Efficacy and safety of plasma exchange in interstitial lung diseases with anti-melanoma differentiation-associated 5 gene antibody positive clinically amyopathic dermatomyositis.

OBJECTIVE: Clinically amyopathic dermatomyositis (CADM) patients frequently develop refractory interstitial lung disease (ILD), with a poor prognosis. We aimed to verify the efficacy and safety of plasma exchange (PE) treatment for ILD in CADM. METHOD: A retrospective case-control study was conducted to compare clinical outcomes with and without PE treatment in CADM-ILD patients refractory to combination therapy of high-dose glucocorticoids, calcineurin inhibitors, and cyclophosphamide. Among 19 enrolled patients, 11 were further treated with PE. We compared survival rates and other clinical characteristics. PE consisted of either fresh-frozen plasma or albumin as a replacement solution. RESULTS: Basal clinical characteristics at diagnosis, including age, gender, serum ferritin, Krebs von den Lungen-6 (KL-6), C-reactive protein, and respiratory function tests, did not differ between the two groups. The survival rate for treatment with PE was higher than for treatment without PE (91% and 50%, respectively, p < 0.05). Among PE-treated patients, anti-melanoma differentiation-associated gene-5 (anti-MDA-5) antibody titre, ferritin, and KL-6 as serological activity markers were sustainably reduced only after initiating PE. Therapeutic intervention with PE reduced the frequency of exacerbation of ILD requiring methylprednisolone pulse therapy. The occurrence of bacterial, fungal, and cytomegalovirus infection did not differ between the groups with and without PE, and adverse events associated with PE resolved with appropriate intervention. CONCLUSION: Combination therapy with PE was associated with an improved survival rate, and may be effective for the management of refractory ILD in CADM patients. A personalized therapeutic strategy including PE could be introduced for fatal rapidly progressive ILD.

Prostaglandin D2 metabolite is not a useful clinical indicator for assessing atopic dermatitis.

Prostaglandin D2 (PGD2 ) plays an important role in atopic dermatitis (AD), and 11,15-dioxo-9α-hydroxy-2,3,4,5-tetranorprostan-1,20-dioicacid (PGDM) is a major metabolite of PGD2 . We investigated the relationship between urinary PGDM levels and severity of paediatric AD. In total, 31 patients with AD and 21 healthy controls (HCs) without AD were recruited, and urinary PGDM levels were measured. Of the 31 patients with AD, 14 were reassessed for urinary PGDM after topical steroid therapy. There was no difference in urinary PGDM levels between patients with AD and HCs. Although there was a significant positive correlation between the SCORing Atopic Dermatitis (SCORAD) index and the serum level of thymus and activation-regulated chemokine (TARC), the urinary PGDM levels did not correlate with either SCORAD or serum TARC. Moreover, both SCORAD and serum TARC were significantly improved by topical steroid therapy; however, urinary PGDM levels were not changed. In conclusion, the level of urinary PGD2 metabolites in children with AD is substantially the same as that in HCs even if the disease is severe.

Oral squamous cell carcinoma arising in a patient with Werner syndrome.

Werner syndrome (WS) is an autosomal recessive disorder characterized by physical signs and symptoms, including premature aging and scleroderma-like skin changes. The gene responsible for WS is the WRN gene. A significant proportion of WS-related malignant tumours are non-epithelial types, and the incidence of oral squamous cell carcinoma (SCC) is rare. A case of oral SCC of the lower alveolus and gingiva arising in a 63-year-old woman with WS is reported here. Biopsy confirmed moderately differentiated SCC. Surgical resection was performed and there was no recurrence or metastasis at the 3-year follow-up. Mutation analysis using next-generation sequencing, detected no mutations in the genes encoding the molecules strongly involved in the development of oral SCC, such as TP53 or PIK3CA. No obvious mutations were detected. Based on the results of the study, the results of mutation analysis suggest that this case might be genetically different from the common mechanisms of SCC in the oral cavity.

Association Between the Fertile Period and Live Birth Post-Kidney Transplantation: A Retrospective Single-Center Cohort Study.

BACKGROUND: Despite restoration of fertility after kidney transplantation, the benefit is limited in female kidney recipients. Our objective is to determine the reasons for this discrepancy. METHODS: We evaluated 315 women who underwent kidney transplantation from 1983 to 2015 (a median of age at transplantation [10th-90th percentile] of 32 years [7-55 years]); 230 recipients between the ages of 15 to 49 years old as of March 2016 were observed. RESULTS: We experienced 10 abortions and 21 live births from our 23 recipients and 2 abortions and 7 live births in 7 recipients from other transplant center. The live birth rate was 8.9 per 1000 female transplant recipients of childbearing age. Seven recipients received either treatments of artificial insemination or in vitro fertilization. Average age at pregnancy was 33.2 ± 3.2 years old, and the fertile period post-transplantation was longer in recipients with live births than those without live births (14.1 ± 7.1 vs 9.9 ± 7.3 years, P < .05). In 42.9% of recipients with live birth, pregnancy-induced hypertension was observed in the last trimester. The gestational age and the average birth weight were 32.8 ± 5.0 months and 2184 ± 632 g, respectively. During follow-up of 14.5 years, there was one case of graft loss, which is a rate of 2.5 per 1000 female recipients. CONCLUSION: Although pregnancy complications are often observed in kidney recipients, graft survival is less influenced by pregnancy. Importantly, kidney disease at childbearing age disrupts pregnancy even after kidney transplantation.

Pharmacokinetic Profile of Twice- and Once-daily Tacrolimus in Pediatric Kidney Transplant Recipients.

BACKGROUND: The aim of this study was to assess the differences in pharmacokinetic (PK) profiles after the 1:1 ratio-based conversion from a twice-daily to a once-daily tacrolimus formulation (TD-TAC and OD-TAC, respectively) in pediatric recipients of kidney transplants. METHODS: TD-TAC was initially administered to 29 pediatric patients who underwent kidney transplantations between April 2010 and September 2015 and were then subsequently switched to OD-TAC. The switch dose ratio was 1:1, and the 24-hour complete PK parameter assessment was performed before and after the regimen was changed from TD-TAC to OD-TAC. RESULTS: The mean total daily dose at baseline was 5.5 ± 2.9 mg (0.18 ± 0.10 mg/kg body weight). Consecutive PK studies revealed no significant difference in the mean time to achieve maximum concentrations and the area under the concentration-time curve from 0 to 24 hours (AUC0-24) of both drug formulations. However, the mean trough concentration (Cmin) and the maximum concentration of OD-TAC were 22% and 6% lower and higher, respectively, than those of TD-TAC. Therefore, a better correlation was observed between the AUC0-24 and Cmin of OD-TAC than between those of TD-TAC. CONCLUSIONS: After the change from TD-TAC to OD-TAC, the AUC0-24 values were equivalent despite a 22% reduction in Cmin. Cmin may therefore be an excellent predictor in the therapeutic drug monitoring of OD-TAC because of its superior correlation with AUC0-24.

Successful Kidney Transplantation in Epstein Syndrome With Antiplatelet Antibodies and Donor-specific Antibodies: A Case Report.

An autosomal dominant hereditary disease, Epstein syndrome (ES) is characterized by sensorineural hearing impairment, macrothrombocytopenia, and hereditary nephritis, and can progress to end-stage kidney disease after puberty. Generally, kidney transplantation is difficult to perform in Epstein syndrome owing to the high risk of perioperative bleeding. Additionally, due to previous platelet transfusions, ES patients sometimes have antihuman leukocyte antigen (HLA) antibodies, including antiplatelet antibodies and donor-specific anti-HLA antibodies (DSA), which may result in refractoriness to platelet transfusion and antibody-mediated rejection (AMR). We report a case of successful kidney transplantation in a patient with ES who had DSA and antiplatelet antibodies. To prevent AMR, we used a desensitization protocol (a combination of plasmapheresis, rituximab, and basiliximab induction). Surveillance biopsy performed at 4 months and 1 year after transplantation showed no pathological findings suggesting AMR. To prevent perioperative bleeding complications, we infused the patient with HLA-matched platelets, thereby maintaining the platelet count at >10.0 × 10(4)/µL, and no postoperative episodes of bleeding occurred.

Suppression of the let-7b microRNA pathway by DNA hypermethylation in infant acute lymphoblastic leukemia with MLL gene rearrangements.

MicroRNAs (miRNAs) regulate cell proliferation and differentiation by controlling the expression of proteins involved in many signaling pathways. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenicity and a poor prognosis in several types of cancers. The miRNA let-7b is one of the severely downregulated miRNAs in mixed-lineage leukemia (MLL)-rearranged acute lymphoblastic leukemia (ALL) patients. In vitro transfection of leukemogenic MLL fusion genes into human embryonic kidney-293 cells suppressed let-7b expression. In leukemic cells with an MLL fusion gene, the regulatory region for let-7b expression was hypermethylated, and its expression was partially recovered after culturing the cells with the demethylating agent 5-azacitidine. These results suggest that loss of let-7b expression may be one of the consequences of oncogenic MLL fusion proteins, and contributes to leukemogenesis possibly through the upregulation of let-7b-regulated target genes with leukemogenic potential in hematopoietic cells. The enforced expression of let-7b in ALL cell lines with an MLL fusion gene inhibited their growth, indicating the possible use of let-7b as a new therapeutic tool for refractory infant ALL with an MLL fusion gene.

Interleukin 33 is induced by tumor necrosis factor alpha and interferon gamma in keratinocytes and contributes to allergic contact dermatitis.

BACKGROUND: Interleukin (IL) 33, a novel member of the IL-1 family, is produced mainly by epithelial cells and endothelial cells in response to various types of stress, including necrosis. The effects of IL-33 on the immune cells involved in allergic contact dermatitis have recently been revealed in vitro. However, in vivo, the induction mechanism and function of IL-33 are not fully understood. OBJECTIVES: Our objectives were to investigate induction of IL-33 in keratinocytes and to evaluate the functions of IL-33 and its inducers in a murine model of allergic contact dermatitis. MATERIAL AND METHODS: KERTr cells, a human keratinocyte cell line, were cultured with various cytokines, including tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. IL-33 expression was detected using quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry, and Western blotting. The functions of IL-33, TNF-a, and IFN-y in allergic contact dermatitis were evaluated using a murine model. RESULTS: TNF-alpha and IFN-gamma induced expression of IL-33 mRNA and protein in KERTr cells. Blockade of IL-33 attenuated swelling in the ears of the experimental mice. Similar effects were noted for blockade of TNF-alpha and IFN-gamma in these mice. CONCLUSIONS: TNF-alpha and IFN-gamma induce expression of IL-33, and IL-33 produced by keratinocytes contributes to allergic contact dermatitis. Blockade of IL-33, TNF-alpha, and IFN-gamma could represent novel and potent strategies to treat allergic contact dermatitis.

A notable case report of May-Hegglin anomaly with immune complex-related nephropathy: a genetic and histological analysis.

May-Hegglin anomaly (MHA) is a rare autosomal dominant disease characterized by macrothrombocytopenia and leukocyte inclusions with microfilaments in the ribosomes. Mutations in the MYH9 gene, encoding non-muscle myosin heavy chain IIA (NMMHC-IIA) have been identified in patients with MHA and other MYH9-related diseases. Two young males (an older and younger brother) presented with macrothrombocytopenia and leukocyte inclusion bodies. Electron microscopy (EM) revealed parallel filaments in leukocyte inclusion bodies characteristic of MHA. Immunofluorescence microscopy (IF) showed NMMHC-IIA antibodies in 1 - 2 leukocyte inclusion bodies. These findings were consistent with MHA and they were identified to express the MYH9 mutation, D1424H. The older brother underwent a renal biopsy because of persistent proteinuria. Histology revealed mesangial proliferative glomerulonephritis with granular deposits of IgG and C1q. EM showed that the dense deposits were located in subendothelial cells, mesangial cells and Bowman's capsule. Immunocytochemistry revealed that NMMHC-IIA antibodies were localized in podocyte and endothelial cells in the glomerulus. Moreover, the expression of nephrin and podocin, slit diagram protein, was normal. An inflammatory mechanism may occur separately from MYH9-related disease. This report presents a case of MHA with immune complex-related nephropathy.

5-lipoxygenase pathway promotes cell proliferation in human glioma cell lines.

OBJECTIVE: 5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes (LTs), that might promote carcinogenesis. We investigated 5-LO expression and examined whether the 5-LO pathway is associated with the proliferation of human brain tumors. METHODS: We immunohistochemically evaluated the profile of 5-LO expression in various types of brain tumors obtained from 42 patients, and examined the proliferative effects of the 5-LO pathway in human glioma cell lines using a proliferation assay. RESULTS: Immunohistochemistry of glioblastomas, astrocytomas, meningiomas, medulloblastomas, craniopharyngiomas, ependymomas, neurinomas, oligodendrogliomas, malignant lymphomas, dysembryoplastic neuroepithelial and metastatic brain tumors revealed 5-LO expression in the cytoplasm and nuclei or nuclear envelopes of tumor cells. The 5-LO inhibitor A861 and the LTA4 hydrolase inhibitor Bestatin dose-dependently suppressed the proliferation of A172 cells, a glioma cell line. CONCLUSIONS: We confirmed the expression of 5-LO in various human brain tumors and demonstrated the partial suppression of tumor growth by inhibitors of the 5-LO-LTA4 hydrolase pathway in human glioma cell lines. The 5-LO-LTA4 pathway might play roles in the proliferation of human glioma cells.

Emedastine difumarate inhibits histamine-induced collagen synthesis in dermal fibroblasts.

BACKGROUND: Mast cell-derived histamine is known to act on dermal fibroblasts and contribute to formation of an intractable chronic allergic dermatitis. Although this fibrotic event may also occur in other organs such as the nasal mucosa, no direct evidence has been reported as to whether responsiveness to histamine by fibroblasts derived from different organs is of the same intensity. Furthermore, while type 1 histamine receptor (H1R) blockers have been shown to be effective for alleviation of the symptoms of allergic diseases, their ability to affect histamine-induced tissue remodeling has not yet been clarified. OBJECTIVE: Our aim was to study the effect of H1R-blockers on histamine-induced tissue remodeling. METHODS: A macroarray assay was used for a comprehensive analysis of histamine-induced gene expression by normal human fibroblasts. Fibroblasts derived from skin or nasal mucosa were cultured in the presence of various concentrations of histamine, and the synthesis of type 1 collagen was measured by means of semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. To determine the effect of H1R blockers, diphenhydramine hydrochloride and emedastine difumarate were investigated in this assay. RESULTS: Histamine induced expression of various kinds of fibrogenic molecules in fibroblasts. Increased type 1 collagen expression was observed in fibroblasts treated with high-dose (0.1 mM to 1 microM) and low-dose (1 pM) histamine. This histamine-induced type 1 collagen synthesis was effectively diminished by emedastine difumarate. While organ specificity seems to be involved, emedastine difumarate is considered to be an effective drug for reversal of such histamine-induced remodeling in the skin. CONCLUSIONS: We found that the expression of fibroblast-derived genes is differentially regulated by different concentrations of histamine and that the robustness of the inhibitory action of H1R blockers is different for skin-derived and nasal mucosa-derived fibroblasts. We believe that our findings may contribute to a better understanding of the mechanisms of histamine-induced tissue remodeling and provide information useful for the management of refractory allergic dermatitis.

Expression and production of aberrant PAX5 with deletion of exon 8 in B-lineage acute lymphoblastic leukaemia of children.

Summary We investigated PAX5 expression in childhood B-lineage acute lymphoblastic leukaemia (ALL). Seven of 21 children with B-lineage ALL had multiple PAX5 variants, while 14 children and healthy controls showed full-length (FL) and one variant PAX5. By Western blotting, healthy controls displayed Pax5-FL, while one short Pax5, derived from the deletion of exon 8 (Pax5-DeltaE8) was produced in 90% of ALL samples, as well as in ALL cell lines. PAX5-DeltaE8 lacked more than 50% of the transactivation domain, indicating that aberrant Pax5 production might lead to the arrest of B-cell differentiation, contributing to the pathogenesis of B-lineage ALL.

Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes.

Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.

dsRNA enhances eotaxin-3 production through interleukin-4 receptor upregulation in airway epithelial cells.

The exacerbation of asthma during viral infections is mainly explained by neutrophils infiltrating into the airways. However, enhanced functions of eosinophils are also observed. The aim of this study was to reveal the mechanism of how eosinophils are activated during and after viral infection of the airways, using a model of viral infection. A synthetic double-stranded RNA, poly inosinic-cytidyric acid (poly(IC)), was transfected to a human airway epithelial cell line (BEAS-2B) and the primary bronchial epithelial cells, to mimic a viral infection. The production of chemokines from the cells was investigated. The transfection of poly(IC), alone, marginally affected the eotaxin-3 production of the cells. However, the transfection of poly(IC) prior to interleukin (IL)-4 stimulation enhanced eotaxin-3 production. Poly(IC) transfection increased mRNA and protein expressions of IL-4 receptor (R)alpha and IL-2Rgamma, components of the IL-4R. In BEAS-2B cells, IL-4-mediated phosphorylation of signal transducer and activator of transcription six was enhanced in poly(IC) transfected cells. This was reversed by the addition of anti-IL-4Ralpha antibody, suggesting the role of an increased number of IL-4 receptors in enhanced IL-4-induced eotaxin-3 production. Poly(IC)-induced upregulation of IL-4Ralpha was inhibited by treatment with cycloheximide or dexamethasone. In conclusion, these results suggest that viral airway infection may enhance interleukin-4-induced eotaxin-3 production through upregulation of the interleukin-4 receptor in airway epithelial cells.

Screening for Menkes disease using the urine HVA/VMA ratio.

Menkes disease is a disorder of copper transport that results in early death. Early therapy with parenteral copper-histidine has been shown to markedly improve outcomes. However, early diagnosis is difficult because patients are asymptomatic in early infancy. In Menkes disease, impaired activity of dopamine beta-hydroxylase, a copper-dependent enzyme, leads to increased urine ratios of homovanillic acid/vanillylmandelic acid (HVA/VMA). Urine HVA/VMA ratios ranged from 4.1 to 69.7 among 15 patients with Menkes disease, whereas only 0.18% of controls had ratios greater than 4.0. Thus, the urine HVA/VMA ratio is a useful screening method for Menkes disease.

High frequency of QPY allele and linkage disequilibrium of granzyme-B in Epstein-Barr-virus-associated hemophagocytic lymphohistiocytosis.

Mediation of Epstein-Barr virus (EBV)-specific cytotoxicity in T lymphocyte via the perforin/granzyme pathway has been demonstrated; therefore, a study involving cytolytic molecules was essential for the clarification of hemophagocytic lymphohistiocytosis (HLH) pathogenesis. This investigation, which analysed the frequency of three allelic mutations of granzyme-B (55Q/R, 95P/A and 247Y/H) in patients with EBV-HLH and infectious mononucleosis, identified the high prevalence of the QPY haplotype in EBV-HLH patients in comparison with healthy controls. A > G polymorphism was also detected in intron 5; furthermore, nearly complete linkage disequilibrium was observed among these polymorphisms. The recessive role of the QPY haplotype of granzyme-B might be responsible for the pathogenesis of EBV-HLH. Cytotoxicity and DNA fragmentation of cytotoxic T lymphocytes did not differ among patients characterized by the QPY/QPY, RAH/RAH and QPY/RAH genotypes. This finding suggested that DNA fragmentation in target cells is mediated not only by granzyme-B but also by other molecules, including other granzymes or Fas.

Silicone gel sheets relieve pain and pruritus with clinical improvement of keloid: possible target of mast cells.

Silicone gel sheet treatment is widely used to treat hypertrophic scars and keloids since it is easily applied and prevents scar pain and itching. We used Cica-Care silicone gel sheets in the conservative treatment of six patients for 24 weeks and recorded pain, itching, redness, and scar elevation every 4 weeks. We also investigated the number of mast cells and Fas antigen expression in the lesional skin (one patient) before and after treatment. The pain and itching clearly decreased after 4 weeks of the silicone gel sheeting and disappeared after 12 weeks. Twelve weeks were required for a reduction in scar redness and elevation. After 24 weeks, a decrease in the number of mast cells and the enhanced expression of Fas antigen by lesional fibroblasts were observed. Thus, silicone gel sheeting is effective and safe, especially with more severe symptoms of pain and itching possibly induced by mediators derived from increased mast cells.

Dexamethasone accelerates catabolism of leukotriene C4 in bronchial epithelial cells.

Leukotriene (LT)C4, a potent chemical mediator in bronchial asthma, is metabolised to the less active LTE4 via LTD4 in two consecutive reactions catalysed by enzymes of the glutamyl transpeptidase and dipeptidase families. The activities of these catabolic enzymes may be influenced by glucocorticosteroids. This study was conducted to examine whether this inactivation of LTC4 is affected by dexamethasone (DEX) in transformed human bronchial epithelial cells and normal human bronchial epithelial cells. After incubation with DEX for 0-5 days, cells were resuspended in the presence of exogenous LTC4, and conversion of LTC4 to LTE4 was measured using high-performance liquid chromatography. Gamma-glutamyl transpeptidase (GGT) and GGT-related enzyme (GGTRE) messenger ribonucleic acid (mRNA) expression were examined using reverse transcriptase-polymerase chain reaction analysis, and GGT activity by enzyme assay. Conversion to LTE4 was accelerated by DEX pretreatment. GGTRE but not GGT mRNA expression was enhanced after incubation with DEX. The results indicate that dexamethasone transcriptionally upregulates the activity of gamma-glutamyl transpeptidase-related enzyme in human bronchial epithelial cells, which accelerates inactivation of leukotriene C4 via conversion to leukotriene E4. This is a novel mechanism of glucocorticosteroids in human bronchial epithelial cells.

Induction of thromboxane A2 synthesizing enzymes in DMSO-induced granulocytic differentiation of HL-60 cells.

Human leukemia (HL)-60 cells were differentiated by several agents, and prostaglandins (PGs) and thromboxane (TX) synthesizing activity increased in response to the differentiation of the cells. We examined the expression of messenger RNA (mRNA) for TX-synthesizing enzymes, cyclooxygenase (COX)-1, COX-2 and TXA(2) synthase, in dimethyl sulfoxide (DMSO)-differentiated HL-60 cells by reverse transcriptase polymerase chain reaction (RT-PCR), and A23187-stimulated TXB(2) production, a stable metabolite of TXA(2), by radioimmunoassay (RIA). A23187-stimulated TXB(2) production, and mRNA abundance for COX-2, were not detected in non-treated HL-60 cells. TXA(2) synthase mRNA were barely detected in non-treated HL-60 cells. DMSO-induced HL-60 cells gained induction of TXB(2) synthesis and mRNA for COX-2 and TXA(2) synthase during granulocytic differentiation. COX-1 mRNA was constitutively expressed. A23187-stimulated TXB(2) production in DMSO-treated cells was inhibited by NS-398, a specific COX-2 inhibitor. These results demonstrated that TXB(2) production in granulocytic HL-60 cells was regulated at both the enzyme level of COX-2 and TXA(2) synthase.