An active-site titration method for lipases.
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A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations. (+info)
Prototypical imidazoline-1 receptor ligand moxonidine activates alpha2-adrenoceptors in bulbospinal neurons of the RVL.
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Moxonidine is an antihypertensive drug that lowers sympathetic vasomotor tone by stimulating either alpha2-adrenergic (alpha2-AR) or imidazoline I1 receptors within the rostral ventrolateral medulla (RVL). In this study, we investigated the effects of moxonidine (10 microM) on RVL neurons in brain stem slices of neonatal rats. We recorded mainly from retrogradely labeled RVL bulbospinal neurons (putative presympathetic neurons) except for some extracellular recordings. Prazosin was used to block alpha1-adrenoceptors. Moxonidine inhibited the extracellularly recorded discharges of all spontaneously active RVL neurons tested (bulbospinal and unidentified). This effect was reversed or blocked by the selective alpha2-AR antagonist SKF 86466 (10 microM). In contrast, the I1 imidazoline ligand AGN 192403 (10 microM) had no effect on the spontaneous activity. In whole cell recordings (holding potential -70 mV), moxonidine produced a small and variable outward current (mean 7 pA). This current was observed in both tyrosine hydroxylase-immunoreactive and other bulbospinal neurons and was blocked by SKF 86466. Excitatory postsynaptic currents (EPSCs) evoked by focal electrical stimulation were isolated by incubation with gabazine and strychnine, and inhibitory postsynaptic currents (IPSCs) were isolated with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moxonidine reduced the amplitude of the evoked EPSCs (EC(50) = 1 microM; 53% inhibition at 10 microM) but not their decay time constant (5.6 ms). The effect of moxonidine on EPSCs persisted in barium (300 microM) and was reduced approximately 80% by SKF 86466. Moxonidine also reduced the amplitude of evoked IPSCs by 63%. In conclusion, moxonidine inhibits putative RVL presympathetic neurons both presynaptically and postsynaptically. All observed effects in the present study are consistent with an alpha2-AR agonist activity of moxonidine. (+info)
Novel polyunsaturated n-alkenes in the marine diatom Rhizosolenia setigera.
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Four previously unknown n-C25 and n-C27 heptaenes of the marine diatom Rhizosolenia setigera were isolated and identified using NMR spectroscopy. They possess six methylene interrupted (Z)-double bonds starting at C-3 and an additional terminal or n-2 (Z)-double bond. Structural and stable carbon isotopic evidence suggests that these polyenes are biosynthesized by chain elongation of the C22:6n-3 fatty acid, followed by decarboxylation and introduction of double bonds at specific positions. (+info)
APHS can act synergically with clinically available HIV-1 reverse transcriptase and protease inhibitors and is active against several drug-resistant HIV-1 strains in vitro.
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OBJECTIVES: The use of multiple drug combinations in current anti-human immunodeficiency virus (HIV) therapy allows lower dosages of individual drugs and results in enhancement of the therapeutic effect due to synergic interactions between different drugs. We have shown that o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently developed non-steroidal anti-inflammatory drug, shows anti-HIV activity in a dose-dependent manner. The first aim of this study was to investigate whether APHS can act synergically with the clinically available reverse transcriptase and protease inhibitors (RTIs and PIs, respectively) in vitro. Because of the increasing prevalence of RTI- and PI-resistant HIV-1 strains, the second aim of this study was to assess the antiviral activity of APHS against drug-resistant HIV-1 strains in vitro. MATERIALS AND METHODS: HIV-infected peripheral blood mononuclear cells (PBMC) were treated for 7 days with different combinations of APHS and RTIs or PIs. The MT-2 cell line was infected with different HIV-1 strains and treated with APHS for 5 days. RESULTS: APHS showed synergic interactions with the RTIs zidovudine, lamivudine and efavirenz and with the PIs indinavir and ritonavir. The 50% inhibitory concentration (IC50) of APHS in this assay dropped from 13 microM when used alone, to 5 micro M after combination with an RTI or PI. In combination with APHS the IC50 of the RTI and PI drugs tested also dropped. APHS inhibits the replication of HIV-1 strains resistant to zidovudine, lamivudine, stavudine, didanosine, zalcitabine and ritonavir. CONCLUSIONS: These results indicate that APHS can be combined with RTIs and PIs and can inhibit several NRTI and PI-resistant HIV-1 strains. (+info)
WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate uptake.
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The pharmacological profile of a novel glutamate transport inhibitor, WAY-855 (3-amino-tricyclo[2.2.1.0(2.6)]heptane-1,3-dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY-855 inhibited glutamate uptake mediated by all three subtypes in a concentration-dependent manner, with preferential inhibition of the CNS-predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 microM, respectively, while EAAT1 activity was inhibited by 50% at 100 microM (IC50 values determined in oocytes were 1.3 microM (EAAT2), 52.5 microM (EAAT3) and 125.9 microM (EAAT1)). Application of WAY-855 to EAAT-expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]d-aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY-855 inhibited d-aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY-855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY-855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes. (+info)
5-Hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3-heptanone: a pancreatic lipase inhibitor isolated from Alpinia officinarum.
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A pancreatic lipase inhibitor, 5-hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3-heptanone (HPH), from the rhizome of Alpinia officinarum (AO) was isolated and its antihyperlipidemic activity was measured. HPH inhibited a pancreatic lipase with an IC(50) value of 1.5 mg/ml (triolein as a substrate). HPH significantly lowered the serum TG level in corn oil feeding-induced triglyceridemic mice, and reduced serum triglyceride (TG) and cholesterol in Triton WR-1339-induced hyperlipidemic mice. However, HPH did not show hypolipidemic activity in high cholesterol diet-induced hyperlipidemic mice. Based on these findings, we propose that PL inhibitors may be effective as hypolipidemic agents. (+info)
Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and l-alpha-threofuranosyl ring systems: interactions with P2Y receptors.
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The ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y(2) and P2Y(4) receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) l-alpha-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y(1) receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a K(i) value of 22.5 nM in binding to the human P2Y(1) receptor, and antagonized the stimulation of PLC by the potent P2Y(1) receptor agonist 2-methylthio-ADP (30 nM) with an IC(50) of 650 nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A(3)). Thus, this ring system afforded some P2Y receptor selectivity. A l-alpha-threofuranosyl bisphosphate derivative 9 displayed an IC(50) of 15.3 microM for inhibition of 2-methylthio-ADP-stimulated PLC activity. l-alpha-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y(2) (EC(50)=9.9 microM) versus P2Y(4) receptors. The P2Y(1) receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking. (+info)
Isolation and absolute configuration determination of aliphatic sulfates as the Daphnia kairomones inducing morphological defense of a phytoplankton.
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2,6-Dimethylheptyl sulfate (1) and 6-methyloctyl sulfate (3) were isolated from Daphnia pulex as the Daphnia kairomones that induced morphological defense of a freshwater phytoplankton Scenedesmus gutwinskii var. heterospina (NIES-802). The absolute stereochemistry at C2 of 1 was determined by (1)H-NMR analysis of the (R)-MTPA ester of alcohol 2. The absolute configuration at C6 of 3 was determined by Ohrui's method applied to alcohol 4. (+info)