Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters.

Human leukotriene C(4) synthase (LTC(4)S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions. It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC(4)S. Here, we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC(4)S. Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.

Gender differences in the long-term outcomes after valve replacement surgery.

OBJECTIVE: To compare the long-term outcomes in women and men after valve replacement surgery. DESIGN: Observational study. SETTING: Postoperative aortic valve replacement (AVR) or mitral valve replacement (MVR). PATIENTS: 3118 patients (1261 women, 1857 men) who underwent AVR or MVR between 1976 and 2006 (2255 AVR, 863 MVR), with mean follow-up of 5.6 (4.5) years. MAIN OUTCOME MEASURES: The independent effect of gender on the risk of long-term complications (reoperation, stroke and death) after valve replacement surgery using multivariate actuarial methods. RESULTS: After implantation of an aortic valve bioprosthesis, women had a significantly lower rate of reoperation compared to men (comorbidity-adjusted hazard ratio (HR) 0.4; 95% confidence intervals (CI) 0.2 to 0.9). In contrast, if an aortic mechanical prosthesis had been implanted, women were more at risk for late stroke compared to men (HR 1.7; CI 1.1 to 2.7). After adjustment for age and co-morbidities, women had significantly better long-term survival compared to men after bioprosthetic AVR (HR 0.5; CI 0.3 to 0.6), but there was no survival difference between genders after mechanical AVR. Trends existed towards better survival for women after bioprosthetic MVR (HR 0.6; CI 0.4 to 1.0) and mechanical MVR (HR 0.8; CI 0.5 to 1.1). CONCLUSION: The long-term outcomes after valve replacement surgery differ between women and men. Although women have more late strokes after valve replacement, they undergo fewer reoperations and have better overall long-term survival compared to men.

Cysteinyl leukotrienes mediate the enhancing effects of indomethacin and aspirin on eosinophil production in murine bone marrow cultures.

BACKGROUND: Prostaglandin E(2) (PGE(2)) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone-marrow cultures. Because cysteinyl leukotrienes (cys-LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone-marrow were due to blockade of PGE(2) production alone, or involved further promotion of cys-LTs production/signalling. EXPERIMENTAL APPROACH: BALB/c liquid bone-marrow cultures were established with IL-5, alone or associated with indomethacin, aspirin, or cys-LTs. The effects of preventing cys-LT production or signalling were assessed. KEY RESULTS: Indomethacin and aspirin counteracted the suppression of eosinophil production by exogenous PGE(2). LTD(4), LTC(4) and LTE(4) enhanced IL-5-dependent eosinophil production and further counteracted the effect of exogenous PGE(2). The 5-lipoxygenase activating protein (FLAP) inhibitor, MK886, a leukotriene synthesis inhibitor, zileuton, the CysLT(1) receptor antagonists, MK571 and montelukast, or inactivation of the LTC(4) synthase gene, abolished effects of indomethacin and aspirin. MK886 and zileuton were ineffective but MK571 and montelukast were effective, against LTD(4). Indomethacin, aspirin and LTD(4) failed to enhance eosinophil production in bone-marrow from CysLT1 receptor-deficient mice. Indomethacin, aspirin and LTD(4) no longer counteracted the effects of exogenous PGE(2) in the presence of MK571 and montelukast. MK886, MK571 and montelukast had no effect by themselves, or in association with PGE(2). CONCLUSIONS AND IMPLICATIONS: Dependence on the FLAP/5-lipoxygenase/LTC(4) synthase pathway and receptor signalling shows that cyclo-oxygenase inhibitors act here through endogenous cys-LTs. While PGE(2) does not act by suppressing cys-LT production, cys-LTs override PGE(2) signalling. Eosinophil production is therefore coordinately regulated by both pathways.

Expression of 5-lipoxygenase and cyclooxygenase pathway enzymes in nasal polyps of patients with aspirin-intolerant asthma.

In aspirin-intolerant subjects, adverse bronchial and nasal reactions to cyclooxygenase (COX) inhibitors are associated with over-production of cysteinyl-leukotrienes (cys-LTs) generated by the 5-lipoxygenase (5-LO) pathway. In the bronchi of patients with aspirin-intolerant asthma, we previously linked cys-LT over-production and aspirin hyper-reactivity with elevated immunoexpression in eosinophils of the terminal enzyme for cys-LT production, LTC4 synthase. We investigated whether this anomaly also occurs in the nasal airways of these patients. Immunohistochemical expression of 5-LO and COX pathway proteins was quantified in nasal polyps from 12 patients with aspirin-intolerant asthma and 13 with aspirin-tolerant asthma. In the mucosa of polyps from aspirin-intolerant asthmatic patients, cells immunopositive for LTC4 synthase were four-fold more numerous than in aspirin-tolerant asthmatic patients (p=0.04). There were also three-fold more cells expressing 5-LO (p=0.037), with no differences in 5-LO activating protein (FLAP), COX-1 or COX-2. LTC4 synthase-positive cell counts correlated exclusively with mucosal eosinophils (r=0.94, p<0.001, n=25). Co-localisation confirmed that five-fold higher eosinophil counts (p=0.007) accounted for the increased LTC4 synthase expression in polyps from aspirin-intolerant asthmatic patients, with no alterations in mast cells or macrophages. Within the epithelium, increased counts of eosinophils (p=0.006), macrophages (p=0.097), and mast cells (p=0.034) in aspirin-intolerant asthmatic polyps were associated only with 2.5-fold increased 5-LO-positive cells (p<0.05), while the other enzymes were not different. Our results indicate that a marked over-representation of LTC4 synthase in mucosal eosinophils is closely linked to aspirin intolerance in the nasal airway, as in the bronchial airways.

A prospective longitudinal study of quality of life after resection of hepatocellular carcinoma.

HYPOTHESIS: Hepatic resection improves quality of life (QOL) in patients with resectable hepatocellular carcinoma (HCC). DESIGN: A prospective longitudinal study. SETTING: A university teaching hospital. PATIENTS: Sixty-six consecutive patients undergoing resection of HCC, and 10 patients with unresectable HCC found after surgical exploration who were subsequently treated with transarterial chemoembolization (control group). MAIN OUTCOME MEASURE: Serial measurements of preoperative and postoperative QOL using the Functional Assessment of Cancer Therapy-General (FACT-G) Questionnaire for up to 2 years after surgery (at 3, 6, 9, 12, 18, and 24 months). RESULTS: Among the 66 patients with resectable HCC, the mean postoperative QOL scores at 3 months after surgery were significantly higher than the mean preoperative QOL scores in domains related to physical, social, and emotional well-being and relationship with physicians. The mean total QOL score increased from 83.5 (SD, 9.4) before surgery to 94.1 (SD, 7.7) at 3 months after surgery (P <.001). No significant change of QOL scores at 3 months after surgery was observed in the control group. Twenty patients in the resected group died of early recurrence within 2 years after surgery, but their mean postoperative QOL scores remained higher than the preoperative QOL scores for most assessment times. In contrast, in the control group, the mean total QOL scores became significantly lower than the preoperative scores, starting 9 months after surgery. Forty-six patients in the resected group completed all QOL assessments. At all postoperative assessments, their mean QOL scores were higher than preoperative scores. Recurrence developed in 13 of the 46 patients within the 2-year study, and there was significant deterioration of QOL over time among these 13 (P <.001), whereas no significant change in QOL over time was observed among the 33 recurrence-free patients. Of various clinicopathologic factors, only advanced pTNM stage was significantly predictive of deterioration of QOL over time after resection of HCC. CONCLUSIONS: Hepatic resection results in significant enhancement of QOL in patients with HCC. Development of recurrence is the main factor leading to deterioration in QOL over time after resection of HCC.

Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase.

Leukotriene C(4) synthase (LTC(4)S), the terminal 5-lipoxygenase pathway enzyme that is responsible for the biosynthesis of cysteinyl leukotrienes, has been deleted by targeted gene disruption to define its tissue distribution and integrated pathway function in vitro and in vivo. The LTC(4)S (-/-) mice developed normally and were fertile. LTC(4)S activity, assessed by conjugation of leukotriene (LT) A(4) methyl ester with glutathione, was absent from tongue, spleen, and brain and > or = 90% reduced in lung, stomach, and colon of the LTC(4)S (-/-) mice. Bone marrow-derived mast cells (BMMC) from the LTC(4)S (-/-) mice provided no LTC(4) in response to IgE-dependent activation. Exocytosis and the generation of prostaglandin D(2), LTB(4), and 5-hydroxyeicosatetraenoic acid by BMMC from LTC(4)S (-/-) mice and LTC(4)S (+/+) mice were similar, whereas the degraded product of LTA(4), 6-trans-LTB(4), was doubled in BMMC from LTC(4)S (-/-) mice because of lack of utilization. The zymosan-elicited intraperitoneal extravasation of plasma protein and the IgE-mediated passive cutaneous anaphylaxis in the ear were significantly diminished in the LTC(4)S (-/-) mice. These observations indicate that LTC(4)S, but not microsomal or cytosolic glutathione S-transferases, is the major LTC(4)-producing enzyme in tissues and that its integrated function includes mediation of increased vascular permeability in either innate or adaptive immune host inflammatory responses.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing.

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT(1)) and CysLT(2) receptors, recently have been characterized and cloned. Because the CysLT(1) receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT(1) receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT(1) receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT(1) receptor mRNA is expressed in lung and skin; and reverse transcription-PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT(1) receptor was mapped to band XD. Leukotriene (LT) D(4) induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT(1) receptor cDNA. This agonist effect of LTD(4) was fully inhibited by the CysLT(1) receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [(3)H]LTD(4); and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD(4) >> LTE(4) = LTC(4) >> LTB(4). Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4.

Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE-stimulated hMCs elaborated minimal cys-LT (0.1 +/- 0.1 ng/10(6) hMCs) and abundant prostaglandin (PG)D(2) (16.2 +/- 10.3 ng/10(6) hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE-dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD(2) (by 2. 5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE-mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD(2) generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C(4) synthase (LTC(4)S) mRNA within 6 h, and increased the expression of LTC(4)S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC(4)S biosynthetic pathway (induction of LTC(4)S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.

5' flanking region polymorphism of the gene encoding leukotriene C4 synthase does not correlate with the aspirin-intolerant asthma phenotype in the United States.

BACKGROUND: Approximately 10% of patients with asthma have a distinct clinical entity in which their symptoms are exacerbated by aspirin and most other nonsteroidal anti-inflammatory agents. These individuals typically have significant basal overproduction of cysteinyl leukotrienes, and within their biosynthetic pathway, the terminal enzyme, leukotriene C(4) synthase (LTC(4)S), is significantly overexpressed. A single nucleotide polymorphism consisting of an adenine (A) to cytosine (C) transversion -444 nucleotides upstream of the ATG translation start site in the LTC(4)S gene has been associated with a relative risk of 3.89 for the aspirin-intolerant phenotype in Polish patients. OBJECTIVE: These studies were undertaken to further investigate the functional effect of this allele in LTC(4)S gene expression and subsequently to determine whether an association between the presence of this polymorphism and aspirin-intolerant asthma existed within patients of the United States. METHODS: Functionality of the C-444 allele was assessed by using promoter-reporter constructs and transient transfection assays in the THP-1 monocytic cell line. Genotyping was performed on 137 unaffected control subjects, 33 patients with aspirin-tolerant asthma, and 61 patients with aspirin-intolerant asthma from the United States. RESULTS: Promoter-reporter constructs containing the C-444 allele revealed no significant upregulatory or downregulatory effects in the transcription of the LTC(4)S gene. The LTC(4)S genotype distribution was consistent with the Hardy-Weinberg equilibrium in patients with aspirin-tolerant asthma and unaffected control subjects but not in patients with aspirin-intolerant asthma; however, the distributions were not significantly different among the phenotype groups. CONCLUSIONS: Our data demonstrate that the C-444 allele in the LTC4S gene is not statistically different among patients with the aspirin-intolerant asthmatic phenotype, patients with the aspirin-tolerant asthmatic phenotype, and unaffected control subjects in the United States. This finding, along with the lack of functionality of this polymorphism, suggest that it is not related to a specific asthma phenotype and may represent a population-stratified polymorphism within patients of eastern European descent.

Matrix-associated transforming growth factor-beta1 primes mouse bone marrow-derived mast cells for increased high-affinity Fc receptor for immunoglobulin E-dependent eicosanoid biosynthesis.

Mast cells at different tissue locations are heterogeneous with respect to histochemical staining characteristics, granule protease and proteoglycan content, and eicosanoid biosynthesis. We used Matrigel, an extract from the Engelbreth-Holm-Swarm mouse sarcoma that is enriched in basement-membrane proteins, to investigate the effect of tissue matrix proteins on the differentiation of mouse mast cells, with particular attention to eicosanoid biosynthesis. Culture of mouse bone-marrow cells in interleukin-3 on Matrigel for 3 to 4 wk provided a population of mast cells with more intense metachromasia and increased safranin counterstaining compared with mast cells derived in the absence of Matrigel (bone marrow-derived mast cells [BMMC]). High-affinity Fc receptor for immunoglobulin E-dependent biosynthesis of prostaglandin D(2) and leukotriene (LT) C(4) was 6- and 11-fold higher, respectively, from mast cells derived in the presence of Matrigel compared with conventional BMMC derived in its absence. BMMC derived in the presence of Matrigel also generated substantial quantities of 6-trans-LTB(4) diastereoisomers and LTB(4), which were minimally generated by conventional BMMC. When conventional BMMC derived in the absence of Matrigel were then cultured on Matrigel for 5 d, eicosanoid biosynthesis was upregulated without any change in granule staining characteristics. This upregulation in eicosanoid biosynthesis was inhibited by neutralizing anti- transforming growth factor (TGF)-beta1-specific antibodies, was reproduced by 1 ng/ml TGF-beta1, and was attributed to increased expression of cytosolic phospholipase A(2).

Cell-specific transcription of leukotriene C(4) synthase involves a Kruppel-like transcription factor and Sp1.

Leukotriene C(4) synthase (LTC(4)S) is responsible for the biosynthesis of cysteinyl leukotrienes that participate in allergic and asthmatic inflammation. We analyzed 2.1 kilobases of the 5'-flanking region of the human LTC(4)S gene, which contains three DNase I hypersensitivity sites, for its transcriptional activity when fused to a promoterless and enhancerless luciferase gene. Deletion analysis revealed a nonspecific basal promoter region between nucleotides -122 and -56 upstream of the translation start site which contains a consensus Sp1 binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of either the Sp1 binding site between nucleotides -120 and -115 or the Inr (CAGAC) between nucleotides -66 and -62 reduced the expression of the reporter gene by approximately 60%, whereas double mutations decreased the expression by approximately 80%. The incubation of nuclear extracts from THP-1 and K562 cells with a (32)P-labeled oligonucleotide containing the Sp1 site or the Inr sequence gave gel-shifted complexes that were blocked by their respective cold oligonucleotides, and antisera specific for Sp1 and Sp3 provided supershifts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides -165 to -125 reduced reporter activity. This region contains a tandem CACCC repeat (at nucleotides -149 to -145 and -139 to -135). An oligonucleotide containing the distal CACCC motif was gel shifted by THP-1 cell nuclear extract and was supershifted by antisera to Sp1 and Sp3. Cotransfection of an Sp1 expression plasmid into Drosophila SL2 cells with a -228 to -3 LTC(4)S reporter construct transactivated the reporter gene, whereas mutations at the CACCC repeat region reduced Sp1 transactivation by approximately 66%. Similarly, the Kruppel-like factor Zf9/CPBP (core promoter-binding protein) transactivated the -228 construct in COS cells but not its CACCC mutant construct. These findings indicate the involvement of Sp1 and an Inr in non-cell-specific regulation and a Kruppel-like transcription factor and Sp1 in the cell-specific regulation of the LTC(4)S gene. These are the first such analyses of a member of a newly recognized superfamily of membrane-associated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.

Minimum graft size for successful living donor liver transplantation.

BACKGROUND: The extension of living donor liver transplantation to adult recipients is limited by the adequacy of the size of the graft. We evaluate the effect of the graft size on the survival of the recipient in order to establish a clinical guide for the minimum requirement. METHODS: The clinical records of 14 adults and 11 children (body weight 6.1-100 kg) who underwent living donor liver transplantation for chronic or acute liver failure were reviewed. The effect of the graft weight ratio (graft weight divided by standard liver weight of recipient) on graft function and survival was studied. RESULTS: The graft weight ratio ranged from 31 to 203%. The overall graft and patient survival rates were 84% at a median follow-up of 29 months. The survival rate was 95% for recipients with a graft weight ratio >40%, and 40% only for those with a ratio < or =40% (P = 0.016). It was 88% (7/8) when the ratio was >100%, 100% (5/5) when the ratio was 71 to 100%, 100% (7/7) when the ratio was 41 to 70%, and 40% (2/5) only when the ratio was < or =40%. When the graft weight ratio was < or =40%, early graft dysfunction was evident and contributed to the causes of death in three patients. CONCLUSIONS: Preoperative computed tomographic measurement of liver size of a living donor is essential. A graft that represented 40% or less of the recipient's standard liver weight should be regarded as a marginal graft with a lower success rate.

Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells.

We have used mice in which the gene for cytosolic phospholipase A2 (cPLA2) has been disrupted to demonstrate the absolute requirement for cPLA2 in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA2 prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D2 generation. These defects occurred with two distinct stimuli, stem cell factor and IgE/antigen, which were, however, sufficient for signal transduction defined by exocytosis of beta-hexosaminidase. Whereas cPLA2 is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD2 generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD2.

Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma.

Aspirin causes bronchoconstriction in aspirin-intolerant asthma (AIA) patients by triggering cysteinyl-leukotriene (cys-LT) production, probably by removing PGE2-dependent inhibition. To investigate why aspirin does not cause bronchoconstriction in all individuals, we immunostained enzymes of the leukotriene and prostanoid pathways in bronchial biopsies from AIA patients, aspirin-tolerant asthma (ATA) patients, and normal (N) subjects. Counts of cells expressing the terminal enzyme for cys-LT synthesis, LTC4 synthase, were fivefold higher in AIA biopsies (11.5+/-2.2 cells/mm2, n = 10) than in ATA biopsies (2.2+/-0.7, n = 10; P = 0. 0006) and 18-fold higher than in N biopsies (0.6+/-0.4, n = 9; P = 0. 0002). Immunostaining for 5-lipoxygenase, its activating protein (FLAP), LTA4 hydrolase, cyclooxygenase (COX)-1, and COX-2 did not differ. Enhanced baseline cys-LT levels in bronchoalveolar lavage (BAL) fluid of AIA patients correlated uniquely with bronchial counts of LTC4 synthase+ cells (rho = 0.83, P = 0.01). Lysine-aspirin challenge released additional cys-LTs into BAL fluid in AIA patients (200+/-120 pg/ml, n = 8) but not in ATA patients (0. 7+/-5.1, n = 5; P = 0.007). Bronchial responsiveness to lysine-aspirin correlated exclusively with LTC4 synthase+ cell counts (rho = -0.63, P = 0.049, n = 10). Aspirin may remove PGE2-dependent suppression in all subjects, but only in AIA patients does increased bronchial expression of LTC4 synthase allow marked overproduction of cys-LTs leading to bronchoconstriction.

Molecular cloning of the gene for mouse leukotriene-C4 synthase.

Leukotriene C4 (LTC4) synthase (LTC4S), an integral membrane protein, catalyzes the conjugation of leukotriene A4 with reduced glutathione to form LTC4, the biosynthetic parent of the additional cysteinyl leukotriene metabolites. An XmnI-digested fragment of a P1 clone from a 129 mouse ES library contained the full-length gene of 2.01 kb for mouse LTC4S. The mouse LTC4S gene is comprised of 5 exons of 122, 100, 71, 82 and 241 nucleotides, with intron sizes that range from 76 nucleotides to 937 nucleotides. The intron/exon boundaries are identical to those of the human genes for LTC4S and 5-lipoxygenase-activating protein (FLAP). Primer extension demonstrated a single transcription-initiation site 64 bp 5' of the ATG translation-start site. Nucleotide sequencing of 1.2 kb of the 5' flanking region revealed multiple putative sites for activating protein-2, CCAAT/enhancer-binding protein, and polyoma virus enhancer-3. Fluorescent in situ hybridization mapped the mouse LTC4S gene to mouse chromosome 11, in a region containing the genes for interleukin 13 and granulocyte/macrophage-colony-stimulating factor, and orthologous to the chromosomal location of 5q35 for the human LTC4S gene. Thus, the mouse LTC4S gene is similar in size, intron/exon organization and chromosomal localization to the human LTC4S gene. Recent mutagenic analysis of the conjugation function of human LTC4S has identified R51 and Y93 as critical for acid and base catalysis of LTA4 and reduced glutathione, respectively. A comparison across species for proteins that possess LTC4S activity reveals conservation of both of these residues, whereas R51 is absent in the FLAP molecules. Thus, within the glutathione S-transferase superfamily of genes, alignment of specific residues allows the separation of LTC4S family members from their most structurally similar counterparts, the FLAP molecules.

Leukotriene C4 synthase: a critical enzyme for the biosynthesis of SRS-A.

Leukotriene (LT) C4 synthase catalyzes the conjugation of LTA4 with reduced glutathione (GSH) to form LTC4, the parent compound of cysteinyl leukotrienes. It is a 18 kDa protein that functions as homodimer. Cloning of LTC4 synthase cDNA reveals amino acid homology with 5-lipoxygenase activating protein (FLAP) and newly identified microsomal glutathione S-transferase II (mGST-II) but not with cytosolic GSTs or mGST-I. LTC4 synthase gene contains 5 exons and four introns. This gene has been localized to the long arm of human chromosome 5 at the region of 5q35 which is in close proximity to the cluster of genes that are involved in inflammation and asthma. Mutagenic studies reveals that amino acid residues Arg-51 and Tyr-93 are critical for catalytic function. Arg-51 was proposed to open the epoxide ring of LTA4 and Tyr-93 to provide the thiolate anion of GSH.