Prostaglandin E2 increases the calcium concentration in rat brown adipocytes and their consumption of oxygen

Effects of prostaglandin E₂ (PGE₁) were examined on the oxygen consumption and intracellular calcium concentration of rat brown adipose tissue (BAT). PGE₂ 0.1 nM-1 μM increased oxygen consumption of the tissue blocks of BAT, with a maximum 2–13 min after PGE2 administration. PGE₂ was most effective at 1 and 10 nM, and the oxygen consumption was elevated for over 40 min. Pretreatment of BAT with indomethacin, a prostaglandin synthesis inhibitor, did not affect the increase in oxygen consumption induced by noradrenaline. PGE₂ at 1–10 nM gradually increased the intracellular calcium concentration of freshly dispersed single brown adipocytes by 3–4 times in 30 min. PGE₂ also increased the intracellular calcium concentration of brown adipocytes in calcium-free medium. These results raise the possibility that PGE₂ and noradrenaline affect heat genesis and metabolism of BAT independently.     

Controlled trial of induction of labor by vaginal suppositories containing prostaglandin E2

A group of 84 women at 39 – 43 weeks of pregnancy were randomly allocated to a blind trial of induction of labor with vaginal suppositories containing inert material or either 0.2 mg or 0.4 mg of prostaglandin E₂. The suppositories were self-administered every two hours during waking hours on two successive days until labor started or 15 had been used. Side-effects were absent. Labor was established within 48 hr of insertion of the first suppository in 9.3% of control patients, 65.4% of those treated with 0.2 mg PGE₂ and 85.7% of those treated with 0.4 mg PGE₂. The mean Apgar scores in the three groups were the same. The mean total dose of PGE₂ were 2.0 mg (0.2 mg group) and 2.3 mg (0.4 mg group). It is concluded that vaginal PGE₂ is an effective and acceptable method of inducing labor at term.     

1α,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1α,25(OH)₂D₃ and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1α,25(OH)₂D₃ regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1α,25(OH)₂D₃-binding protein ERp60, phospholipase A₂ (PLA₂), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1α,25(OH)₂D₃ (10⁻⁸ M), which binds ERp60, activating PLA₂, and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-β1 stored in the ECM as large latent TGF-β1 complexes, consisting of latent TGF-β1 binding protein, latency associated peptide, and latent TGF-β1. Others have shown that MMP-2 specifically activates TGF-β2. TGF-β1 regulates 1α,25(OH)₂D₃-production, providing a mechanism for local control of growth factor activation. 1α,25(OH)₂D₃ activates PKCα in the PM via ERp60-signaling through PLA₂, lysophospholipid production, and PLCβ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCζ. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1α,25(OH)₂D₃, PKCζ activity is decreased and PKCα is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.     

Prostaglandin E2 produced by inducible COX-2 and mPGES-1 promoting cancer cell proliferation in vitro and in vivo

Aim

Many cancers originate and flourish in a prolonged inflammatory environment. Our aim is to understand the mechanisms of how the pathway of prostaglandin E₂ (PGE₂) biosynthesis and signaling can promote cancer growth in inflammatory environment at cellular and animal model levels.

Main methods

In this study, a chronic inflammation pathway was mimicked with a stable cell line that over-expressed a novel human enzyme consisting of cyclooxygenase isoform-2 (COX-2) linked to microsomal (PGE₂ synthase-1 (mPGES-1)) for the overproduction of pathogenic PGE₂. This PGE₂-producing cell line was co-cultured and co-implanted with three human cancer cell lines including prostate, lung, and colon cancers in vitro and in vivo, respectively.

Key findings

Increases in cell doubling rates for the three cancer cell types in the presence of the PGE₂-producing cell line were clearly observed. In addition, one of the four human PGE₂ subtype receptors, EP₁, was used as a model to identify PGE₂-signaling involved in promoting the cancer cell growth. This finding was further proven in vivo by co-implanting the PGE₂-producing cells line and the EP₁-positive cancer cells into the immune deficient mice, after that, it was observed that the PGE₂-producing cells promoted all three types of cancer formation in the mice.

Significance

This study clearly demonstrated that the human COX-2 linked to mPGES-1 is a pathway that, when mediated by the EP, is linked to promoting cancer growth in a chronic inflammatory environment. The identified pathway could be used as a novel target for developing and advancing anti-inflammation and anti-cancer interventions.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Effects of prostaglandin E2 on the micronucleus formation in mouse bone marrow cells by various mutagens

The effects of prostaglandin E₂ (PGE)₂, as trigger of erythroid progenitor cells into the cell cycle, were studied on the induction of micronucleu by various mutagens; with mitomicin C (MMC) the optimal protocol was established. dose-response relationship between PGE₂ doses and micronucleus frequency were observed 30 h after injection of MMC to mice administered PGE₂ 24 h previously. Sensitazion by PGE₂ pretreatment was also found for other mutagens, such as vincristine, 5-fluorouracil, benzo[a]pyrene, 1,1-dimethylhydrazine and 2-naphthylamine. These results support the hypothesis that accelerating the erythropoiesis increases the frequency of micronucleic induced by mutagens.     

Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

The use of oral prostaglandin E2 in the management of intrauterine fetal death

12 otherwise healthy patients with intrauterine fetal death 1 to 6 weeks earlier were treated with oral prostaglandin E₂. 9 of the 12 patients delivered within 48 hours after treatment began. 2 others delivered within 48 hours after unsuccessful treatment ceased. In a third patient the cervix relaxed after treatment, and the uterine contents were removed by curettage. No serious complications, such as hemorrhage occurred. The uterus seemed surprisingly responsive to oral prostaglandin E₂ in cases of intrauterine fetal death.     

Sn-S and Ru-Ru bonds cleavage reactions between [Ph3SnS(CH2)3SSnPh3] and Ru3(CO)12: X-ray crystal structures of [Ph3SnS(CH2)3SSnPh3] and trans-[Ru(CO)4(SnPh3)2]

The organotin complex [Ph₃SnS(CH₂)₃SSnPh₃] (1) was synthesized by PdCl₂ catalyzed reaction between Ph₃SnCl and disodium-1,3-propanedithiolate which in turn was prepared from 1,2-propanedithiol and sodium in refluxing THF. Reaction of 1 with Ru₃(CO)₁₂ in refluxing THF affords the mononuclear complex trans-[Ru(CO)₄(SnPh₃)₂] (2) and the dinuclear complex [Ru₂(CO)₆(μ-κ²-SCH₂CH₂CH₂S)] (3) in 20 and 11% yields, respectively, formed by cleavage of Sn-S bond of the ligand and Ru-Ru bonds of the cluster. Treatment of pymSSnPPh₃ (pymS = pyrimidine-2-thiolate) with Ru₃(CO)₁₂ at 55-60 °C also gives 2 in 38% yield. Both 1 and 2 have been characterized by a combination of spectroscopic data and single crystal X-ray diffraction analysis.     

Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE₂ (KH₂-PGE₂) which has a longer half-life in blood. We examined the effects of PGE₂ infusion and alterations in lipolysis , and of clotting, prolonged storage and hemolysis , on KH₂-PGE₂ immunoreactivity in unextracted human plasma and serum samples. Indeed KH₂-PGE₂ levels rose several hundred fold during infusions of PGE₂ at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH₂-PGE₂ levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH₂-PGE₂ levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH₂-PGE₂ levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH₂-PGE₂ measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores or from disrupted membrane phospholipids . Unextracted plasma appears to be unsatisfactory for use in this RIA.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

Cellular senescence involves an intracrine prostaglandin E2 pathway in human fibroblasts

Cyclooxygenase 2 and release of prostaglandin E₂ are involved in many responses including inflammation and are upregulated during cellular senescence. However, little is known about the role of lipid inflammatory mediators in senescence. Here, we investigated the mechanism by which the COX-2/PGE₂ axis induces senescence. Using the NS398 specific inhibitor of COX-2, we provide evidence that reactive oxygen species by-produced by the COX-2 enzymatic activity are negligible in front of the total senescence-associated oxidative stress. We therefore investigated the role of PGE₂ by invalidating the PGE₂ synthases downstream of COX-2, or the specific PGE₂ receptors, or by applying PGE₂ or specific agonists or antagonists. We evaluated the effect on senescence by evaluating the senescence-associated proliferation arrest, the percentage of senescence-associated β-galactosidase-positive cells, and the expression of senescent molecular markers such as IL-6 and MCP1. We show that PGE₂ acting on its EP specific receptors is able to induce both the onset of senescence and the maintenance of the phenotype. It did so only when the PGE₂/lactate transporter activity was enhanced, indicating that PGE₂ acts on senescence more via the pool of intracellular EP receptors than via those localized at the cell surface. Treatment with agonists, antagonists and silencing of the EP receptors by siRNA revealed that EP3 was the most involved in transducing the intracrine effects of PGE₂. Immunofluorescence experiments confirmed that EP3 was more localized in the cytoplasm than at the cell surface. Taken together, these results suggest that COX-2 contributes to the establishment and maintenance of senescence of normal human fibroblasts via an independent-ROS and a dependent-PGE₂/EPs intracrine pathway.     

PGE2 and PGF2α release by human peritoneal macrophages in endometriosis

Objective: To test for differences in the amount and activity of peritoneal macrophages present in the peritoneal fluid of women with, and without endometriosis using prostaglandin release by macrophages in culture as a marker.Patients: Women of reproductive age undergoing laparoscopy for infertility or chronic pelvic pain with postoperative diagnosis of endometriosis and women undergoing laparoscopy for sterilization.Methods: Peritoneal fluid was aspirated during laparoscopy, volume was recorded, macrophages were isolated via a Ficoll Paque gradient and kept in primary culture. PGE₂ and PGF_2α release of the cells were measured before and after stimulation with zymosan.Results: Women with endometriosis had significantly more peritoneal macrophages than controls. Peritoneal macrophages of women with endometriosis released significantly more PGE₂ than those of the control group: 8.4 ± 2.0 versus 1.4 ± 0.4 ng/ml/10⁶cells (mean ± SEM, p=0.0005) and PGF_2α : 10 ± 4.3 (endometriosis) versus 1.8 ± 0.4 (control) ng/ml/10⁶cells (mean ± SEM, p = 0.045).Conclusion: There is a significant increase in the amount of prostaglandins released by peritoneal macrophages from women with endometriosis. These prostaglandins might alter uterine and tubal contractility, thereby affecting fertility.     

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E₂ 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E₂ titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E₂ or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E₂ raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Kinetic studies of the reduction of hexaaquacobalt(III) perchlorate by a cobaloxime, [Co(dmgBF2)2(H2O)2]

The reaction of with Co(dmgBF₂)₂(H₂O)₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 9.23 ± 0.14 × 10² s⁻¹. The [H⁺] dependence at lower temperatures shows some saturation effect that allowed an estimate of the hydrolysis constant for as K_a = 9.5 × 10⁻³ M at 10 and 15 °C. Marcus theory and the known self-exchange rate constant for Co(OH₂)₅OH^2+/+ were used to estimate an electron self-exchange rate constant of k₂₂ = 1.7 × 10⁻⁴ M⁻¹ s⁻¹ for .     

Prostaglandin I2 as a potentiator of acute inflammation in rats

Prostaglandin I₂ potentiated the paw swelling induced by carrageenin in rats. Prostaglandin I₂ (0.1 μg) showed similar activity to PGE₁ (0.01 μg). This potentiating property disappeared in 60 minutes and was completely abolished by diphenhydramine (25 mg kg⁻¹, i.p.). In vascular permeability tests, PGI₂ itself (2.5 × 10⁻¹⁰ mol, 88 ng) caused no dye leakage reaction, but PGE₁ (2.5 × 10⁻¹⁰ mol, 88.5 ng) caused a significant dye leakage. This effect of PGE₁ was statistically significant compared with vehicle- or PGI₂-treated group (p<0.05). Prostaglandin I₂ potentiated the increased vascular permeability induced by 5-hydroxytriptamine (2.5 × 10⁻¹⁰ mol), bradykinin (5 × 10⁻¹⁰ mol) and histamine (2 × 10⁻¹⁰ to 2 × 10⁻⁸ mol). The potentiation was the most evidence in the case of histamine.     

A borreliacidal factor in Amblyomma americanum saliva is associated with phospholipase A2 activity

Previous work in our laboratory described the in vitro killing of Borrelia burgdorferi when co-cultured with saliva from adult Amblyomma americanum. Borreliacidal activity was not evident using Ixodes scapularis saliva. Mixing trypsin with saliva eliminated the borreliacidal activity of A. americanum saliva, while incorporating a trypsin inhibitor restored all borreliacidal activity, indicating this factor was of protein or peptide origin. One-dimensional PAGE indicated at least 7 major protein differences between I. scapularis and A. americanum saliva. To determine the borreliacidal factor, A. americanum saliva was fractionated by gel filtration and subsequent killing of B. burgdorferi was associated with a single fraction. Two-dimensional gel analysis indicated protein and/or peptide(s) in borreliacidal fractions running between 38 and 64 kDa. Finally, admixing saliva with the phospholipase A₂ inhibitor oleyloxyethyl phosphorylcholine completely eliminated the ability of A. americanum saliva to kill B. burgdorferi. These studies indicate the borreliacidal activity found in A. americanum saliva is likely due to phospholipase A₂ enzymatic activity.     

Transesterification of sunflower oil to biodiesel on ZrO2 supported La2O3 catalyst

ZrO₂ supported La₂O₃ catalyst prepared by impregnation method was examined in the transesterification reaction of sunflower oil with methanol to produce biodiesel. It was found that the catalyst with 21 wt% loaded La₂O₃ and calcined at 600 °C showed the optimum activity. The basic property of the catalyst was studied by CO₂-TPD, and the results showed that the fatty acid methyl ester (FAME) yield was related to their basicity. The catalyst was also characterized by TG–DTA, XRD, FTIR, SEM and TEM, and the mechanism for the formation of basic sites was discussed. It was also found that the crystallite size of support ZrO₂ decreased by loading of La₂O₃, and the model of the solid-state reaction on the surface of La₂O₃/ZrO₂ catalyst was proposed. Besides, the influence of various reaction variables on the conversion was investigated.     

A novel hepatopancreatic phospholipase A2 from Hexaplex trunculus with digestive and toxic activities

A marine snail digestive phospholipase A₂ (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A₂, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 °C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6 mM CaCl₂. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases.Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA₂. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A₂ from venoms and another class of non toxic pancreatic phospholipase A₂ from mammals.