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International Journal of Molecular... Mar 2016The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia...
The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy.
Topics: Animals; Anti-Allergic Agents; Cell Line, Tumor; Phorbol Esters; Plant Extracts; Rats; Seeds; Thymelaeaceae
PubMed: 27007372
DOI: 10.3390/ijms17030398 -
The Journal of Biological Chemistry Sep 1986The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors...
The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors of protein kinase C (Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). After 24 h, low concentrations of sphinganine (1-5 microM blocked both cell adherence and the inhibition of growth in response to phorbol 12-myristate 13-acetate, as measured by cell number and acid phosphatase activity. Sphinganine and sphingosine decreased adherence by 50% at 1-3 microM; other long-chain bases were effective in parallel to their inhibition of protein kinase C. Sphinganine decreased the binding of [3H]phorbol dibutyrate by the phorbol receptor of HL-60 cells, protein kinase C, and inhibited the response of HL-60 cells to dioctanoylglycerol, a cell permeable activator of this enzyme. Long-chain base uptake by HL-60 cells was demonstrated with [3-3H]sphinganine and within 1-3 days much had been converted to ceramides. By day 3, most of the cells had recovered the ability to adhere and exhibited macrophage characteristics, whereas cells in suspension did not differentiate. The level of free sphinganine in HL-60 cells was determined to be 12.3 +/- 1.2 pmol/10(6) cells. These results establish that sphingoid bases inhibit protein kinase C in HL-60 cells and may function physiologically as negative effectors of this enzyme.
Topics: Acid Phosphatase; Binding, Competitive; Cell Adhesion; Cell Differentiation; Cell Line; Humans; Kinetics; Leukemia, Myeloid, Acute; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Sphingosine; Structure-Activity Relationship; Tetradecanoylphorbol Acetate
PubMed: 3462189
DOI: No ID Found -
British Journal of Pharmacology Feb 1987Miniature endplate potentials and nerve-evoked endplate potentials were studied in a phrenic nerve-diaphragm preparation of the mouse. A phorbol ester, phorbol-12 beta,...
Miniature endplate potentials and nerve-evoked endplate potentials were studied in a phrenic nerve-diaphragm preparation of the mouse. A phorbol ester, phorbol-12 beta, 13 alpha-dibutyrate, and a diacylglycerol, 1-oleyl-2-acetyl-sn-glycerol, both increased the frequency of miniature endplate potentials. The effect of the phorbol ester on the frequency was still significant when the preparation was incubated in a calcium-deficient solution. The phorbol ester, but not the diacylglycerol, caused a significant increase in the amplitude of the miniature potentials. The phorbol ester also increased the amplitude of the endplate potentials in the presence of (+)-tubocurarine. The mechanisms of action of phorbol ester and diacylglycerol are discussed in relation to the control of acetylcholine release and action at the neuromuscular junction.
Topics: Animals; Benzylamines; Calcium; Diaphragm; Diglycerides; Electric Stimulation; Glycerides; In Vitro Techniques; Magnesium; Membrane Potentials; Mice; Motor Endplate; Neuromuscular Junction; Pargyline; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Phrenic Nerve; Stereoisomerism; Tubocurarine
PubMed: 3470104
DOI: 10.1111/j.1476-5381.1987.tb08962.x -
Journal of Neurochemistry Aug 2003Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or...
Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or interleukin-1beta. In the present study, phorbol-12,13-dibutyrate (PDBu) was found to induce catalytically active CYP2E1 more than fourfold in cortical glial cultures. Little induction was seen up to 12 h, and full effects only at 21-24 h of PDBu treatment. CYP2E1 expression in PDBu-treated cells was enriched in a subset of astrocytes. The protein kinase C inhibitors, staurosporine and calphostin C, and the tyrosine kinase inhibitor genistein, but not its inactive analogue daidzein, prevented the induction of CYP2E1 by PDBu. It is suggested that CYP2E1, together with interleukin-6 and ciliary neurotrophic factor, is part of a response of astrocytes to cellular stress elicited by, e.g. cerebral injury, cytokines or phorbol ester, and mediated in part through protein kinase C.
Topics: Animals; Astrocytes; Cells, Cultured; Cerebral Cortex; Chlorzoxazone; Cytochrome P-450 CYP2E1; Enzyme Induction; Enzyme Inhibitors; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Rats
PubMed: 12887687
DOI: 10.1046/j.1471-4159.2003.01897.x -
The Journal of Biological Chemistry Jan 1985The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by...
The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.
Topics: Amiloride; Carrier Proteins; Cell Adhesion; Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Pyrazines; Receptors, Cell Surface; Receptors, Transferrin; Sodium-Hydrogen Exchangers
PubMed: 2981834
DOI: No ID Found -
The Journal of Physiology Dec 19911. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac...
1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2. 2. Application of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/- 34% (n = 8) (p less than 0.02). The rate of change of cell length during contraction (i.e. +dL/dt) increased from 67.2 +/- 8.7 microns/s to 127.7 +/- 14.1 microns/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55.8 +/- 7.4 microns/s to 118.9 +/- 12.1 microns/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/- 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p less than 0.05) enhanced by 15.1 +/- 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p less than 0.05) by an average of 59.1 +/- 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (which does not activate protein kinase C). 5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na+ then it is suggested that, like pHi regulation in sheep heart Purkinje fibres, pHi recovery in rat cardiac ventricular myocytes is mainly through sarcolemmal Na(+)-H+ exchange. We suggest that in the presence of TPA the Na(+)-H+ exchange is stimulated. 6. The relationship between pHi and cell shortening is non-linear as has been observed by others in whole tissue preparations. The presence of TPA shifts the relationship upwards such that at any one pHi, cell shortening is greater.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Animals; Calcium; Cardiotonic Agents; Fluorescent Dyes; Guinea Pigs; Heart; Heart Ventricles; Hydrogen-Ion Concentration; Male; Myocardial Contraction; Myocardium; Phorbol Esters; Rats; Rats, Inbred Strains
PubMed: 1822559
DOI: 10.1113/jphysiol.1991.sp018889 -
Proceedings of the National Academy of... Sep 1992The regulation of the Erk (extracellular-signal-regulated kinase) gene-encoded protein kinase activity by reversible phosphorylation has been reported to involve either...
The regulation of the Erk (extracellular-signal-regulated kinase) gene-encoded protein kinase activity by reversible phosphorylation has been reported to involve either an activator of autophosphorylation or an upstream protein kinase. In this communication we describe assays utilizing the Erk-1 protein fused to glutathione S-transferase that permit the identification of protein kinase(s) that phosphorylate and activate the myelin basic protein kinase activity encoded by the Erk-1 gene. A phorbol ester-stimulated protein kinase activity was identified that phosphorylated a kinase-negative Erk-1 gene product on tyrosine and threonine. The protein kinase phosphorylated and activated wild-type protein expressed in bacteria from 20- to 50-fold. The activation of the Erk-1-encoded myelin basic protein kinase required ATP and correlated directly with the degree of phosphorylation on the same amino acid residues previously shown to be phosphorylated in vivo. Conversion of the tyrosine site of phosphorylation to phenylalanine yielded an Erk-1 gene product that could not be activated. Similar results were obtained when the threonine site was mutated to valine. It is likely that the phorbol ester-stimulated protein-tyrosine/threonine kinase(s) is an up-stream target for multiple extracellular signals.
Topics: Animals; Base Sequence; Cells, Cultured; In Vitro Techniques; Mice; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Peptide Mapping; Phorbol Esters; Phosphorylation; Phosphothreonine; Protein Kinases; Protein-Tyrosine Kinases; T-Lymphocytes
PubMed: 1518847
DOI: 10.1073/pnas.89.17.8200 -
The Journal of Biological Chemistry Aug 2002Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator...
Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs.
Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-1-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.
Topics: Base Sequence; Cell Line; Chloramphenicol O-Acetyltransferase; Collagenases; DNA Primers; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Insulin; Malate Dehydrogenase; Mutagenesis; Oligodeoxyribonucleotides; Phorbol Esters; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins; Transcription Factor AP-1; Transcription Factors; Transfection; beta-Galactosidase
PubMed: 12032154
DOI: 10.1074/jbc.M203682200 -
The Journal of Neuroscience : the... Aug 2008Diacylglycerol (DAG) and phorbol esters strongly potentiate transmitter release at synapses by activating protein kinase C (PKC) and members of the Munc13 family of... (Comparative Study)
Comparative Study
Diacylglycerol (DAG) and phorbol esters strongly potentiate transmitter release at synapses by activating protein kinase C (PKC) and members of the Munc13 family of presynaptic vesicle priming proteins. This PKC/Munc13 pathway has emerged as a crucial regulator of release probability during various forms of activity-dependent enhancement of release. Here, we investigated the relative roles of PKC and Munc13-1 in the phorbol ester potentiation of evoked and spontaneous transmitter release at the calyx of Held synapse. The phorbol ester phorbol 12,13-dibutyrate (1 microM) potentiated the frequency of miniature EPSCs, and the amplitudes of evoked EPSCs with a similar time course. Preincubating slices with the PKC blocker Ro31-82200 reduced the potentiation, mainly by affecting a late phase of the phorbol ester potentiation. The Ro31-8220-insensitive potentiation was most likely mediated by Munc13-1, because in organotypic slices of Munc13-1(H567K) knock-in mice, in which DAG binding to Munc13-1 is abolished, the potentiation of spontaneous release by phorbol ester was strongly suppressed. Using direct presynaptic depolarizations in paired recordings, we show that the phorbol ester potentiation does not go along with an increase in the number of readily releasable vesicles, despite an increase in the cumulative EPSC amplitude during 100 Hz stimulation trains. Our data indicate that activation of Munc13 and PKC both contribute to an enhancement of the fusion probability of readily releasable vesicles. Thus, docked and readily releasable vesicles are a substrate for modulation via intracellular second-messenger pathways that act via Munc13 and PKC.
Topics: Animals; Calcium; Drug Synergism; Excitatory Postsynaptic Potentials; Indoles; Mice; Mice, Knockout; Nerve Tissue Proteins; Neurotransmitter Agents; Organ Culture Techniques; Phorbol Esters; Presynaptic Terminals; Protein Kinase C; Rats; Rats, Wistar
PubMed: 18701688
DOI: 10.1523/JNEUROSCI.0550-08.2008 -
Drug News & Perspectives Oct 2005The persistence of latent reservoirs of human immunodeficiency virus type 1 (HIV-1) represents a major barrier to virus eradication in patients on combination... (Review)
Review
The persistence of latent reservoirs of human immunodeficiency virus type 1 (HIV-1) represents a major barrier to virus eradication in patients on combination antiretroviral therapy. It has been suggested that treating infected individuals simultaneously with highly active antiretroviral therapy (HAART) and agents that activate cells to express HIV-1 might eliminate these latent reservoirs. The phorbol ester prostratin, used in Western Samoa as an ethno-botanical treatment for viral hepatitis, was isolated at the National Cancer Institute in 1992. Prostratin represents a distinct subclass of protein kinase C activators, since unlike other phorbol esters it does not induce tumor formation. Prostratin upregulates expression of viral products from latently infected cells such as U1, ACH-2 and peripheral blood mononuclear cells from patients on HAART with undetectable plasma viremia. It also inhibits HIV infection and viral spread at the entry/fusion step of viral life cycle. The lack of tumor promotion of prostratin coupled with its ability to upregulate latent HIV-1 provirus expression and inhibition of viral infection are important features that could be exploited as effective therapy to eliminate latent reservoirs.
Topics: Animals; Anti-HIV Agents; Antiretroviral Therapy, Highly Active; HIV Infections; HIV-1; Humans; Phorbol Esters; Protein Kinase C; Virus Activation; Virus Latency
PubMed: 16391719
DOI: 10.1358/dnp.2005.18.8.944543