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Thiopurine methyltransferase (TPMT) enzymes play a crucial role in azathioprine metabolism. Mutations in the enzyme initiate generation of excess thioguanine, which causes suppression of various cell lineages. The objectives of this study were to investigate the prevalence of TPMT mutation in Bangladeshi patients with systemic lupus erythematosus (SLE) requiring azathioprine therapy and its relation to the development of myelosuppression. 250 patients with SLE were enrolled, then monitored for myelosuppression adverse events for 4 months. TPMT*3C (rs1142345), TPMT*3B (rs1800460), and TPMT*2 (rs1800462) polymorphisms were analyzed using polymerase chain reaction–restriction fragment length polymorphism. The risk of myelosuppression (i.e., leukopenia, thrombocytopenia, and anemia) was estimated as the odds ratio (OR) with 95% confidence intervals (CIs) and p values. Of the 250 patients, 17 (6.8%) were heterozygous for the TPMT*3 mutation and 233 (93.2%) were homozygous for the wild type; no patients carried a homozygous mutation. Grade III/IV leukopenia, thrombocytopenia, and anemia occurred in 12.0%, 12.0%, and 27.9% of wild-type TPMT patients respectively; corresponding rates in heterozygous TPMT*3C patients were 70.6%, 64.7%, and 5.9%. Patients with Grade III/IV azathioprine-induced leukopenia were significantly more likely to have the heterozygous TPMT*3C genotype than the wild-type variant (OR 17.6; 95% CI 5.8–53.6; p

We previously reported that prostaglandin (PG)D2 and its isosteric analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), promote adipogenesis in 3T3-L1 cells during the maturation phase. Focusing on the differentiation phase, although both PGs inhibited adipogenesis, this effect was canceled out by PGI2 and PGJ2 derivatives. Thus, PGD2 and 11d-11m-PGD2 play different roles during the phases, but do not affect PGI2- and PGJ2-derivative-induced adipogenesis.

Prostaglandin (PG) D2 is relatively unstable and dehydrated non-enzymatically into PGJ2 derivatives, which are known to serve as pro-adipogenic factors by activating peroxisome proliferator-activated receptor (PPAR) γ, a master regulator of adipogenesis. 11-Deoxy-11-methylene-PGD2 (11d-11m-PGD2) is a novel, chemically stable, isosteric analogue of PGD2 in which the 11-keto group is replaced by an exocyclic methylene. Here we attempted to investigate pro-adipogenic effects of PGD2 and 11d-11m-PGD2 and to compare the difference in their ways during the maturation phase of cultured adipocytes. The dose-dependent study showed that 11d-11m-PGD2 was significantly more potent than natural PGD2 to stimulate the storage of fats suppressed in the presence of indomethacin, a cyclooxygenase inhibitor. These pro-adipogenic effects were caused by the up-regulation of adipogenesis as evident with higher gene expression levels of adipogenesis markers. Analysis of transcript levels revealed the enhanced gene expression of two subtypes of cell-surface membrane receptors for PGD2, namely the prostanoid DP1 and DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)) receptors together with lipocalin-type PGD synthase during the maturation phase. Specific agonists for DP1, CRTH2, and PPARγ were appreciably effective to rescue adipogenesis attenuated by indomethacin. The action of PGD2 was attenuated by specific antagonists for DP1 and PPARγ. By contrast, the effect of 11d-11m-PGD2 was more potently interfered by a selective antagonist for CRTH2 than that for DP1 while PPARγ antagonist GW9662 had almost no inhibitory effects. These results suggest that PGD2 exerts its pro-adipogenic effect principally through the mediation of DP1 and PPARγ, whereas the stimulatory effect of 11d-11m-PGD2 on adipogenesis occurs preferentially by the interaction with CRTH2.

We attempted to evaluate the contents and distribution of antioxidants in the whole seeds, seed shells, and peeled seeds of the Japanese horse chestnut. The seed shells exhibited the highest antioxidant activities due to the presence of highly polymeric proanthocyanidins as we have reported recently. On the other hand, the peeled seeds predominantly contained flavonols such as quercetin and kaempferol at a high level of 66.7% of total polyphenols, also contributing to the predominant antioxidant activities. The instrumental analysis of the extract from the whole seeds revealed the identification of eight flavonol O-glycosides, including six compounds with quercetin and two species with kaempferol as aglycones. The isolated species exhibited different antioxidant activities depending on the types of aglycones, glycosides, and acylated moieties. The results indicate that the peeled seeds are a good source of flavonol O-glycosides serving as antioxidants to be used for food additives and dietary supplements.

Polygonum tinctorium Lour. (family Polygonaceae), known as indigo plant, has been useful as a medicinal or edible plant abundant in polyphenolic compounds. We have recently shown that flavonol O-glycosides with 3,5,4′-trihydroxy-6,7-methylenedioxyflavone (TMF) are predominant flavonoids in indigo leaves. However, no study has been performed regarding changes in the levels of flavonoid species during the germination and growth of indigo plant. Here, we attempted to determine the individual constituents of flavonol O-glycosides and the changes in their contents of the seeds, sprouts, and aerial parts. These results revealed that only the seeds predominantly contained flavonol O-(acetyl)-rhamnosides with quercetin or kaempferol as an aglycone. During the development of the sprouts and aerial parts, flavonol O-glycosides with TMF as an aglycone became mainly detectable and accounted for 79.4% and 74.9% of total flavonol O-glycosides from the extracts of aerial parts harvested in 2016 and 2017, respectively. Of the plant organs tested, the aerial parts exhibited the highest antioxidant activities concomitant with greatly increased levels of total polyphenols. Thus, we were able to conduct the identification and quantification of flavonol O-glycosides from the seeds, sprouts, and aerial parts of indigo plant and to evaluate antioxidant activities of their extracts. Taken together, our findings clearly provide the evidence that the aerial parts of indigo plant are a rich source of flavonol O-glycosides with TMF and exhibit much higher antioxidant activities, indicating the usefulness for the application to food and nutraceutical purposes.

μ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the trans-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of Arabidopsis thaliana VSR4 in the sorting of VSR4. The results obtained showed the increased localization of VSR4 at the plasma membrane or vacuoles by mutations in CRSs including the tyrosine-sorting motif YMPL or acidic dileucine-like motif EIRAIM, respectively. Interaction analysis using the bimolecular fluorescence complementation (BiFC) system, V10-BiFC, which we developed, indicated an interaction between VSR4 and AP1M2, AP1M type 2, which was attenuated by a YMPL mutation, but not influenced by an EIRAIM mutation. These results demonstrated the significance of the recognition of YMPL in VSR4 by AP1M2 for the post-Golgi sorting of VSR4.

Persicaria tinctoria (Aiton) Spach, also called Polygonum tinctorium Lour., (family Polygonaceae) for indigo plant has been traditionally useful as a medicinal or edible plant with a variety of biological activities. Of these, much attention has been paid to their anti-inflammatory activities. We have recently demonstrated that indigo leaves contain high levels of flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) as an aglycone. In this study, we attempted to evaluate anti-inflammatory activities of TMF-O-glycosides and free TMF prepared from indigo leaves after extraction with hot water. Free TMF was found to appreciably down-regulate the gene expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, inducible nitric oxide, and tumor necrosis factor-α in cultured macrophage cells stimulated with lipopolysaccharide while up-regulating the expression of anti-inflammatory IL-10. However, no study has been conducted regarding in vivo anti-inflammatory activities of TMF-O-glycosides and free TMF until now. Here, we assessed in vivo anti-inflammatory effects of these dietary compounds on ulcerative colitis in a murine model of inflammatory bowel disease by the induction with dextran sulfate sodium (DSS). Histological evaluation revealed that both TMF-O-glycosides and free TMF effectively protected against DSS-induced ulcerative colitis. The analysis of digested products by liquid chromatography and mass spectrometry led us to detect free TMF as a predominant metabolite in the feces of mice fed with TMF-O-glycosides. Moreover, free TMF was later detected as glucuronyl conjugates of TMF in the liver of mice fed with both fractions. These results indicate the effective digestion of TMF-O-glycosides and the subsequent absorption of free TMF in the gut of mice for exerting anti-inflammatory effects. Taken together, our findings suggest that dietary TMF-O-glycosides could be promising natural sources for the utilization as herbal medicine and nutraceuticals to expect in vivo anti-inflammatory activities.

Intestinal bacteria produce skatole (3-methylindole) from tryptophan in dietary proteins and ingesting large quantities of animal protein is associated with increased fecal skatole concentrations. Although possibly associated with disrupted intestinal homeostasis, the influence of skatole on intestinal epithelial cellular function has not been characterized in detail. The present study aimed to determine whether skatole induces intestinal epithelial cell (IEC) dysfunction. We found that skatole dose-dependently caused IEC death and time-dependently induced IEC apoptosis. Since skatole directly interacts with aryl hydrocarbon receptors (AhR), we investigated whether these receptors influence the skatole-induced death of IEC. In addition to increased AhR transcriptional activity induced by skatole, the AhR antagonist CH223191 partially suppressed of skatole-induced IEC death. Extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) are mitogen-activated protein kinases (MAPK) induced by skatole. None of them were repressed by CH223191, whereas the p38 inhibitor SB203580 promoted skatole-induced IEC death. These findings together indicated that skatole induces both AhR-dependent activation pathways and the AhR-independent activation of p38, consequently regulating the amount of IEC death. Accumulating evidence indicates that consuming large amounts of animal protein is associated with the pathogenesis and progression of inflammatory bowel diseases (IBD). Thus, intestinal skatole production induced by large amounts of dietary animal protein might be associated via IEC death with intestinal pathologies such as IBD.

Protein–protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC–CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.

Polygonum tinctorium Lour (indigo plant) has been regarded as a useful medicinal plant for traditional herbal medicine. The polyphenolic fraction of indigo leaves exhibited anti-inflammatory activities as determined by the suppressed synthesis of nitric oxide (NO) in cultured RAW264 macrophage cells. The acid hydrolysate of the fraction showed much more potent effect than the unhydrolyzed one. In sharp contrast, those fractions of indigo stems had almost no effect. 3,5,4′-Trihydroxy-6,7-methylenedioxyflavone (TMF)-O-glycosides and tryptanthrin were detected exclusively in the extracts of the leaves. The isolated flavonol species were furthermore tested for their anti-inflammatory activities against the synthesis of NO and prostaglandin E2 in the cultured macrophage cells. More potent anti-inflammatory effects were recognized with different aglycones of flavonols than their flavonol O-glycosides. Although the inhibitory effects of TMF were less effective than those of tryptanthrin, the levels of flavonol O-glycosides with TMF were much more abundant than those of tryptanthrin in the leaves. Oral administration of the fraction containing flavonol O-glycosides with TMF into mice revealed the detection of free TMF in the blood circulation, indicating that the aglycone moiety can be cleaved by digestive enzymes and absorbed in the gut. Alternatively, the assay of hydrophilic oxygen radical absorbance capacity revealed that the isolated species of flavonol O-glycosides with TMF and their aglycone had appreciable antioxidant activities. Taken together, our findings suggest that the predominant flavonol O-glycosides with TMF as an aglycone could be promising natural agents for the application to herbal medicine, nutraceuticals, and food additives.

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.

The indigo plant (Polygonum tinctorium Lour) has been used traditionally as a medicinal plant with a variety of biological effects. Of these, polyphenolic ingredients are postulated to contribute to these activities. However, the identification and quantification of polyphenolic compounds in indigo plants have not been conducted comprehensively until now. This study was undertaken to identify the related ingredients by combined instrumental analyses using ultra-performance liquid chromatography electrospray-ionisation mass spectrometry and gas chromatography-mass spectrometry after the extracts of plant tissues were fractionated by absorption column chromatography. These analyses allowed the identification of kaempferol, quercetin-3-O-glucuronide, quercetin, kaempferol-3-O-glucopyranoside, caffeic acid, chlorogenic acid and tentative 3,5,4′-trihydroxy-6,7-methylenedioxyflavone. Furthermore, predominant polyphenolic compounds were quantified by reverse-phase high-performance liquid chromatography and capillary gas chromatography, revealing the higher proportions of kaempferol, quercetin-3-O-glucuronide and quercetin among them. The results indicate that the indigo plant is a promising source for flavonoids and the related compounds with beneficial medicinal effects.

We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I2 called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.

Prostaglandin (PG) J(2) series including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is the dehydration products of PGD(2) that are biosynthesized through the cyclooxygenase (COX) pathway. These prostanoids are active ligands for peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis in adipocytes. Here we investigated whether PGJ(2) derivatives can modulate the gene expression of monocyte chemoattractant protein-1 (MCP-1), a pro-inflammatory chemokine, during the maturation phase of adipocytes. Each of selective or nonselective inhibitors for COX isoforms suppressed significantly the accumulation of fats by interfering the induced expression of the PPARγ gene. Immunological assays of PGJ(2) series revealed higher production of Δ(12)-PGJ(2) than 15d-PGJ(2) by cultured adipocytes, implicating the contribution of endogenous PGJ(2) series to the stimulated adipogenesis. In addition, the increased transcription of MCP-1 was detectable at later maturation phase of adipogenesis, which was prevented by co-incubation with aspirin. Although 15d-PGJ(2) was more potent than Δ(12)-PGJ(2), both PGJ(2) derivatives series had similar effects to rescue dose-dependently the expression of the MCP-1 gene attenuated by aspirin. These findings suggest that the expression of MCP-1 involved in adipocyte inflammation could be positively regulated by the PGJ(2) series during adipogenesis in adipose tissue.

15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD2. To determine the endogenous synthesis of 15d-PGJ2, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ2. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ2 was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ2. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ2 from 0.5 pg to 9.7 ng with 71 pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ2 with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ2 in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ2 became more evident after several hours of incubation with authentic PGD2 at 37 °C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ2 and its roles in various cells and tissues.

Compared with oligomeric proanthocyanidins, highly polymeric proanthocyanidins have more difficulty to analyze the molecular sizes and the mode of interflavan linkages of flavan-3-ol units through doubly linked A-type bonds and single B-type bonds. Recently, we have shown that seed shells of the Japanese horse chestnut contain highly polymeric proanthocyanidins as dominant polyphenolics that can be separated into two fractions according to the difference in the molecular sizes. Here, we tried to perform the structural characterization of them in terms of the molecular sizes and the proportions of A-type linkages relative to B-type linkages. The results were compared with those of the corresponding preparations with variations in the sizes from fruits of blueberry and cranberry. Gel permeation chromatography revealed that the molecular sizes of them were higher in the order of blueberry > cranberry > seed shells of the Japanese horse chestnut when they are compared between the respective fractions. For the analysis of terminal and extension units of those proanthocyanidins, the isolated fractions were subjected to the thiolytic cleavage of the B-type linkages using 1-dodecanethiol, and the resulting degradation products were identified by ultra-performance liquid chromatography electrospray-ionization mass spectrometry (UPLC-ESI/MS). These analyses provided fast and good resolution of the degradation products and revealed higher proportions of A-type linkages compared with B-type linkages in the both isolated fractions in the order of the seed shells > cranberry > blueberry. Moreover, the isolated fractions with higher molecular sizes and those more abundant in the proportions of A-type linkages were found to be more effective in the inhibition of pancreatic lipase activity. The results suggest that higher molecular sizes and more abundance of A-type bonds in polymeric proanthocyanidins are promising key factors for the attenuation of lipid digestion as dietary supplements.

The purpose of this study was to evaluate the effects of polyphenols from seed shells of Japanese horse chestnut (JHP) on methotrexate (MTX)-induced intestinal injury in rats. MTX application caused intestinal morphological injury and increase in malondialdehyde (MDA) levels, decrease in levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in small intestine. However, oral administration of JHP ameliorated MTX-induced intestinal injury and inhibited the increase in MDA and the decrease in GSH and GSH-Px activity in small intestine. In conclusion, our results indicated that oral administration of JHP alleviated MTX-induced intestinal injury through its antioxidant properties.

Recently, we have shown that seed shells contain a large amount of highly polymeric proanthocyanidins having a series of heteropolyflavan-3-ols with doubly linked A-type linkages as well as single B-type bonds without gallic acid esterified to them. Here, we attempted to evaluate in vivo anti-obesity effects of the polymerized proanthocyanidins in mice. An oral starch or glucose tolerance test in mice revealed that the isolated two fractions of highly polymerized proanthocyanidins with the different degree of polymerization suppressed effectively the elevation of blood glucose from oral starch, but not from oral glucose, suggesting the preferential inhibition of the digestive enzymes of carbohydrates. Moreover, in vivo anti-obesity effects of the total fraction containing the proanthocyanidins as a drink were investigated in mice fed a high-fat diet. Their anti-obesity effects became more evident after 9 weeks as determined by the attenuation of the elevation in body weight, the mass of peritoneal adipose tissues, and the plasma levels of total cholesterol and leptin. Furthermore, the increased size of hepatocytes and the generation of steatosis with micro- and macrovesicles in liver were normalized by the dietary supplementation of the total proanthocyanidin fraction. The findings suggest the usefulness of highly polymeric proanthocyanidins from seed shells in the application to food as a dietary supplement with anti-obesity effects in vivo through the inhibition of digestive enzymes of carbohydrates and fats.

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE 2 involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE 2 associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE 2 and PGF 2α , during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is one of the isoprostanes that are mainly generated nonenzymatically in vivo from arachidonic acid through free radical-induced lipid peroxidation. To assess oxidative stress in vivo, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) for 8-iso-PGF2alpha. A sensitive calibration curve allowed the quantification of the amounts from 0.23 pg to 98.4 pg with 4.7 pg of 50% displacement in one assay. The ELISA method was applied to the measurement of the plasma levels of 8-iso-PGF2alpha in young rats (4-8 weeks of age) and aged rats (106-123 weeks). The average level of esterified form in the plasma from aged rats was 30.6-fold higher than that in the plasma from young rats, reflecting the enhanced status of oxidative stress in aged animals. In addition, the aged rats exhibited higher levels of this F2-isoprostane esterified to lipids from liver and kidney, suggesting local oxidative injury in specific organs. These results indicate the utility and accuracy of our ELISA for 8-iso-PGF2alpha as a biomarker in vivo to assess systemic oxidative stress in animals or humans as well as oxidative injury at local sites.

トチノキ種皮からの熱水抽出物をDiaion HP-20カラムとChromatorex ODS 1024Tカラムで分画した後，Sephadex LH-20のカラムクロマトグラフィーで3つの画分，すなわちF1，F2，F3に分離した．それぞれの画分について消化酵素のリパーゼに対する阻害活性を測定した．その阻害活性は，平均分子量13000のF2と31000のF3に回収された．F2より高分子のF3の方が強い阻害活性を示した．F2とF3は，B-タイプとA-タイプのインターフラバン結合を有するプロアントシアニジンである．プロアントシアニジン類を含む画分を用いて，マウスによる油脂負荷試験を行った結果，マウス体重1kgあたり200mgおよび500mg投与したところ，無投与群に対して有意に血中トリアシルグリセロール濃度の上昇を抑制した．同様のプロアントシアニジン類の標品を用いて単回投与毒性試験を行った結果，最小致死量は2000mg/kg以上であることが確認された．肝毒性の指標である血中のGOTとGPTの測定結果は，異常な値を示さなかった．以上より，トチノキ種皮に豊富に存在する高分子プロアントシアニジン類は，動物体内の消化管におけるリパーゼ活性阻害による脂肪吸収抑制を目的とした健康食品素材として期待できる．

（1） トチノキ種皮よりポリフェノールを熱水で抽出し，Diaion HP-20, Chromatorex ODS 1024T, そしてSephadex LH-20のカラムクロマトグラフィーで分離したF1，F2，F3のそれぞれの画分をMALDI-TOF/MSで分析した．その結果，主成分は，縮合型タンニンであり，F2で19mer, F3で23mer以上のポリマーとして存在することが明らかになった．（2） トチノキ種皮のタンニンは，単結合のBタイプの架橋に加えて，二重結合により架橋したAタイプのインターフラバン結合を多数，有していた．また，構成単位は，ガロイル基により修飾されていない（＋）-カテキン，もしくは（−）-エピカテキンであった．（3） トチノキ種皮に由来するポリフェノール成分について，糖質消化酵素に対する阻害作用を検討した．その分画成分は，α-グルコシダーゼおよびα-アミラーゼに対して強い阻害活性を示した．これらは，血糖値の急激な上昇を緩和する機能性食品素材として期待できる．

Seed shells of the Japanese horse chestnut (Aesculus turbinata BLUME) contain high levels of polyphenolic antioxidants. These compounds were extracted, fractionated, and finally separated into three fractions, F1, F2, and F3, according to their degrees of polymerization. The structures of the isolated fractions were characterized by a combination of mass spectrometric analyses. F1 contained mainly low molecular weight phenolic substances, including procyanidin trimers. The predominant fractions F2 and F3 consisted of polymeric proanthocyanidins having a series of heteropolyflavan-3-ols, (+)-catechin/(-)-epicatechin units, and polymerization degrees of 19 and 23, respectively. The polyphenol polymers had doubly linked A-type interflavan linkages in addition to single B-type bonds without gallic acid esterified to them. The isolated polyphenolic compounds exhibited potent antioxidative activities comparable to monomeric (+)-catechin and (-)-epicatechin, or more efficacious than those monomers. The results suggest the potential usefulness of polyphenol polymers from seed shells as a source for nutraceutical factors.

Adipocytes serve not only as a storage depot of fats but also as endocrine cells secreting adipocytokines including tumor necrosis factor alpha (TNFalpha). Using preadipogenic 3T3-L1 cells, we attempt to determine the response of adipocytes at different stages of the life cycle to TNFalpha with respect to the gene expression of the arachidonate cyclooxygenase (COX) pathway and the role of endogenous prostaglandins (PGs). The gene expression analysis of the COX pathway revealed the marked increase in mRNA and protein levels of COX-2 in response to TNFalpha in preadipocytes, whereas COX-1 was expressed constitutively. Moreover, the cells at different cycle stages exhibited the specific gene expression of isoformic enzymes of prostaglandin (PG) synthases for PGs of the D(2), E(2), and F(2alpha) series upon exposure to TNFalpha. The treatment of preadipocytes with TNFalpha along with calcium ionophore A23187 resulted in the stimulated formation of PGE(2) and PGF(2alpha), attenuating the apoptotic cell death induced by TNFalpha alone. The response of adipocytes to synthesize these PGs declined during the differentiation and maturation phases. The cells during the differentiation phase were the most sensitive to TNFalpha in terms of the decrease in adipogenesis without the mediation of endogenous PGs. TNFalpha was also effective in suppressing adipogenesis during the maturation process. Taken together, TNFalpha can control cell number of preadipocytes as well as the size of fat storage in mature adipocytes. The action of TNFalpha on preadipocytes can be modulated by the production of endogenous PGs through the induction of COX-2.

A plant allene oxide synthase (AOS) reacting with 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HPOT), a lipoxygenase product of alpha-linolenic acid, provides an allene oxide which functions as an intermediate for jasmonic acid (JA) synthesis, making AOS a key enzyme regulating the JA level in plants. Although AOSs in various plants have been investigated, there is only limited information about AOSs in soybean (Glycine max). In this study, we cloned and characterized two soybean AOSs, GmAOS1 and GmAOS2, sharing 95% homology in the predicted amino acid sequences. GmAOS1 and GmAOS2 were composed of 564 and 559 amino acids respectively, with predicted N-terminal chloroplast-targeting signal peptides. Both AOSs expressed in Escherichia coli were selective for 13S-hydroperoxides of alpha-linolenic and linoleic acids, suggesting the potential of GmAOS1 and GmAOS2 to contribute to JA synthesis. GmAOS1 and GmAOS2 were expressed in leaves, stems, and roots, suggesting broad distribution in a soybean plant.

Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation phase without IBMX on adipogenesis after the maturation phase.

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) has been identified as a natural ligand for peroxisome proliferator-activated receptor (PPAR) gamma to promote adipogenesis. However, it remains elusive about the ability of PPARgamma-expressing adipocytes to produce PGJ(2) series and the role in the life cycle of adipocytes. Here, we developed an enzyme-linked immunosorbent assay specific for 15d-PGJ(2). The analysis using this method revealed the increase in the endogenous synthesis of immunoreactive 15d-PGJ(2) in cultured adipocytes during the maturation phase. Further studies using cyclooxygenase inhibitors clarified the contribution of endogeous 15d-PGJ(2) produced by mature adipocytes to upregulation of fat storage in an autocrine manner.

Potato (Solanum tuberosum) plants are rich in 9-lipoxygenase, which converts linoleic acid and alpha-linolenic acid to 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPOD) and 9S-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid (9-HPOT) respectively. The allene oxide synthase (AOS) involved in 9-HPOD/9-HPOT metabolism in potato, however, has not been characterized in detail. We cloned a cDNA encoding a novel AOS from potato sprouts by reverse transcriptase-PCR based on a partial sequence in the EST database. This AOS was successfully expressed in the yeast Pichia pastoris, and purified using Ni-NTA resin. The recombinant enzyme metabolized 9-HPOD, 9-HPOT, 13-HPOD, and 13-HPOT with reaction efficiencies of 2.5 x 10(7), 1.0 x 10(7), 2.5 x 10(6), and 7.6 x 10(6) M(-1) s(-1) respectively. The alpha-ketol formed from 9-HPOD was composed mainly of the 9R-enatimomer (90%). Besides sprouts, the mRNA of this AOS was detected in buds, flowers, and stems, but not in leaves, tubers, or roots of mature plants, suggesting that this enzyme has a tissue-specific function.

Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF2alpha and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX-1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2alpha (cPLA2alpha) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF2alpha following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2alpha and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF2alpha, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF2alpha and the specific action of these prostanoids at different cycle stages of adipocytes.

（1）あく抜き処理前，あるいは，あく抜き処理後のトチノミ由来の各サポニン成分の単離物を用いて，マウスでの糖負荷試験を行ったところ，あく抜き処理後の食用トチノミに存在する4種類のデアセチルエスシンは，いずれも同程度の血糖値上昇の抑制活性を示した．エスシン関連物質の血糖値の上昇抑制活性は，エスシン類＞デアセチルエスシン類＞デサシルエスシン類の順に高い傾向になった．このことより，エスシン類の21位のアシル基と22位のアセチル基が，血糖値上昇の抑制活性の強さに大きく関係していることが明らかとなった．（2）エスシン，デアセチルエスシン，および，デサシルエスシンはα-アミラーゼに対して弱い阻害活性を示したが，小腸のα-グルコシダーゼ（マルターゼ，スクラーゼ）に対しては，ほとんど阻害効果は示さなかった．（3）あく抜き処理前の天然トチノミに由来するサポニン画分に比較して，あく抜き処理後の食用トチノミに由来するサポニン画分では，同一濃度において明らかに苦味の低減化が認められた．（4）食用トチノミに含まれるエスシンの有機酸エステルが加水分解されたサポニン類は，エスシンに比べて苦味が大きく低減化されており，また，血糖値の上昇抑制作用を保持しているので，機能性食品素材としてより好ましい．

Lipoxygenase is a dioxygenase recognizing a 1-cis,4-cis-pentadiene of polyunsaturated fatty acids. The enzyme oxygenates various carbon atoms of arachidonic acid as a substrate and produces 5-, 8-, 12- or 15-hydroperoxyeicosatetraenoic acid with a conjugated diene chromophore. The enzyme is referred to as 5-, 8-, 12- or 15-lipoxygenase, respectively. Earlier we found two isoforms of 12-lipoxygenase, leukocyte- and platelet-type enzymes, which were distinguished by substrate specificity, catalytic activity, primary structure, gene intron size, and antigenicity. Recently, the epidermis-type enzyme was found as the third isoform. Attempts have been made to find isozyme-specific inhibitors of 12-lipoxygenase, and earlier we found hinokitiol, a tropolone, as a potent inhibitor selective for the platelet-type 12-lipoxygenase. More recently, we tested various catechins of tea leaves and found that (−)-gallocatechin gallate was a potent and selective inhibitor of human platelet 12-lipoxygenase with an IC50 of 0.14 μM. The compound was much less active with 12-lipoxygenase of leukocyte-type, 15-, 8-, and 5-lipoxygenases, and cyclooxygenases-1 and -2.

We recently found that 12-O-tetradecanoyl phorbol-beta-acetate (TPA) induced apoptosis in cultured Madin-Darby canine kidney (MDCK) cells. The present study shows that the apoptosis was mediated by the activation of caspases including caspase-3 and -7. Moreover, nordihydroguaiaretic acid (NDGA), a general lipoxygenase (LOX) inhibitor, synergistically stimulated the TPA-induced apoptosis despite no activation with NDGA alone. TPA preferentially increased the transcription of cyclooxygenase (COX)-2 in MDCK cells, whereas the expression of LOXs was almost negligible. These findings suggested that the effect of NDGA was independent of the inhibition of LOXs. The study using a cell-permeable 2',7'-dichlorofluorescin diacetate confirmed the more remarked production of reactive oxygen species at 6 h after the cells were treated with a mixture of TPA and NDGA. Calcium ionophore A23187 was markedly effective to attenuate the TPA-induced apoptosis, indicating that elevated endogenous prostaglandins (PGs) served as survival factors through not only the activation of phospholipase A(2) by A23187 but also the induction of COX-2 by TPA. Consistent with this indication, exogenous addition of PGF(2alpha), a predominant prostanoid in MDCK cells, was the most potent to protect the cells from the apoptosis induced by a mixture of TPA and NDGA.

Adipocytes function not only as in the storage and mobilization of lipids but also as endocrine cells by secreting tumor necrosis factor-alpha (TNF-alpha), free fatty acids, and other cytokines. To study the effects of dietary lipids and metabolic factors on the control of the life cycle of adipocytes, we utilized mouse 3T3-L1 preadipocytes that could be induced to differentiate into adipocytes. To evaluate the role of endogenous prostaglandins (PGs) in the adipogenic changes, we examined the effect of specific inhibitors of cyclooxygenase (COX). SC-560, a specific COX-1 inhibitor, suppressed adipogenesis dose dependently, suggesting a role of constitutive COX-1 in the endogenous synthesis of PGs, including PGJ2 derivatives formed by mature adipocytes with the ability to promote adipogenesis. NS-398, a COX-2 inhibitor, had little influence on the maturation processes. Both COX inhibitors were effective in stimulating apoptosis of preadipocytes induced by TNF-alpha, indicating that both PGE2 and PGF2alpha produced by preadipocytes through the action of both COX isoforms serve as survival factors. However, the effect of both inhibitors was negligible for the proliferation of preadipocytes. Moreover, conjugated linolenic acid from bitter gourd at lower concentrations that was without effects by itself synergistically stimulated TNF-alpha-induced apoptosis. Therefore, dietary lipid factors are capable of controlling the life cycle of adipocytes together with metabolic factors.

(1) 食品として使用されているあく抜き処理したトチノミには,乾燥固形重量あたり0.3%のサポニン類が含まれていた.(2) あく抜き処理トチノミ中の主要なサポニンは,エスシン類のC22位のアセチル基が脱離した構造を持つ4種類の成分であった.(3) あく抜き処理したトチノミより分離したサポニン画分を用いて,マウスを用いた糖負荷試験を行ったところ,300mg/kg添加群は,糖負荷後0.5時間と1時間でコントロールの0mg/kg群に比較して,有意に血糖値上昇の抑制作用を示した.(4) 木灰液によるあく抜き処理の食用トチノミは,食品加工中に生成した新規の機能性食品因子を含むことを見い出した.

Both cyclooxygenase (COX) and lipoxygenase (LOX), as well as their metabolites, are suggested to play a dual role in regulating cell survival and apoptosis in various types of cells. However, their mechanism remains unclear. When Madin–Darby canine kidney (MDCK) cells were treated with 12-O-tetradecanoylphorbol β-acetate (TPA) and nordihydroguaiaretic acid (NDGA), a LOX inhibitor, we detected a synergistic stimulation of apoptosis as determined by hallmarks such as DNA ladder formation and chromatin condensation. The apoptosis was slightly induced by TPA alone. The effect of TPA was markedly stimulated along with NDGA. NDGA on its own did not have the ability to induce apoptosis. Aspirin, a COX inhibitor, was not effective. Alternatively, hydrogen peroxide-induced apoptosis was not promoted by the addition of NDGA. These two types of apoptosis would be regulated by a different mechanism. Although NDGA-induced apoptosis might be associated with the depletion of intracellular reduced glutathione in some kinds of tumor cells, N-acetyl cysteine (NAC), a thiol agent, had no inhibitory effect on the NDGA-induced apoptosis. To determine the further mechanism of NDGA-induced apoptosis in MDCK cells, we are examining the involvement of apoptosis-specific signaling molecules and the effect of lipoxygenase metabolites on NDGA-induced apoptosis.

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ(12,14)-PGJ2 and Δ(12)-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.

Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), an active phorbol diester, to quiescent cultured Madin-Darby canine kidney (MDCK) cells caused a maximal stimulation of phosphatidylethanol formation within 1-2 h in the presence of 1% ethanol, indicating the activation of phospholipase D (PLD). The specificity of phorbol diesters for the activation of PLD activation was confirmed by the fact that phorbol 12,13-dibutyrate (PDBu) was effective, whereas 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) was without effect. Down-regulation caused by the long-term pretreatment of the cells with active phorbol diesters significantly decreased the production of phosphatidylethanol. Staurosporine, a well known protein kinase (PK)C inhibitor at 1 microM, decreased the activation of PLD. Taken together, these observations suggested the involvement of PKC in the activation of PLD. The cellular PLD activity was found to be selectively localized in the particulate fraction by centrifugation at 12,000 x g. The particulate PLD showed the selective substrate specificity for phosphatidylcholine rather than phosphatidylethanolamine. In response to the addition of 100 nM PMA, 1,2-diacylglycerol (DG) increased in a biphasic fashion. In view of the time course of the activation of PLD, the second increase in the 1,2-DG around 20 min was contributed by the activation of PLD. In response to the simultaneous addition of 100 nM PMA and 100 nM A23187, the cultured MDCK cells activated the arachidonate cascades to form prostaglandin (PG)E2 and PGF2 alpha as major products, requiring slower 24 h to reach maximal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ(12)-PGJ(2) and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ(12)-PGJ(2). The assay provided a sensitive calibration curve for Δ(12)-PGJ(2), allowing us to determine a range from 0.16 pg to 0.99 ng with a value of 13 pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ(2) and 15d-PGJ(2) were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ(12)-PGJ(2) in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ(12)-PGJ(2) in a range from 10 to 200 pg/mL. The application of our ELISA revealed that the formation of Δ(12)-PGJ(2) became more pronounced after several hours of incubation of PGD(2) at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ(12)-PGJ(2) during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ(12)-PGJ(2), reflecting the biosynthesis of unstable PGD(2) in the culture system of adipocytes.

Lipoxygenases (EC 1.13.11.12, linoleate:oxygen, oxidoreductases, LOXs) which are widely found in plants, fungi, and animals, are a large monomeric protein family with non-heme, non-sulphur, iron cofactor containing dioxygenases that catalyze the oxidation of polyunsaturated fatty acids (PUFA) as substrate with at least one 1Z, 4Z-pentadiene moiety such as linoleic, linolenic and arachidonic acid to yield hydroperoxides (Gardner, 1991).

15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15d-PGJ₂) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD₂. To determine the endogenous synthesis of 15d-PGJ₂, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ₂. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ₂ was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ₂. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ₂ from 0.5 pg to 9.7 ng with 71 pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ₂ with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ₂ in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ₂ became more evident after several hours of incubation with authentic PGD₂ at 37 °C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ₂ and its roles in various cells and tissues.