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Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD(2) and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD(2) synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE(2) was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense L-PGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE(2) and PGF(2α), while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- Δ(12,14)-prostaglandin J(2). These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ(2) series.

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E 2 and PGF 2α involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ 2 interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ 12 -PGJ 2 and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ 2 . Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ 2 or PDTC although 15d-PGJ 2 alone has no stimulatory effect. Moreover, 15d-PGJ 2 did not block the inhibitory effects of exogenous PGE 2 and PGF 2α on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ 2 can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D 2 can be produced in adipocytes and dehydrated to J 2 series of PGs including 15-deoxy-Δ 12,14 -PGJ 2 (15d-PGJ 2 ) and Δ 12 -PGJ 2 , which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ 12 -PGJ 2 has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ 12 -PGJ 2 . According to the standard curve, the amount of Δ 12 -PGJ 2 can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ 2 , while it only showed the cross-reaction of 28% with unstable PGJ 2 . This immunological assay was applied to the determination of the endogenous formation of Δ 12 -PGJ 2 in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ 12 -PGJ 2 increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ 2 . Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ 12 -PGJ 2 in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ 2 series at various doses on adipogenesis during the maturation phase. Although Δ 12 -PGJ 2 was slightly less potent than 15d-PGJ 2 , each of these PGJ 2 series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ 12 -PGJ 2 contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ 2 during the maturation phase of cultured adipocytes.

The synergistic stimulation of arachidonic acid release and prostaglandin (PG) E 2 production was observed when quiescent Madin-Darby canine kidney (MDCK) cells were exposed to phorbol 12-myristate 13-acetate (PMA) and Ca 2+ ionophore, A23187. Addition of PMA or A23187 by itself was not effective. When cells were treated with 50 n m PMA and 0.1 μ m A23187 at a suboptimal concentration, there was a marked increase in the production of PGE 2 . In the presence of higher concentrations of A23187 (5–10 μ m ), 50 n m PMA was only synergistic in potentiating the liberation of free arachidonic acid, but failed to stimulate the PGE 2 production. The amount of free arachidonic acid liberated by 50 n m PMA and 10 μ m A23187 reached a maximum level within several hours, whereas PGE 2 synthesis induced by 50 n m PMA and 0.1 μ m A23187 proceeded with a slower process requiring more than 24 h to reach a maximum. The stimulated PGE 2 synthesis was blocked by transcription and translation inhibitors. The addition of 50 n m PMA alone or the mixture of 50 n m PMA and 0.1 μ m A23187 was found similarly to increase the cellular PG endoperoxide synthase activity, suggesting that PMA was responsible for the increased enzyme activity. However, these agents failed to enhance the activities of phospholipases and PGE 2 synthase from PGH 2 . Northern blot analysis confirmed the increased level of PG endoperoxide synthase mRNA in the cells treated with PMA. The effect of PMA was mimicked by other protein kinase C activators. The pretreatment with PMA caused a down-regulation in the PGE 2 production. The stimulated PGE 2 production was abolished in the presence of selective protein kinase C inhibitors such as staurosporine and H-7. In addition, sphingosine, dihyrosphingosine, and psychosine, recently found to be protein kinase C inhibitors, blocked the effect of PMA in intact MDCK cells. Thus, the results indicate that the synergistically stimulated PGE 2 production with phorbol diesters and 0.1 μ m A23187 occurred principally through the de novo synthesis of PG endoperoxide synthase, also implying a role for protein kinase C.

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE(2) upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and troglitazone as peroxisome proliferator-activated receptor gamma (PPARgamma) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARgamma and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.

Recently, we have identified novel saponins from edible seeds of Japanese horse chestnut ( Aesculus turbinata BLUME) after processing the natural seeds with wood ashes to remove bitterness. We attempted to determine anti-obesity effects of those saponins from edible seeds as well as natural seeds. The purified individual components of saponins from natural and edible seeds inhibited pancreatic lipase in vitro. The potency was in the order of escins > desacylescins > deacetylescins. Escins Ib and IIb as well as deacetylescins Ib and IIb with the angeloyl moiety were more potent than the corresponding Ia and IIa series with the tigloyl moiety. Moreover, in vivo anti-obesity effects of the saponin fractions were monitored for 8 weeks in mice fed high-fat diets. Saponin fractions from both seeds significantly attenuated the elevation in body weight, the mass of peritoneal adipose tissues, and plasma triacylglycerol, which was accompanied by higher contents of undigested fats in feces without changes in food intake, indicating the effective inhibition of fat digestion in vivo. Taken together, saponin fractions including desacylescins and deacetylescins from edible seeds are potentially useful for the development of nutraceutical foods with anti-obesity effects and more attenuated bitter taste.

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-alpha. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-alpha induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-alpha. Antioxidants effectively reduced TNF-alpha-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-alpha-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E2 and PGF 2alpha. TNF-alpha preferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-alpha-induced apoptosis, which was reversed by exogenous PGE2 and PGF 2alpha. These results indicate that endogenous PGE2 and PGF 2alpha synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-alpha.

Natural seeds of Japanese horse chestnut (Aesculus turbinata Blume) contain large amounts of mixed triterpenoidal saponins called escins. Recent studies have shown that escins have several biological activities including anti-inflammatory action and inhibitory effects on the absorption of ethanol and glucose. For the edible utilization of the seeds, natural seeds are usually treated with wooden ashes to remove harshness. Here, we found the novel compounds derived from escins in the edible seeds after the food processing with wooden ashes. The instrumental analyses revealed the chemical structures of escins and the derivatives. These compounds are identified as four types of deacetylescins Ia, IIa, Ib, and IIb as well as two types of desacylescins I and II. To determine their biological activity, the purified compounds were tested for their potential nutraceutical activity. The oral glucose tolerance test in mice revealed that a single oral administration of the isolated components of deacetylescins at a dose of 100 mg/kg was clearly effective in attenuating the elevation of blood glucose levels. The inhibitory effects of escins and their derivatives were in the order of escins>deacetylescins>desacylescins. Moreover, we found the inhibitory activity of those compounds on pancreatic lipase. Escins were the most potent in inhibiting the enzyme activity, and followed by desacylescins and then deacetylescins. Taken together, our results suggest the potential usefulness of novel saponins including deacetylescins and desacylescins from edible seeds as novel sources for nutraceutical foods with anti-obese effects.

Mouse 8S-lipoxygenase (8-LOX) metabolizes arachidonic acid (AA) specifically to 8S-hydroperoxyeicosatetraenoic acid (8S-HPETE), which will be readily reduced under physiological circumstances to 8S-hydroxyeicosatetraenoic acid (8S-HETE), a natural agonist of peroxisome proliferator-activated receptor alpha (PPAR alpha). Here, we investigated whether 8-LOX could further oxygenate AA and whether the products could activate PPARs. The purified recombinant 8-LOX converted AA exclusively to 8S-HPETE and then to (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid (8S,15S-diHPETE). The kcat/Km values for 8S-HPETE and AA were 3.3x10(3) and 2.7x10(4) M(-1) s(-1), respectively. 8-LOX also dioxygenated 8S-HETE and 15S-H(P)ETE specifically to the corresponding 8S,15S-disubstituted derivatives. By contrast, 15-LOX-2, a human homologue of 8-LOX, produced 8S,15S-diH(P)ETE from 8S-H(P)ETE but not from AA nor 15S-H(P)ETE. 8S,15S-diHETE activated PPAR alpha more strongly than 8S-HETE did. The present results suggest that 8S,15S-diH(P)ETE as well as 8S-H(P)ETE would contribute to the physiological function of 8-LOX and also that 8-LOX can function as a potential 15-LOX.

Potato tubers are shown to contain a unique lipoxygenase pathway to form 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) from linoleic acid. Here, we report the metabolic pathway of 9-HPODE in the cytosolic fraction and the characterization of enzymes involved in the conversion of metabolites. The analysis of enzymatic reaction products at pH 5.5 revealed the formation of 9-keto-10,12-octadecadienoic acid, 9-hydroxy-10,12-octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid. The cytosolic enzymes were separated by anion-exchange chromatography into two fractions E1 and E2, having molecular masses of 66 and 54 kDa, respectively. The enzyme fraction E1 only produced 9-keto-10,12-octadecadienoic acid, whereas E2 formed other products. The enzyme E1 showed higher reactivity with 13- and 9-hydroperoxide of alpha-linolenic acid than 9-HPODE, but no reaction with hydroxy fatty acids. In contrast, the enzyme E2 showed the highest reactivity with 9-HPODE, followed by hydroperoxides of alpha-linolenic acid and arachidonic acid. We also evaluated the antibacterial activity of hydroxy fatty acids against Erwinia carotovora T-29, a bacterium infecting potato tubers. Growth of the bacteria was suppressed more potently with 9- or 13-hydroxy fatty acids than dihydroxy or trihydroxy fatty acids, suggesting a role for the metabolites in the resistance of bacterial infection.

The arachidonate cascade generates a series of lipid mediators to regulate various biological events through cyclooxygenase (COX) and lipoxygenase (LOX) pathways [[3],[5]]. In addition, both COX and LOX metabolites were reported to play a dual role in regulating cell survival and death depending on certain types of cells or signalling molecules [1,[4]]. However, the cellular mechanism by which these eicosanoids mediate the induction or suppression of apoptosis remains still unclear.

Obesity is a risk factor to cause complicated diseases such as diabetes and others. Obesity accompanies an increase in the number or size of adipocytes, which should involve the changes in the life cycle of fat cells. During these processes, peroxisome proliferator-activated receptor (PPAR)γ, which is a member of nuclear receptors, plays an important role in the differentiation and the progression of insulin resistance in adipocytes [4]. PPARγ was originally recognized by the fact of the activation by antidiabetic drugs. Later, fatty acids and eicosanoids were found to activate PPARγ as endogenous ligands [1,3].

Plant phospholipase D (PLD) has been implicated in many cellular processed such as lipid deterioration under senescence and stress, lipid turnover during normal growth and development, and lipid-derived signaling pathways [Wang, 2000]. Plant PLD activity is also known to be stimulated during germination, but the molecular mechanism for stimulation of PLD activity remains unclear. PLD pathway is considered to be linked to PLC pathway because these metabolites are enzymatically interconverted and phospholipase C (PLC) increases intracellular levels of Ca2+ to induce activation of Ca2+-dependent enzyme such as PLD. As light stimulated turnover of phosphatidyl inositol catalyzed by PLC in various types of plants [Morse, et al., 1987], light might be a trigger for stimulation of PLD activity during germination

The arachidonate cascade includes the cyclooxygenase (COX) pathway to form prostanoids and the lipoxygenase (LOX) pathway to generate several oxygenated fatty acids, collectively called eicosanoids. Eicosanoids are suggested to play a dual role in regulating cell survival and apoptosis in various types of cells through an unknown mechanism. We found apoptosis in cultured Madin-Darby canine kidney (MDCK) cells treated with 12-O-tetradecanoylphorbol beta-acetate (TPA), a potent tumor promoter, and nordihydroguaiaretic acid (NDGA), a LOX inhibitor. The effect of TPA was synergistically stimulated along with NDGA. Aspirin, a COX inhibitor, was not effective. The target of NDGA might be different from the mechanism involving a LOX activity in some kinds of carcinoma cells because the increased expression of 12-LOX was not detected in MDCK cells treated with TPA. Caspase and poly(ADP-ribose) metabolites were found to be involved in the signal transduction pathway of the TPA- and NDGA-induced apoptosis in MDCK cells. Alternatively, hydrogen peroxide-induced apoptosis was not affected by NDGA. Thus, the TPA-induced response involved the mechanism independent of the oxidative stress. Obesity is a risk factor for severe diseases including noninsulin-dependent diabetes and atherosclerosis characterized by the changes of cell properties of adipocytes. We found that conjugated linolenic acid from bitter gourd was able to induce apoptosis in mouse preadipogenic 3T3-L1 cells. The findings provide the potential use of conjugated fatty acids to regulate obesity.

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh; Center for Integrated Research in Science, Shimane University; United Graduate School of Agricultural Sciences, Tottori University; Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University; Department of Clinical Pharmacy, Faculty of Pharmacy, Tokushima Bunri University

Madin-Darby canine kidney (MDCK) cells were modified with dietary unsaturated fatty acids. The effects on the fatty acid composition in each phospholipid class and the formation of prostanoids upon stimulation were studied, from which the specificity of metabolism of individual unsaturated fatty acids and the regulation of arachidonate cascades in the modified cells were discussed. C18 unsaturated fatty acids were preferentially incorporated into phosphatidylcholine (PC) over phosphatidylethanolamine (PE), but arachidonic acid (20:4(n-6)) derived from gamma-linolenic acid (18:3(n-6)) was much more predominant in PE than PC. The fatty acid level in PE ranged from about 26-28% when the cells were modified with 20:4(n-6) or 5,8,11,14,17-eicosapentaenoic acid (20:5(n-3)), indicating the limitation of the storage of the eicosapolyenoic acids. The extra amounts appeared to be stored in PC. 18:3(n-6) was comparable to 20:4(n-6) to raise the level of 20:4(n-6) in PE, but not in PC which had half of 20:4(n-6) in PE. The supplementation of linoleic acid (18:2(n-6)). 18:3(n-6), and 20:4(n-6) caused significant increases in the synthesis of prostaglandin (PG)E2 up to almost the same levels when the modified cells were stimulated with 50 nM PMA and 100 nM A23187 for 24h. The cultured cells modified with eicosapolyenoic acids including 20:3(n-6), 20:4(n-6), and 20:5(n-3) were found to be inhibitory for the induction of PGE2 synthetic activity involving de nova synthesis of PG endoperoxide synthase, suggesting negative feedback regulation of the modified cells.

13,14-Dihydro-15-ketoprostaglandin (PG) F 2α was separated completely from PGD 2 , PGE 2 and PGF 2α , within 10–15 min by gas chromatography—selected-ion monitoring on a capillary column after conversion to their methyl ester— n -butyloxime—dimethylisopropylsilyl (ME—nBO—DMiPS) ether or ME—nBO—cyclic diethylsilylene (DES) derivatives. The mass spectra were characterized by their inherent ions of [M — 43] + at m/z 596 for the ME—nBO—DMiPS ether and of [M — 73] + at m/z 450 for the ME—nBO—DES derivatives, respectively. The appearance of the characteristic ions in the high-mass region with relatively high intensity and the use of these ions in selected-ion monitoring in a high-resolution mode ( M/ΔM = 3500) made it possible to enhance the selectivity in the detection of 13,14-dihydro-15-keto-PGF 2α in biological fluids. The detection limit of the ME—nBO—DES derivative of 13,14-dihydro-15-keto-PGF 2α was found to be 2 pg with a signal-to-noise ratio of 4:1 when the ion of [M — 73] + was monitored at m/z 450.30.

A solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and beta-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound beta-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2,3-Dinor- and 2,3,4,5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80% and the results correlated well with the values obtained by radioimmunoassay (r = 0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2,3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.

Lipid molecular species compositions of chloroplast thylakoid membranes of mesophyll cells from Spinacia oleracea, Glycine max, Oryza sativa and Zea mays and of bundle sheath cells from Zea mays have been quantitatively determined. No significant difference in the lipid molecular species composition was found among the five membrane sources. The predominant molecular species of monogalactosyldiacylglycerol was the 1-linolenoyl parallel to 2-linolenoyl species. The 1-linolenoyl parallel to 2-linoenoyl and 1-palmitoyl parallel to 2-linolenoyl species were the major molecular species of digalactosyldiacylglycerol. 6-Sulfoquinovosyldiacylglycerol was mainly composed of the palmitoyl parallel to linolenoyl and palmitoyl parallel to lineolyl species. Almost all of the C-2 position of phosphatidylglycerol were esterified with the palmitoyl or delta 3-trans-hexadecenoyl residue. The molecular species compositions of phosphatidylcholine and phosphatidylinositol were basically similar to those of membranes in non-photosynthetic tissues.

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.

A solid-phase enzyme immunoassay of 6-ketoprostaglandin F1 alpha (a stable degradation product of prostaglandin I2) was developed in which the hapten molecule was labeled with beta-galactosidase. The antiserum was bound to a polystyrene tube, and the enzyme-labeled and unlabeled 6-ketoprostaglandin F1 alpha were allowed to react in a competitive manner with the immobilized antibody. The activity of the immunologically bound beta-galactosidase was assayed by fluorometry, and correlated with the amount of unlabeled 6-ketoprostaglandin F1 alpha. According to a calibration curve 6-ketoprostaglandin F1 alpha was detectable in a range of 30 fmol-10 pmol. When 6-ketoprostaglandin F1 alpha was extracted from human serum by using an octadecylsilyl silica column (Sep-Pak C18) and the crude extract was subjected to the enzyme immunoassay, the content of 6-ketoprostaglandin F1 alpha was 50-60 pmol/ml of human serum. An endogenous substance(s) which disturbed the immunoreaction and gave such an apparently high concentration of 6-ketoprostaglandin F1 alpha separated from the endogenous 6-ketoprostaglandin F1 alpha by HPLC. With the purified 6-ketoprostaglandin F1 alpha fraction there was a good correlation (r = 0.994) between enzyme immunoassay and radioimmunoassay. The validity of the enzyme immunoassay was confirmed by gas chromatography-selected ion monitoring.

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.

Abietic acid is one of the terpenoids, which are multifunctional natural compounds. It has been reported that abietic acid suppresses effects on inflammation. However, the mechanism underlying the anti-inflammatory effects remains unclear. The present work indicates that abietic acid suppresses the protein expression of tumor necrosis factor-alpha and cyclooxygenase 2, which are involved in inflammation, in lipopolysaccharide-stimulated macrophages. Moreover, this effect resembles that of thiazolidinedione, a synthetic peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand. Indeed, abietic acid activates PPARgamma in luciferase reporter assays. The activity of abietic acid induces PPARgamma target gene expression in RAW264.7 macrophages and 3T3-L1 adipocytes. These data indicate that abietic acid is a PPARgamma ligand and that its anti-inflammatory effect is partly due to the activation of PPARgamma in stimulated macrophages. The present work suggests a novel possibility that abietic acid, a naturally occurring compound, can be used not only for anti-inflammation but also for regulating lipid metabolism and atherosclerosis.

The effect of recombinant interleukin 1 Beta (IL-1(beta)) was investigated on osteoblastic cell line MC3T3-E1 cloned from mouse calvaria. IL-1(beta) stimulated cell proliferation which increased cell number and caused dose-related stimulation of DNA synthesis, with a maximal effect at a concentration of 12.5 U/ml; suppressed alkaline phosphatase activity and collagen synthesis maximally at 0.5 and 62.5 U/ml, respectively; and increased the amount of free [3H] hydroxyproline in the cultures, but the amount was quite low. Prostaglandin E2 synthesis was also stimulated dose dependently by the presence of IL-1(beta), with a maximal increase at 2.5 U/ml, at which concentration the prostaglandin E2 level in the medium was 1.61 +/- 0.10 ng/ml. The increased prostaglandin E2 synthesis did not affect either the IL-1(beta)-mediated change in DNA or collagen synthesis or alkaline phosphatase activity. These results extend the possibility that IL-1(beta) is to act as a regulator of bone formation.

A sensitive heterologous enzyme immunoassay for prostaglandin E2 was developed using 9-deoxy-9-methylene-prostaglandin F2 alpha as a stable prostaglandin E2 mimic. beta-Galactosidase was conjugated to the hapten mimic. Anti-prostaglandin E2 IgG was bound to a polystyrene tube. The enzyme-labeled hapten mimic mixed with unlabeled prostaglandin E2 was allowed to react in a competitive manner with the immobilized antibody. Then, the beta-galactosidase specifically bound to the antibody was assayed fluorometrically, and the enzyme activity was correlated with the amount of unlabeled prostaglandin E2. According to the calibration curve thus obtained, prostaglandin E2 could be determined in a range of 1.2-430 fmol. Prostaglandin E2 was extracted from human urine by the use of an octadecylsilyl silica column. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high content of prostaglandin E2. The interfering substance was separated from prostaglandin E2 by reverse-phase high-performance liquid chromatography. The purified urinary extract was examined by the enzyme immunoassay for prostaglandin E2, and the validity of the results was confirmed by gas chromatography-selected ion monitoring.