koyama, S. et al. (1994) Roles of neutrophil elastase and superoxide anion in leukotriene B4-induced lung injury in rabbit. J. Appl. Physiol., 76, 91–96. Zicari, A., Ticconi, C., Pontieri, G. et al. (1997) Effects of glucocorticoids and progesterone on prostaglandin E2 and leukotriene B4 release by human fetal membranes at term gestation. Prostaglandins, 54, 539–547. Received on November 20, 1998; accepted on April 8, 1999

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