Toxicologic studies of dihydro-5,6-dehydrokawain and 5,6-dehydrokawain.
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Animals↗
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Results for “Pyrones”
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Metabolism of swertiamarin from Swertia japonica by human intestinal bacteria.
The biotransformation of swertiamarin [1, a seco-iridoid glucoside isolated from Swertia japonica (Schult.) Makino] by human intestinal bacteria was investigated. Three metabolites were isolated and identified as erythrocentaurin (2), 5-hydroxymethylisochroman-1-one (3), and gentianine (4) by spectroscopic methods. Through screening of various defined strains of intestinal bacteria (25 species), it was found that all these species had the ability to metabolize 1 to 2 and 3, whereas only a few species had the ability to produce 4. This is the first report to show that one of the metabolic intermediates of the secoiridoid compound is further transformed to a nitrogen-containing compound through metabolic processes by human intestinal bacteria.
Feces↗
The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women.
Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.
Androstenediols↗
The efficacy of Cavain in patients suffering from anxiety.
The therapeutical efficacy of Cavain should be proved in the treatment of patients suffering from abnormal anxiety, psychosomatic complaints and psychoreactive disorder. Thus two randomized groups of patients (26 each) were treated in double-blind technique with either 2 x 200 mg daily Cavain or placebo for a period of 28 days. Prior to the beginning of the investigations and within 14 days intervals the Hamilton Anxiety Scale (HAMA) and the Adjective Check List (Janke and Debus) were applied. The global therapeutical improvement and compatibility were documented after 14 and 28 days. A significant superiority of Cavain in comparison to placebo could be found. Cavain acted anxiolytically and promotive on the subjective vitality-related performance. Therapeutical conclusions are discussed.
Adult↗
Superficial micropeels: glycolic acid and alpha-hydroxy acid with kojic acid.
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Chemexfoliation↗
Asymmetric catalysis in complex target synthesis.
This article describes three distinct strategies by which stereochemically complex molecules are synthesized and the ways asymmetric catalysis can impact on all three. The development of general methods to prepare synthetically useful building blocks leads to an expanded "chiral pool" of potential starting materials for asymmetric synthesis. The possibility of discovering new reactions to access new types of building blocks is particularly attractive and serves to help define the frontiers of the field. Asymmetric catalysis can also be applied to diastereoselective synthesis such that the stereochemistry of the catalyst, and not that of the substrate, determines the relative configuration of the product. Finally, in reactions where multiple stereocenters are generated simultaneously or in tandem, catalyst and substrate control can operate in a complementary manner to achieve one of many possible stereochemical outcomes selectively.
Alkaloids↗
Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.
Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an ars operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The ars operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.
Azotobacter vinelandii↗
Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity.
Type III polyketide synthases (PKS) generate an array of natural products by condensing multiple acetyl units derived from malonyl-CoA to thioester-linked starter molecules covalently bound in the PKS active site. One strategy adopted by Nature for increasing the functional diversity of these biosynthetic enzymes involves modifying polyketide assembly by altering the preference for starter molecules. Chalcone synthase (CHS) is a ubiquitous plant PKS and the first type III PKS described functionally and structurally. Guided by the three-dimensional structure of CHS, Phe-215 and Phe-265, which are situated at the active site entrance, were targeted for site-directed mutagenesis to diversify CHS activity. The resulting mutants were screened against a panel of aliphatic and aromatic CoA-linked starter molecules to evaluate the degree of starter molecule specificity in CHS. Although wild-type CHS accepts a number of natural CoA thioesters, it does not use N-methylanthraniloyl-CoA as a substrate. Substitution of Phe-215 by serine yields a CHS mutant that preferentially accepts this CoA-thioester substrate to generate a novel alkaloid, namely N-methylanthraniloyltriacetic acid lactone. These results demonstrate that a point mutation in CHS dramatically shifts the molecular selectivity of this enzyme. This structure-based approach to metabolic redesign represents an initial step toward tailoring the biosynthetic activity of plant type III PKS.
Acetic Acid↗
Identification of a small molecule inhibitor of Sir2p.
Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.
Amino Acid Sequence↗
A common pharmacophore for cytotoxic natural products that stabilize microtubules.
Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel-i.e., the stabilization of microtubules leading to mitotic arrest-is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [3H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure-activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity.
Alkaloids↗
An anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria.
Infections with parasitic helminths are important causes of morbidity and mortality worldwide. New drugs that are parasite specific and minimally toxic to the host are needed to counter these infections effectively. Here we report the finding of a previously unidentified compound, nafuredin, from Aspergillus niger. Nafuredin inhibits NADH-fumarate reductase (complexes I + II) activity, a unique anaerobic electron transport system in helminth mitochondria, at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep. Thus, our study indicates that mitochondrial complex I is a promising target for chemotherapy, and nafuredin is a potential lead compound as an anthelmintic isolated from microorganisms.
Administration, Oral↗
Inhibition of macrophage Ca(2+)-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones.
A novel Ca(2+)-independent phospholipase A2 (PLA2) has recently been purified from the murine macrophage-like cell line P388D1 (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF3), arachidonyl trifluoromethyl ketone (AACOCF3), and a bromoenol lactone (BEL). Both PACOCF3 and AACOCF3 were found to inhibit the macrophage PLA2 in a concentration-dependent manner. PACOCF3 was found to be approximately 4-fold more potent than AACOCF3, with IC50 values of 3.8 microM (0.0075 mol fraction) and 15 microM (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF3.PLA2 complex rapidly dissociated upon dilution. BEL was also found to inhibit the macrophage PLA2 in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40 degrees C. Inhibition was not reversed after extensive dilution of the enzyme into assay buffer. Treatment of the PLA2 with BEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF3. In addition, PLA2 treated with [3H]BEL resulted in the covalent labeling of a major band at M(r) 80,000. Inactivation of the PLA2 by 5,5'-dithiobis(2-nitrobenzoic acid) prior to treatment with [3H]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a M(r) 80,000 band rather than a M(r) 40,000 band upon treatment with [3H]BEL distinguishes the macrophage Ca(2+)-independent PLA2 from a previously identified myocardial Ca(2+)-independent PLA2 and provides strong evidence that the M(r) 80,000 protein is the catalytic subunit.
Animals↗
Distinguishing between folate receptor-alpha-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells.
L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTXrA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTXrA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTXrA-TF1, which constitutively expresses high levels of FR-alpha. MTXrA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-alpha and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 microM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a approximately 20-fold lower influx Kb and Vmax for MTXrA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTXrA-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 microM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTXrA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTXrA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTXrA-TF1 cell lines; however, inhibition in MTXrA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-alpha. The data indicate that the reduced folate carrier and FR-alpha function independently and exhibit distinct properties. FR-alpha expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.
Alkenes↗
Nitric oxide activates the glucose-dependent mobilization of arachidonic acid in a macrophage-like cell line (RAW 264.7) that is largely mediated by calcium-independent phospholipase A2.
Herein, we demonstrate that nitric oxide is a potent (> 20% release) and highly selective inducer of [3H]arachidonic acid mobilization in the macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one resulted in the inhibition of the large majority (86%) of nitric oxide-induced [3H]arachidonic acid release into the medium (IC50 < 0.5 microM) and the concomitant inhibition of in vitro measurable calcium-independent phospholipase A2 activity (92% inhibition) without demonstrable effects on calcium-dependent phospholipase A2 activity. Since nitric oxide is a potent stimulator of glycolysis (and therefore glycolytically derived ATP) and since cytosolic calcium-independent phospholipase A2 exists as a catalytic complex comprised of ATP-modulated phosphofructokinase-like regulatory polypeptides and a catalytic subunit, we examined the role of glucose in facilitating nitric oxide-mediated arachidonic acid release. Nitric oxide-induced release of [3H]arachidonic acid possessed an obligatory requirement for glucose, was highly correlated with the concentration of glucose in the medium, and was dependent on the metabolism of glucose. Thus, [3H]arachidonic acid release is coupled to cellular glucose metabolism through alterations in the activity of calcium-independent phospholipase A2. Collectively, these results identify a unifying metabolic paradigm in which the generation of lipid second messengers is coordinately linked to the signalstimulated acceleration of glycolytic flux, thereby facilitating integrated metabolic responses to cellular stimuli.
Animals↗
Lineage-specific induction of B cell apoptosis and altered signal transduction by the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV).
Protein tyrosine phosphorylation is known to play key roles in lymphocyte signal transduction, and phosphotyrosine phosphatases (PTP) can act as both positive and negative regulators of these lymphocyte signals. We sought to examine the role of PTP further in these processes by characterizing the effects of bis(maltolato)-oxovanadium(IV) (BMLOV), previously known to be a nontoxic insulin mimetic agent in vivo. BMLOV was found to be a potent phosphotyrosine phosphatase inhibitor. BMLOV induced cellular tyrosine phosphorylation in B cells in a pattern similar to that observed following antigen receptor stimulation, whereas little tyrosine phosphorylation was induced in T cells. In B cells, BMLOV treatment resulted in tyrosine phosphorylation of Syk and phospholipase C gamma 2, while sIgM-induced signals were inhibited. By contrast, T cell receptor signals were moderately increased by BMLOV, and the cells displayed greater induction of IL-2 receptor without toxicity. The compound selectively induced apoptosis in B cell lymphoma and myeloid leukemia cell lines, but not in T cell leukemia or colon carcinoma cells. Interleukin-4 plus anti-CD40 antibody treatment of normal human peripheral B cells rescued the cells from BMLOV-induced death. These results suggest that phosphotyrosine phosphatase inhibitors can activate B cell signal pathways in a lineage-specific manner, resulting in desensitization of receptor-mediated signaling and induction of apoptosis.
Animals↗
Characterization of a CD43/leukosialin-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells.
The monoclonal antibody (mAb) J393 induces apoptosis in Jurkat T-cells. NH2-terminal amino acid sequence analysis identified the 140-kDa surface antigen for mAb J393 as CD43/leukosialin, the major sialoglycoprotein of leukocytes. While Jurkat cells co-expressed two discrete cell-surface isoforms of CD43, recognized by mAb J393 and mAb G10-2, respectively, only J393/CD43 signaled apoptosis. J393/CD43 was found to be hyposialylated, bearing predominantly O-linked monosaccharide glycans, whereas G10-2/CD43 bore complex sialylated tetra- and hexasaccharide chains. Treatment with soluble, bivalent mAb J393 killed 25-50% of the cell population, while concomitant engagement of either the CD3.TcR complex or the integrins CD18 and CD29 significantly potentiated this effect. Treatment of Jurkat cells with mAb J393 induced tyrosine phosphorylation of specific protein substrates that underwent hyperphosphorylation upon antigen receptor costimulation. Tyrosine kinase inhibition by herbimycin A diminished J393/CD43-mediated apoptosis, whereas inhibition of phosphotyrosine phosphatase activity by bis(maltolato)oxovanadium-IV enhanced cell death. Signal transduction through tyrosine kinase activation may lead to altered gene expression, as J393/CD43 ligation prompted decreases in the nuclear localization of the transcriptional regulatory protein NF-kappaB and proteins binding the interferon-inducible regulatory element. Since peripheral blood T-lymphocytes express cryptic epitopes for mAb J393, these findings demonstrate the existence of a tightly regulated CD43-mediated pathway for inducing apoptosis in human T-cell lineages.
Antibodies, Monoclonal↗
Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages.
Bromoenol lactone (BEL) has previously been identified as a potent, irreversible, mechanism-based phospholipase A2 (PLA2) inhibitor that possesses greater than 1000-fold selectivity for inhibition of Ca2+-independent PLA2 (iPLA2) versus the Ca2+-dependent ones. Thus, this compound has been used as a selective tool for studies aimed at elucidating the role of iPLA2 in certain cellular functions. Herein we report that BEL also inhibits cellular phosphatidic acid phosphohydrolase (PAP) activity in intact P388D1 macrophages with an IC50 of about 8 microM, which is very similar to that previously found for inhibition of iPLA2 under the same experimental conditions. This results in the blockage of the incorporation of exogenous arachidonate and palmitate into diacylglycerol and triacylglycerol. Thus, inhibition of PAP by BEL blocks triacylglycerol biosynthesis in P388D1 cells due to decreased diacylglycerol availability. Because two forms of PAP activity exist in mammalian cells, differential assays were performed to identify which of these forms was inhibited by BEL. The results of these experiments revealed that BEL selectively inhibits the cytosolic, Mg2+-dependent enzyme. No apparent effect of BEL on the membrane-bound Mg2+-independent PAP form could be detected. Collectively, the results reported herein establish that BEL inhibits two cellular phospholipases, namely iPLA2 and Mg2+-dependent PAP, with similar potency. Therefore, the inhibitory effect of BEL on Mg2+-dependent PAP might explain several cellular functions previously attributed to iPLA2.
Animals↗
Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.
A novel Ca2+-independent phospholipase A2 (iPLA2) has recently been purified and characterized from P388D1 macrophages (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions. Also an iPLA2 from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575). We report herein that the cloned CHO iPLA2 is equivalent to the mouse enzyme purified from P388D1 cells. Polymerase chain reaction amplification of cDNA fragments from P388D1 cells using primers based on the CHO iPLA2 sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D1 cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions. Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D1 enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate. Additionally, covalent modification of the CHO enzyme by [3H]bromoenol lactone is dependent on active enzyme as is the P388D1 iPLA2. Finally, both enzymes have the same specific activities under identical experimental conditions.
Amino Acid Sequence↗