[Marine fungus Stilbella aciculosa as a potential producer of prostaglandins].

The amount and composition of fatty acids in the fungus Stilbella aciculosa associated with the marine macroorganism Apostichopus japonica (trepang) were determined by gas-liquid chromatography and gas chromatography-mass spectrometry. In the culture liquid of S. aciculosa, prostaglandins (PG) of groups E and F were revealed by UV spectroscopy. This finding was confirmed by the presence of direct precursors of PG, polyunsaturated eicosapentaenoic and docosahexaenoic acids, in the culture liquid. The biomass of this fungus contained PG of group B.

Anti-inflammatory constituents of the red alga Gracilaria verrucosa and their synthetic analogues.

A chemical study on the anti-inflammatory components of the red alga Gracilaria verrucosa led to the isolation of new 11-deoxyprostaglandins ( 1- 4), a ceramide ( 5), and a C 16 keto fatty acid ( 6), along with known oxygenated fatty acids ( 7- 14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds 1- 5 were determined by Mosher's method. The anti-inflammatory activity of the isolated compounds ( 1- 14) was evaluated by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most potent activity. In the evaluation of these two compounds and derivatized analogues ( 15- 40), the anti-inflammatory activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential anti-inflammatory leads for the first time.

Synthesis and action of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), in Dictyostelium discoideum.

The cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is synthesized from prostaglandin E (PGE) and activated inositol phosphate (n-IP) in the presence of ATP by an enzyme of rat liver plasma membranes. Extracts of the slime mould Dictyostelium discoideum contain this activated inositol phosphate and D. discoideum cells convert [3H]PGE1 to [3H]cyclic PIP. This extracted polar [3H]product co-chromatographed with cyclic PIP from rat liver on gel filtration, anion exchange- and adsorption chromatography. Starving D. discoideum cells show cyclic AMP-induced oscillations, which can be inhibited by cyclic PIP (0.4 x 10(-7) M), but not by its phosphomonoester prostaglandylinositol phosphate (PIP) (1.4 x 10(-7) M). AMP and ADP at much higher concentrations (1 mM) antagonized these oscillations. The time needed for aggregation and fruiting body formation of starving D. discoideum cells is extended by cyclic PIP (1.4 x 10(-7) M) up to 3-fold, whereas its phosphomonoester (1.9 x 10(-7) M) showed a 9-fold weaker effect, and AMP and ADP even at 1 mM concentration showed no effect.

The endogenous cyclic AMP antagonist, cyclic PIP: its ubiquity, hormone-stimulated synthesis and identification as prostaglandylinositol cyclic phosphate.

This report shows that the cyclic AMP antagonist cyclic PIP is present in all organs and tissues of the rat so far examined: brain, heart, lung, intestine, kidney, liver, spleen, skeletal muscle and fat. The synthesis of cyclic PIP is stimulated by insulin or noradrenaline (alpha-adrenergic action) in a dose-dependent fashion. Increasing cyclic PIP synthesis with increasing insulin concentrations matches the insulin receptor binding curves. Cyclic PIP levels in blood serum remain low after hormonal stimulation and no cyclic PIP can be detected in urine. As an indication of its ubiquity, cyclic PIP was even detected in yeast. Prostaglandin E (as shown by incorporation of [3H]PGE into cyclic PIP and demonstration of a constant specific activity), myo-inositol (as shown by acid hydrolysis of the dephosphorylated cyclic PIP and mass spectrometric identification of the products) and one phosphate (as shown by the ionic nature of cyclic PIP and its inactivation by phosphodiesterase plus phosphatase) are components of cyclic PIP. Chemical derivatization experiments of cyclic PIP suggest the phosphate to be bound to myo-inositol and the myo-inositol phosphate to the prostaglandin E by its C15-hydroxyl group.

Biochemical and immunohistochemical demonstration of a tightly bound form of prostaglandin E2 in the rat brain.

Basal levels of prostaglandin E2 in the rat brain were determined by radioimmunoassay to be 0.68-0.79 pmol/g brain. About one-third of the prostaglandin E2 (0.23-0.28 pmol/g) was resistant to extraction with ethanol, but could be recovered with a mixture of ethanol and 1 N HCl (9:1, v/v), indicating that a tightly bound form of prostaglandin E2 exists in the brain. The amount of the bound form of prostaglandin E2 was almost unchanged by pentylenetetrazole-induced convulsion or by transcardial perfusion with a formaldehyde solution, although these treatments resulted in 40- to 80-fold increases in prostaglandin E2 content extracted with ethanol at neutral pH. A polyclonal antibody against prostaglandin E2-albumin conjugates recognized the bound form of prostaglandin E2, giving a punctate appearance in many neuronal cell bodies in the brain. Although almost all of the neuronal perikarya were immunoreactive for prostaglandin E2, intense immunoreactivity was observed in the mitral cell layer of the olfactory bulb, layer V of the cerebral neocortex, anterodorsal and reticular nuclei of the thalamus, supraoptic, paraventricular, accessory neurosecretory and lateral mammaillary nuclei of the hypothalamus, mesencephalic trigeminal nucleus, nucleus of the trapezoid body and deep cerebellar nuclei. When the cerebral neocortical regions were observed electron microscopically, immunoreaction products were seen as fine granules which were clustered into small patches in the cytoplasm of neuronal cell bodies and proximal dendrites. No immunoreaction products were seen in glial cells or endothelial cells. These results suggest that prostaglandin E2 is involved in fundamental processes of neurons.

The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture.

The abnormal uterine activity associated with dietary n-3 fatty acids may result from competitive inhibition of PG2 production. Uterine synthesis of 2- and 3-series prostaglandins F(PGF) and E(PGE) was studied using mass spectrophotometry in rats fed diets containing predominantly n-3 fatty acid, n-6 fatty acid, or control pelleted diet. Mass spectra of PGF (Me, TMS and Me, TBDMS derivatives) synthesised by uteri of n-3 fed rats were characterised by 8 ions containing the n-3 double bond, and m.i.d. of the 651/653 ions of PGF-Me, TBDMS indicated PGF3 alpha synthesis (44 +/- 8% and 13 +/- 2% of PGF release by uteri incubated + or -5 micrograms/ul calcium ionophore A23187 respectively). In uteri from the control diet group incubated with ionophore, PGF3 alpha ions were detected and PGF 3 alpha represented 9.5 +/- 1.0% of PGF alpha release. Similarly, analysis of PGE from uteri of n-3 fed rats indicated that PGE3 (16 +/- 6% of PGE) was released in the presence of ionophore A23187. Synthesis of 3-series PG by rat uteri was detected after only 3 weeks of n-3 diet. The capacity to synthesise 3-series PG increased at intracellular calcium concentrations which mimicked cell calcium during decidual autolysis at parturition. These experiments suggest that uterine synthesis of 3-series PG is regulated by the specifity of enzymes incorporating fatty acids, rather than by the cyclooxygenase enzyme.

[Cyclic nucleotides in a high molecular weight aminoacyl-tRNA synthetase complex from the liver of normal and irradiated rats]. / Tsyklichni nukleotydy u skladi vysokomolekyliarnogo kompleksu aminoatsyl-tRNK-syntetaz iz pechinky normal'nykh ta oprominenykh shchuriv.

The content of cAMP and cGMP in the composition of the high-molecular complex aminoacyl-tRNA-synthetase from the rat liver has been studied. The ionizing radiation decreases the level of the cyclic nucleotides in the composition of the complex. A problem on interregulation of the cyclic nucleotides and prostaglandins in the complex has been examined.

Rapid and slow hydroxylators of seminal E prostaglandins.

Human seminal fluid contains prostaglandin (PG) E1, PGE2, 19-hydroxy-PGE1 and 19-hydroxy-PGE2 in large and variable amounts. 19-Hydroxy-PGE1 and 19-hydroxy-PGE2 are formed from PGE1 and PGE2 by prostaglandin 19-hydroxylase, a cytochrome P-450 enzyme, in seminal vesicles. The hypothesis that genetic polymorphism of this enzyme might contribute to the variable concentrations of PGE1, PGE2, 19-hydroxy-PGE1 and 19-hydroxy-PGE2 was examined by analysis of seminal fluid of 40 normal men. E prostaglandins were measured with 17-phenyl-PGE2 as an internal standard by high-performance liquid chromatography on beta-cyclodextrin silica. Using the ratios of substrate/product, i.e., R1 = PGE1/19-hydroxy-PGE1 and R2 = PGE2/19-hydroxy-PGE2, as indicators of prostaglandin 19-hydroxylase capacity, a bimodal distribution of R values was found: nine men (23%) were slow hydroxylators (R1 greater than 0.45 and R2 greater than 0.45), while the remaining men were rapid hydroxylators (both R1 and R2 less than 0.45). Semen of slow hydroxylators and semen of the five most rapid hydroxylators (both R1 and R2 less than 0.10) differed in absolute amounts of PGE1 and PGE2 but not in 19-hydroxy-PGE1 and 19-hydroxy-PGE2. 20-Hydroxy-PGE1 and 20-hydroxy-PGE2 are formed from PGE1 and PGE2 by cytochrome P-450 in the vesicular glands and the ampullae of deferent ducts of the ram. Seminal fluid of five rams was analyzed for PGE1, PGE2, 20-hydroxy-PGE1 and 20-hydroxy-PGE2, and a large variation in substrate/product ratios was found. Polymorphism of cytochrome P-450 might contribute to variations in seminal prostaglandins in man and in sheep.

Isolation of an immunosuppressive trauma peptide and its relationship to fibronectin.

The purpose of this study was to characterize a suppressive active glycopeptide (SAP) using affinity chromatography (AFFI) and explore its similarity to fibronectin (FN) degradation products. It is postulated that SAP is a degradation fragment of a large serum-borne protein, possibly FN. Human trauma serum (HTS) and elastase-degraded human FN were run over an AFFI prepared with monoclonal antibody to SAP. Bound protein was eluted with 2M NaCl and dialyzed to remove salt. Immunosuppressive activity of HTS and FN eluates was monitored using inhibition of neutrophil chemotaxis (CTX). Active fractions were compared to the starting material and controls with 15% PAGE. AFFI eluates of HTS revealed a high molecular weight protein band (450 kd) with no inhibitory CTX activity and singular low molecular weight protein (LMW) band (less than 20,000) with 93% +/- 5% CTX suppression. Elastase-digested purified human FN eluates run under identical conditions over the same AFFI revealed two bands with an identical elution profile as HTS eluates. CTX suppression 95 +/- 5% was seen only with the LMW band. Incubation of enriched LWF fragments of digested FN and HTS with whole FN reversed CTX suppression. This suggests affinity purification of a single LMW suppressive glycopeptide which may bind to or be a degradation product of plasma or cellular fibronectin.

Generation and activity of suppressor peptides following traumatic injury.

Severe trauma is known to produce pathophysiologic changes leading to the generation of immunosuppressive compounds. With recent advances in biotechnology, a number of these factors have been identified and characterized. Many of these substances have been found to be degradation products of normal serum and tissue proteins. These degradation products have profound biologic activity both in vivo and in vitro. This report briefly focuses on a number of these factors and summarizes the current work involved in the determination of the identity and mechanisms of a previously reported suppressor-active peptide isolated from the serum of trauma patients.

Prostaglandylinositol cyclic phosphate, an antagonist to cyclic AMP.

The existence of a low molecular weight intracellular regulator has been demonstrated. It is composed of PGE, myoinositol, and one phosphate and has been named cyclic PIP. Its synthesis is stimulated by hormones such as insulin in a dose-dependent manner, it is therefore suggested that cyclic PIP is a second messenger equivalent to cyclic AMP in its potency but antagonistic in its action.

Human alveolar macrophages suppress interleukin-1 (IL-1) activity via the secretion of prostaglandin E2.

It has been suggested that human alveolar macrophages have a limited capacity to release interleukin-1 (IL-1). To determine whether this apparent defect in cell function is related to the release of factors that inhibit the response of lymphocytes to IL-1, we evaluated the capacity of human alveolar macrophages to release prostaglandin E2 (PGE2), a factor that is known to suppress the response of lymphocytes to IL-1. The amount of PGE2 released by alveolar macrophages was dependent on the amount of LPS present in the cultures and the amount of time the cells were present in culture. After 24 h in culture, the alveolar macrophage supernatants contained sufficient amounts of PGE2 to significantly suppress PHA-induced lymphocyte proliferation (p less than 0.01), IL-1-induced thymocyte proliferation (p less than 0.001), but not IL-2-induced lymphocyte proliferation (p greater than 0.2). Consistent with these observations, only small amounts of IL-1 activity could be detected in 24-h supernatants of LPS-stimulated alveolar macrophages using thymocyte proliferation as an assay for IL-1. Using a more sensitive assay for IL-1, however, it was demonstrated that the supernatants of LPS-stimulated macrophages contained amounts of IL-1 that were not significantly different from those present in supernatants of LPS-stimulated monocytes. Indomethacin (1 microgram/ml) completely suppressed the release of PGE2 by alveolar macrophages. These observations suggest that the apparent defect in the release of IL-1 by human alveolar macrophages may be due in part to the release of large amounts of PGE2, which suppresses various lymphocyte functions.

Identification and biological activity of a novel natural prostaglandin, 5,6-dihydro-prostaglandin E3.

A novel natural E-prostaglandin was detected by HPLC among the endogenous prostaglandins extracted from ram seminal vesicles. The corresponding precursor - all-cis-eicosa-8, 11, 14, 17-tetraenoic acid was isolated from bovine liver lipids and the preparative biosynthesis with the microsomal fraction of ram seminal vesicles was performed. The isolated product was purified by HPLC and identified by GC-MS as 5,6-dihydro-PGE3. The results of in vitro tests demonstrate that 5,6-dihydro-PGE3 is 14 times less active uterine stimulant than PGE1, at the same time retaining 75% of the anti-aggregatory potency of PGE1. Thus, 5,6-dihydro-PGE3 meets the requirements of a selective antithrombotic agent more than PGE1.

Separation and quantification of prostaglandins E1 and E2 as their panacyl derivatives using reverse phase high pressure liquid chromatography.

Separation and quantification of prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were achieved using reverse phase high performance liquid chromatography (HPLC). Panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) (PAB) derivatives of PGE2 and PGE1 were prepared. Reverse phase HPLC using a linear gradient of 56% to 80% acetonitrile in water containing 0.10% acetic acid gave baseline resolution of the two derivatives. A 3 um diameter particle, C18 column provided good resolution and reproducible recoveries. Human synovial tissue cells were incubated with the precursor fatty acids for PGE1 or PGE2 and stimulated with a crude Interleukin 1 (IL-1) preparation. Cells grown in the presence of dihomogammalinolenic acid (DGLA), the precursor for PGE1, made significantly more PGE1 than cells grown in control medium or in the presence of arachidonic acid, precursor for PGE2. PGE2 synthesis was reduced when DGLA was added to cells (resting or IL-1-stimulated).

Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid.

Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.

A fast, nondestructive purification scheme for prostaglandin H2 using a nonaqueous, bonded-phase high-performance liquid chromatography system.

Arachidonic acid metabolism produces several biologically important compounds including the leukotrienes and prostaglandins. Prostaglandin H2 (PGH2) is the first metabolite in the arachidonic acid cascade leading to all other prostaglandins. Pivotal to our understanding of PGH2's biology is the ability to separate it in pure form from the numerous other arachidonic acid metabolites produced in a biological milieu. The extensive literature on PGH2 biology and metabolism has relied almost exclusively on the traditional method of separation using gravity flow silicic acid columns. In our hands, such PGH2 preparations were found to contain varying amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), PGE2, PGF2 alpha and other minor impurities as determined by further chromatographic and mass spectral analyses. Analytical separation of PGH2 and other arachidonic acid metabolites has been accomplished using reversed-phase HPLC. However, the labile nature of this molecule in aqueous systems makes such techniques unacceptable for preparative isolation of high purity PGH2 and has necessitated the development of a totally nonaqueous separation. To this end, we attempted several stationary phases and found that the cyano-bonded phase showed the best selectivity for resolving PGH2 from its major contaminants. Separations were performed on self-packed columns using a hexane-isopropanol gradient. Peaks were detected both by liquid scintillation counting and uv spectrophotometry (214 nm). Structure assignments were made by chromatographic comparison with authentic standards (PGF2 alpha, PGE2), biological activity (PGH2--platelet aggregation), and by ammonia direct chemical ionization mass spectrometry (HHT, hydroxy-5,8,10,14-eicosatetraenoic acid, PGH2, PGE2, PGF2 alpha). The latter technique, which by its very nature volatilizes all organic material in the sample, was particularly useful in determining not only that the PGH2 preparations were free from the aforementioned side products, but that they were also free from lipid, protein, and other potential residues frequently found in biological preparations.

Isolation of two novel E prostaglandins in human seminal fluid.

cis-8,11,14,17-[1-14C]Eicosatetraenoic acid was incubated with microsomes of ram seminal vesicles and 1 mM glutathione for 3 min at 37 degrees C. The main metabolite was identified as 17,18-dehydroprostaglandin E1 by capillary column gas chromatography-mass spectrometry. Human seminal fluid was analyzed for the presence of 17,18-dehydroprostaglandin E1 and prostaglandin E3. Whereas prostaglandin E3 could be demonstrated by capillary gas chromatography-mass spectrometry, 17,18-dehydroprostaglandin E1 could not be found under these conditions. However, human seminal fluid contained two compounds with a similar polarity on reversed phase high performance liquid chromatography as 17,18-dehydroprostaglandin E1 and prostaglandin E3. The two compounds were identified as 18,19-dehydroprostaglandin E1 and 18,19-dehydroprostaglandin E2 by gas chromatography-mass spectrometry, by UV analysis after conversion to the corresponding prostaglandin B compounds, and by ozonolysis. The amount of each of the two prostaglandins in human seminal fluid seemed to be in the same order of magnitude as the amount of prostaglandin E3.

Solubilization and characterization of prostaglandin E2 binding protein from porcine cerebral cortex.

The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 4 degree C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time-dependent and the equilibrium was reached at around 60 min at 37 degrees C. By dilution of the reaction mixture, the binding site-[3H]PGE2 complex formed after 5-min incubation slowly dissociated, whereas that formed after 60-min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 greater than PGE1 much greater than PGF2 alpha greater than PGE2 methyl ester greater than PGA2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high- and low-affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion-exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.

Isolation and identification of prostaglandins of the E and F series in testes and semen of lowland-black-white breed of bulls.

The prostaglandins of E and F series were obtained from testes and semen of sexually mature bulls of lowland-black-white breed. From 1 g of fresh testes tissue we obtained 7.01 X 10(-9) M prostaglandin of the F series (PGF) and 20.65 X 10(-9) M prostaglandin of the E series (PGE): from 11 of semen 3.28 X 10(-6) M of PGF and 10.58 X 10(-6) M of PGE were obtained as well. The prostaglandins thus obtained displayed biological activity in experiments on the isolated small intestine of the rabbit.

Mass spectrometric fragmentation patterns for the syn and anti isomers of PGE2 and PGD2-methyloxime methyl esters and their analogs.

A systematic study of the mass spectral fragmentation of the methyl ester-methyloxime-trimethylsilyl ether derivatives of D and E prostaglandins and selected omega-chain analogs is presented. Fragments from the omega-chain analogs are shifted the appropriate mass when compared with the parent PGD2 or PGE2. NMR data of the methyloxime methyl ester of PGE2 have permitted assignment of the syn and anti isomers (relative to the alpha chain) to the fast and slow eluting gas chromatographic peaks, respectively.