Prostaglandins E and F2α in the house cricket and other insects

Prostaglandin E (PGE₁ and PGE₂) and prostaglandin F_2α (PGF_2α) are widely distributed in insects belonging to seven species representing six orders. Whole body concentrations range from 0.2 to 16 pg/mg protein. PGE and PGF_2α are found in muscle but are not abundant in testicular tissue. In the house cricket, Acheta domesticus (L.), high prostaglandin levels in the testes and the female spermatheca are reduced in the presence of the prostaglandin synthesis inhibitors aspirin, acetaminophen and indomethacin. An interaction of the prostaglandins with cyclic GMP and cyclic AMP in cricket tissues was not observed using these inhibitors. The house cricket appears to be a model for studying prostaglandin function in insects.     

Structure–function analysis of hepatitis C virus envelope glycoproteins E1 and E2

Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205–319 of E1 to aa421–716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is ₂₈₅FLVGQLFTFSPRRHW₂₉₉ which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches.     

Prostaglandin E2 modifies SMAD2 and promotes SMAD2–SMAD4 complex formation

We report that PGE₂ promotes Smad2–Smad4 complex formation and this phenomenon could be blocked by DIDS, an anion transporter inhibitor. Our data suggest that PGE₂ had no effects on Smad2 phosphorylation, suggesting that PGE₂-mediated Smad2–Smad4 complex formation is independent of TGF-β signaling and that PGE₂ induced Smad2 modification which is different from TGF-β-mediated phosphorylation. We demonstrate that in primary human glomerular mesangial cells PGE₂ caused modification of Smad2 as detected by Smad2N antibody, raised against a peptide near the N-terminus of Smad2. We hypothesize that Smad2 protein is post-translationaly modified by PGE₂. Direct evidence of Smad2 modification by PGE₂ was achieved by avidin pulldown assay which showed that endogenous Smad2 and recombinant Smad2 protein were attached by biotin-labeled PGE₂. Taken together, our results provided evidence that post-translational modification of Smad2 could be a mechanism for the action of PGE₂ in the pathogenesis of human pathologies.     

2′-(E)-O-p-coumaroylgalactaric acid and 2′-(E)-O-feruloylgalactaric acid in citrus

2′-(E)-O-p-Coumaroyl- and 2′-(E)-O-feruloylgalactaric acids, hitherto unknown in nature, have been isolated and identified from orange peel.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E₂ (PGE₂) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE₂ pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE₂ receptors) is essential. We therefore examined the production of PGE₂ in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE₂ on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE₂ receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE₂ synthesis was determined by mass spectrometry, cell proliferation by DNA [³H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE₂ into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE₂-dependent proliferation. Exogenously added PGE₂ stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10^-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE₂ was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE₂ release, which stimulates cell proliferation via the EP1 receptor.     

Effect of prostaglandins E2 and F2α on in vitro development and hatching of caprine blastocysts

Prostaglandin E₂ has been shown to increase the ovine embryo hatching rate, and PGF_2α to reduce the development of rabbit, bovine, and rat embryos. The objective was to determine the effects of PGE₂ and PGF_2α on development of caprine embryos. Estrus was synchronized in does (n = 25) with medroxyprogesterone acetate (MAP) intravaginal sponges for 12 days, and superovulated with 20 units of FSH. On day 6 following estrus, embryos were flushed (n = 128) and incubated individually per well in 25 μl droplets of TCM-199 and BSA (8 mg/ml) for 6 days at 38.5 °C in a 5% CO₂: air with one of the following treatments: (1) control (0.0002% EtOH), (2) PGE₂ (7 ng/ml), (3) PGF_2α (7 ng/ml), (4) low PGE₂:high PGF_2α (3.5 ng/ml:14 ng/ml), (5) balanced PGE₂:PGF_2α (7 ng/ml:7 ng/ml), or (6) high PGE₂:low PGF_2α (14 ng/ml:3.5 ng/ml). Treatment with PGE₂ alone reduced (P < 0.05) the hatching rate (1/15; 7%). The hatching rate of embryos treated with PGF_2α alone (9/18; 50%), low PGE₂:high PGF_2α (8/16; 50%), and balanced PGE₂:PGF_2α (11/16; 69%) were similar to control (6/18; 33%). In contrast, the hatching rate was non-significantly increased (13/18; 72%) with the high PGE₂:low PGF_2α treatment. None of the treatments affected development from the morula to blastocyst stage. From the current data, it can be concluded that PGE₂ alone reduced hatching rate, and PGF_2α alone had no effect on the development of caprine embryos. High concentrations of PGE₂ with PGF_2α improved the hatching rates. Thus, uterine concentrations of PGE₂ may need to reach a threshold level to improve embryo hatching, as previously reported, while increased uterine concentrations of PGF_2α during early pregnancy would not affect development of the embryo.     

Prostaglandin E2 and T cells: friends or foes?

Our understanding of the key players involved in the differential regulation of T-cell responses during inflammation, infection and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. With respect to this, the inhibitory role of the lipid mediator prostaglandin E(2) (PGE(2)) in T-cell immunity has been documented since the 1970s. Studies that ensued investigating the underlying mechanisms substantiated the suppressive function of micromolar concentrations of PGE(2) in T-cell activation, proliferation, differentiation and migration. However, the past decade has seen a revolution in this perspective, since nanomolar concentrations of PGE(2) have been shown to potentiate Th1 and Th17 responses and aid in T-cell proliferation. The understanding of concentration-specific effects of PGE(2) in other cell types, the development of mice deficient in each subtype of the PGE(2) receptors (EP receptors) and the delineation of signalling pathways mediated by the EP receptors have enhanced our understanding of PGE(2) as an immune-stimulator. PGE(2) regulates a multitude of functions in T-cell activation and differentiation and these effects vary depending on the micro-environment of the cell, maturation and activation state of the cell, type of EP receptor involved, local concentration of PGE(2) and whether it is a homeostatic or inflammatory scenario. In this review, we compartmentalize the various aspects of this complex relationship of PGE(2) with T lymphocytes. Given the importance of this molecule in T-cell activation, we also address the possibility of using EP receptor antagonism as a potential therapeutic approach for some immune disorders.     

Induction of Abortion by Extra-amniotic Administration of Prostaglandins E2 and F2 α

The use of prostaglandins E₂ and F₂α, administered by extra-amniotic instillation, for the induction of abortion was studied in 94 patients in the first and second trimesters of pregnancy. Abortion was successfully induced in 87% of patients within 36 hours and in 94% within 48 hours. The mean abortion time was 22·4 hours. In 60% of patients abortion was complete.Though the differences were not statistically significant, on average multigravid patients aborted more quickly than primigravidae, while the mean abortion time in PGE₂-treated patients was less than in those receiving PGF₂α.No serious complications occurred. Some side effects were observed. Occasional vomiting was the commonest symptom but the incidence of side effects was lower than with alternative routes of administration. A leucocytosis was often noted but there were no significant instances of infection.The method has proved a safe and effective means of terminating pregnancies in the second trimester.     

The association of prostaglandins E2 and F2α with erythropoiesis regulatory factors

Prostaglandins E₂ and F_2α have regulatory roles, respectively, in the potentiation and inhibition of erythropoietin. Antisera to PGF_2α and EIF, an erythropoiesis inhibitory factor, can be used to resolve the reason for improvement in erythropoiesis following renal dialysis. Concurrent loss of PGE₂ with Ep during treatment with neuraminidase suggests that PGE₂ is bound to Ep by way of sialic acid.     

The concentrations of urinary prostaglandins E and plasma prostaglandins F2α and E during induction of ovulation with human gonadotropins

Plasma prostaglandins F₂α and E (PGF₂α, PGE) and urinary PGE were measured in 10 women treated with human gonadotropins (HMG) and subsequently with human chorionic gonadotropins (HCG). Five women became pregnant (6 pregnancies). There was no correlation between concentrations of plasma PGF₂α or PGE and plasma estradiol or progesterone. Urinary PGE concentrations showed a positive correlation with estradiol before HCG and a negative correlation with progesterone after HCG, only in women who subsequently became pregnant.Higher urinary PGE concentrations before HCG suggest that either HMG or rising estradiol levels stimulate PGE renal production. The significant negative correlation.between urinary PGE and progesterone concentrations, after HCG, in those patients who became pregnant suggests that ovarian production of progesterone may decrease renal production of PGE.     

Effect of prostaglandins F2α and E2 on sensitivity of rabbit cortical neurons to acetylcholine and noradrenalin

The effect of prostaglandins F₂ and E₂ on unit activity and sensitivity of somatosensory cortical neurons of rabbits to acetylcholine and noradrenalin was investigated by microiontophoresis. Prostaglandin F₂ predominantly inhibited, whereas E₂ increased the spike activity of the neurons. F₂ usually modified the sensitivity of the neurons to acetylcholine, E₂ to noradrenalin. The influence of F₂ on the excitatory effects of acetylcholine, and of E₂ on the inhibitory effects of noradrenalin as a rule was characterized by weakening of the action of the mediators or a change in the sign of the initial responses. It is suggested that prostaglandins of the F group participate in cholinergic, and those of the E group in adrenergic transmission. The prostaglandins studied evidently exert their action through selective inhibition of the activity of adenylate or guanylate cyclases, leading to a decrease in the synthesis of secondary transmitters, namely cyclic AMP and cyclic GMP.B. K. Anokhin Research Institute of Normal Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 3, pp. 239–245, May–June, 1980.     

Variable synthesis and expression of E β and A e (E β ) Ia polypeptide chains in mice of differentH-2 haplotypes

The genetic and molecular requirements for cell-surface expression of Ia antigens precipitated by anti-I-E subregion sera have been examined. Inbred mice of thed, k, p, andr haplotypes synthesize and express on their lymphocytes the two I-region products normally found in anti-I-E-subregion immunoprecipitates, E and A_e (E ). Cells from mice of theb ands haplotypes fail to synthesize E chains but do synthesize A_e chains, which remain in the cytoplasm as partially glycosylated precursors. Cells of thef andq haplotypes fail to synthesize either the A_e or E polypeptide chains, as shown by both genetic complementation tests and analyses of total cell proteins by two-dimensional polyacrylamide gel electrophoresis. The patterns of expression of the intact E :A_e complex are consistent with the theory that both the A_e and E polypeptide chains must be present in the cells for either chain to be expressed in normal amounts on the cell surface. The implications of these observations for the genetics ofI-region-controlled functions are discussed.     

Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP

The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.     

Localization of E1–E2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling

Summary Tryptic peptides of Ca-ATPase in E_t and E₂ conformational states (Andersen, J. P., Jørgensen, P. L.,J. Membrane Biol. 88:187–198 (1985)) have been isolated by size exclusion high performance liquid chromatography in sodium dodecyl sulfate. This permitted unambiguous localization of a conformational sensitive tryptic split at Arg 198 by N-terminal amino acid sequence analysis. Other splits at Arg 505 and at Arg 819-Lys 825 were insensitive to E₁–E₂ transitions. Tryptic cleavage of Ca-ATPase after phosphorylation by inorganic phosphate showed that this enzyme form has a conformation similar to that of the vanadate-bound E₂ state, both in membranous and in soluble monomeric Ca-ATPase.Hydrophobic labeling of Ca-ATPase in sarcoplasmic reticulum vesicles with the photoactivable reagent trifluoromethyl-[¹²⁵I]iodophenyl-diazirine indicated that E₂ and E₂V states are more exposed to the membrane phase than E₁ and E₁P (Ca²⁺-occluded) states. The preferetial hydrophobic labeling in E₂ forms was found to be localized in the A₁ tryptic fragment.     

E2→E1 transition and Rb(+) release induced by Na(+) in the Na(+)/K(+)-ATPase. Vanadate as a tool to investigate the interaction between Rb(+) and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).     

Changes in prostaglandin E2 and F2α during vitellogenesis in the florida crayfish Procambarus paeninsulanus

While the role of eicosanoids in reproduction in vertebrate species has been well established, the role of these fatty acid derivatives in invertebrate species has not been as well characterized. The purpose of this study was to investigate changes in prostaglandins E₂ and F₂ during vitellogenesis in the crayfish Procambarus paeninsulanus. In homogenates of crayfish ovaries taken at various stages of development, the rate of prostaglandin synthesis and the concentrations of prostaglandins E₂ and F₂ increased during the final stages of yolk production just prior to ovulation. A gradual increase in prostaglandin E₂ amounts was observed throughout the progression of vitellogenesis. The data suggests the possible involvement of prostaglandins in regulatory events associated with vitellogenesis and the induction of ovulation in Procambarus paeninsulanus.Abbreviations EIA enzyme immunoassay kit - GIH gonad inhibiting hormone - HPLC high-pressure liquid chromatography - PGE₂ prostaglandin E₂ - PGF₂ prostaglandin F₂ - RIA radioimmuno assay - SNK Student-Newman-Keuls multiple t-test - TLC thin-layer chromatography