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Silica gel supported pyrolysis of an azido-homo-oxa steroid led to rearrangement, presumably by a mechanism similar to that of solution phase Schmidt fragmentation, to produce a group of novel inhibitors for the oncogenic cell cycle regulator Cdc25A phosphatase. Cyano-containing acid 17, one of the best inhibitors in this group, inhibited the activity of Cdc25A protein phosphatase reversibly and noncompetitively with an IC(50) value of 2.2 microM. Structure-activity relationships revealed that a phosphate surrogate such as a carboxyl or a xanthate group is required for inhibitory activity, and a hydrophobic alkyl chain, such as the cholesteryl side chain, contributes greatly to the potency. Without the cyano group, acid 26 and xanthate 27 were found to be more selective over Cdc25A (IC(50) = 5.1 microM and 1.1 microM, respectively) than toward CD45 (IC(50) > 100 microM, in each case), a receptor protein tyrosine phosphatase. Several of these inhibitors showed antiproliferative activities in the NCI 60-human tumor cell line screen. These steroidal derived Cdc25 inhibitors provide unique leads for the development of dual-specificity protein phosphatase inhibitors.

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Many functions of the immune and inflammatory responses are inhibited by agents that increase intracellular levels of cAMP. Recent investigations have revealed that cAMP levels in inflammatory cells are regulated by cyclic nucleotide phosphodiesterases (PDEs) belonging to the PDE4 family (cAMP-specific PDEs). At least four different genes are known to encode PDE4 isozymes, which are characterized by their selectivity for cAMP over cGMP and their sensitivity to the antidepressant drug rolipram. The aim of our studies was to investigate whether monocytic cells could regulate PDE4 activity and whether certain PDE4 isozymes were expressed preferentially over others. Our results showed that treatment of peripheral blood monocytes or closely related Mono Mac 6 cells with dibutyryl-cAMP or other cAMP-elevating agents transiently increased rolipram-sensitive PDE4 activity 2-3-fold, without concomitant increases in cGMP-inhibited PDE (PDE3) activity. PDE4 activity was predominantly cytosolic, whereas PDE3 activity was localized to the particulate fraction. Our Northern and Western blot studies with reagents recognizing three distinct PDE4 gene products (PDE4A, PDE4B, and PDE4D) revealed that their expression is transcriptionally regulated in monocytic cells. Although none of the three isozymes was detectable under normal culture conditions, all of these were up-regulated when Mono Mac 6 cells were exposed to dibutyryl-cAMP. Distinct differences were observed in their temporal patterns of expression. Endotoxin lipopolysaccharide, a potent monocyte stimulus, also enhanced PDE4 activity in monocytic cells. These data indicate that monocytic cells may express different PDE4 isozymes, depending on their state of activation or differentiation. These isozymes could thus regulate intracellular cAMP levels at various stages of monocyte activation and could thereby be important in limiting the inflammatory response.

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Elevation of cAMP downregulates certain functions of inflammatory cells, including the release of TNF alpha and IL-1 beta by macrophages. Intracellular cAMP levels can be modulated pharmacologically by adding cell-permeable cAMP analogs, by stimulating adenylate cyclase or by inhibiting degradation of cAMP by cAMP-phosphodiesterases (cAMP-PDE). Multiple forms of cAMP-PDEs have been identified in various tissues and cells using both biochemical characterization and selective inhibitors. Therefore, we wanted to determine which of these different PDE isoforms was present in human monocytes and whether this isoform could regulate cytokine release from human monocytes by a mechanism similar to that seen with dbcAMP or PGE1. Our results demonstrate that selective inhibitors of type IV cAMP-PDE, such as rolipram and Ro20-1724, are clearly the most effective compounds at enhancing cAMP levels and inhibiting the release of TNF alpha and IL-1 beta in these cells. The type III cAMP-PDE-selective inhibitors C1930 and cilostamide and the nonselective PDE inhibitors IBMX and pentoxifylline were significantly less potent. In agreement with these data, cAMP-PDE activity in cytosolic extracts from human monocytes was also much more sensitive to inhibition by rolipram than by cilostamide. Additionally, rolipram dramatically reduced TNF alpha mRNA accumulation, which supports previous findings that cAMP regulates TNF alpha at the transcriptional level. Surprisingly, rolipram, rolipram, dbcAMP or PGE1 increased IL-1 beta was reduced, which indicates that cAMP can have both positive and negative effects on the regulation of IL-1 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

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The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.

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Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into its corresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. 1-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3 microM upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 microM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 microM-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.

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The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.

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The biochemical events underlying the ability of thrombin to enhance the metabolism of inositol phospholipids in human platelets have been investigated using platelets prelabeled with [3H]inositol. Thrombin treatment caused rapid formation of radioactive inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) with less marked and more variable changes in the levels of radioactive inositol phospholipids. Formation of IP2 and IP3 could be detected 5 s after exposure to thrombin and before IP levels increased. Low doses of thrombin which produced only shape change in human platelets also caused significant formation of IP2 and IP3 but not IP. These results suggest that thrombin-induced platelet activation may be mediated through hydrolysis of polyphosphoinositides. The majority of IP formed presumably arises from the hydrolysis of IP2. Prostacyclin inhibited thrombin-induced formation of all three inositol phosphates.

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Neutrophils isolated from the pleural cavity of rats 3 hr after the intrapleural injection of carrageenan metabolize exogenously added arachidonic acid via cyclooxygenase and lipoxygenase. In addition, these cells esterify arachidonic acid to produce diarachidonyl diglyceride. The structure of the diglyceride was determined with the use of various chemical and enzymatic digestions, gas chromatography-mass spectrometry, and 252Cf plasma-desorption mass spectrometry. The formation of this unique diglyceride is stimulated by the presence of nonsteroidal anti-inflammatory drugs. Some of the possible consequences of diarachidonyl diglyceride production are discussed.

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The human promyelocytic leukemia cell line HL60 can be induced to differentiate into mature granulocytes by exposure to Me2SO. [1-14C]Arachidonic acid incubated overnight with these cells was incorporated mainly into membrane phospholipids. Stimulation of these cells with the calcium ionophore, A23187, resulted in a rapid release of esterified arachidonic acid from phosphatidylethanolamine and phosphatidylcholine. The released arachidonic acid was metabolized via both the cyclooxygenase and lipoxygenase pathways into three major hydroxylated products, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 5(S)-hydroxy-6,8,11,14-icosatetraenoic acid (5-HETE), and 5-(S),12(R)-dihydroxy-6,8,10,14-icosatetraenoic acid (leukotriene B). Arachidonic acid was also incorporated into triacylglycerols and phosphatidylinositol. The lipoxygenase product, 5-HETE, was rapidly esterified into cellular lipids. Thirty minutes after ionophore stimulation, 55% of the total 5-HETE synthesized was esterified into phospholipids and 35% incorporated into acylglycerols. In contrast, the other hydroxylated derivatives of arachidonic acid (HHT and leukotriene B) were not incorporated into acylglycerols or phospholipids. Esterification of hydroxylated metabolites of arachidonic acid into membrane phospholipids may serve to regulate a number of granulocyte functions.

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Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.

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The enzymes of arachidonate metabolism via the lipoxygenase pathway in human platelet cytosol have been characterized and partially purified. The lipoxygenase activity has a pH optimum of 7.3 and reaches half-maximal activity at an arachidonate concentration of 80 microM. The oxidation of arachidonate by these enzymes is inhibited by reagents that modify sulfhydryl groups. Two separable lipoxygenase activities can be detected by chromatography of platelet cytosol on Sephadex G-150 and of partially purified preparations on DEAE-Sephadex. One of these has an apparent Mr of 100,000. A second enzyme species behaves as a Mr 160,000 entity containing, in addition to lipoxygenase, a peroxidase activity that catalyzes the conversion of 12L-hydroperoxy-5,8,10,14-icosatetraenoic acid (HPETE) to 12L-hydroxy-5,8,10,14-icosatetraenoic acid (HETE). Aspirin, indomethacin, sodium salicylate, phenylbutazone, ibuprofen, naproxen, and sulindac, but not acetaminophen or phenacetin, give rise to increased levels of HPETE in the lipoxygenase pathway. This increase in HPETE levels is the result of the ability of these drugs to inhibit directly the enzymatic conversion of HPETE to HETE.

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Aspirin, indomethacin, and sodium salicylate are anti-inflammatory, analgesic, and antipyretic. Whereas aspirin and indomethacin inhibit prostaglandin synthetase (cyclo-oxygenase; 8,11,14-eicosatrienoate, hydrogen-donor: oxygen oxidoreductase, EC 1.14.99.1), salicylate does not. However, all three drugs affect the metabolism of arachidonate via the lipoxygenase pathway by inhibiting the conversion of 12-hydroperoxy- to 12-hydroxy-5,8,10,14-eicosatetraenoic acid.

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Uncoupler-adapted Euglena gracilis have a greatly impaired capacity to take up and incorporate some exogenously supplied amino acids and sugars. The degree of inhibition varies widely from >90% in the case of valine or glucose to none in the case of histidine. The inhibition is due to a decreased activity of the transport mechanism itself and is not due to either a lesion in the control mechanism for endogenous amino acid or sugar synthesis nor to a direct inhibition of the transport mechanism by uncouplers. No preferential labeling of mitochondrial membranes by [14C]amino acids occurs during the process of adaptation, a time when no cell division occurs. Apparently, during the long time required for adaptation, there occurs no major modification of mitochondrial proteins which could explain the subsequent resistance to uncouplers.

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