Sapatoxins,aliphatic ester tigliane diterpenes from Sapium indicum

From the unripe fruits of Sapium indicum, three aliphatic esters of the tigliane nucleus were isolated. These compounds were derivatives of 4-deoxyphorbol. Sapatoxin A was identified as 12-O-[n-deca-2,4,6-trienoyl]-4-deoxyphorbol-13-acetate, B as 12-O-[n-deca-2,4,6-trienoyl]-4-deoxy-5-hydroxyphorbol-13-acetate and C as 12-O-[n-deca-2,4,6-trienoyl]-4,20-dideoxy-5-hydroxyphorbol-13-acetate, by spectroscopic analysis and hydrolysis reactions.     

Phorbol derivatives from Sapium insigne

From an ether extract of the twigs and leaves of Sapium insigne four new diterpene esters were isolated. They were identified as 12-O-(2′E, 4′E-decadienoyl)-4-deoxy-16-hydroxyphorbol-13-acetate, 12-O-hexanoyl-4α-deoxy-phorbol-13-acetate, 12-O-hexanoyl-4α-deoxy-16-hydroxyphorbo-1-13-acetate and 12-O-dodecanoyl-4α-deoxy-16-hydroxyphorbol-13-acetate by spectroscopic and chemical methods.     

Piscicidal Constituents and Related Diterpene Esters from Aleurites fordii

Piscicidal constituents, A and B, related diterpene esters, C and D, were isolated from the leaves of Aleurites fordii (Euphorbiaceae). A was identified as 12-O-palmitoyl-16-hydroxyphorbol-13-acetate (1). B, C, and D, new diterpene esters, were shown to be 12-O-palmitoyl-4-deoxy-4β-16-hydroxyphorbol-13-acetate (2), 12-O-palmitoyl-4-deoxy-4α-16-hydroxyphorbol-13-acetate (3), and 12-O-palmitoyl-4-deoxy-16-hydroxylumiphorbol-13-acetate (4), respectively.     

Tigliane diterpene esters with IFN γ-inducing activity from the leaves of Aleurites fordii

Bioactivity-guided fractionation on the leaves of Aleurites fordii led to the isolation of a new tigliane diterpene ester, 12-O-hexadecanoyl-7-oxo-5-ene-16-hydroxyphorbol-13-acetate (1) along with four known compounds, 12-O-hexadecanoyl-7-oxo-5-ene-phorbol-13-acetate (2), 12-O-hexadecanoyl-phorbol-13-acetate (3), 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (4), and 12-O-hexadecanoyl-4-deoxy-4α-16-hydroxyphorbol-13-acetate (5). The structures of these compounds were determined by interpretation of NMR (1D and 2D) spectroscopic data and MS data. All the isolates were evaluated for their effects on the induction of IFN-γ in NK92 cells. Compounds 3 and 4 exhibited the most potent responses in IFN-γ induction, comparable to the positive control, phorbol 12-myristate 13-acetate (PMA).     

Changes in polypeptide synthesis and glycosylation in mouse embryonic fibroblast C3H/10T1/2 Cl 8 cells caused by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate

The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate.     

Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.     

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.     

Glycosylation with N-Troc-protected glycosyl donors

N-Troc-protected (Troc = 2,2,2-trichloroethoxycarbonyl) glucosamine and galactosamine glycosyl donors (1-O-acetyl sugar, bromo sugar, and thioglycoside) were compared with the corresponding N-Phth-protected derivatives in glycosylations of 2-(trimethylsilyl)ethanol, 2-bromoethanol, methyl 3-mercaptopropionate, N-Fmoc-protected serine, and 2-(trimethylsilyl)ethyl

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. The N-Troc-protected donors gave pure β-glycosides in somewhat higher yields than the N-Phth-protected counterparts. The N-Troc protecting group can be removed by reduction with zinc, which allows selective N-deprotection in oligosaccharides containing both N-Troc and N-Phth groups.     

Synthesis of 2-(2-adamantyl)piperidines and structure anti-influenza virus A activity relationship study using a combination of NMR spectroscopy and molecular modeling

The 2-(2-adamantyl)piperidines 13 and 15a-c were synthesized and evaluated for anti-influenza virus A and B activity. The parent N-H compound 13 was 3–4 times more active than amantadine and rimantadine against H₂N₂ influenza A. N-alkylation of 13 resulted in derivatives 15a-c that were devoid of biological activity. This dramatic reduction in biological activity may be attributed to the different conformational properties between N-H and N-alkyl piperidines, as deduced from the combination of computational chemistry and NMR spectroscopy.     

Esters and amides of 2,3-dimethoxy-8,9-methylenedioxy-benzo[i]phenanthridine-12-carboxylic acid: potent cytotoxic and topoisomerase I-targeting agents

The exceptional topoisomerase I-targeting activity and antitumor activity of 5-(2-N,N-dimethylamino)ethyl-8,9-dimethoxy-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one (ARC-111, topovale) prompted studies on similarly substituted benzo[i]phenanthridine-12-carboxylic ester and amide derivatives. Among the benzo[i]phenanthridine-12-carboxylic esters evaluated, the 2-(N,N-dimethylamino)ethyl, 2-(N,N-dimethylamino)-1-methylethyl, and 2-(N,N-dimethylamino)-1,1-dimethylethyl esters possessed similar cytotoxicity, ranging from 30 to 55 nM in RPMI8402 and KB3-1 cells. Several of the carboxamide derivatives possess potent topoisomerase I-targeting activity and cytotoxicity. The 2-(N,N-dimethylamino)ethyl, 2-(N,N-diethylamino)ethyl, and 2-(pyrrolidin-1-yl)ethyl amides were among the more cytotoxic benzo[i]phenanthridine-12-carboxylic derivatives, with IC₅₀ values ranging from 0.4 to 5.0 nM in RPMI8402 and KB3-1 cells.     

Uptake of Lucifer Yellow by plant cells in the presence of endocytotic inhibitors

Summary Lucifer Yellow (LY), a membrane-impermeant anion, was able to enterArabidopsis thaliana cells. LY was taken up by fluid-phase endocytosis and a plasmalemmal anionic carrier mechanism. Both mechanisms were shown to be concentration-dependent. At 0.1 mg/ml, LY was mainly taken up via fluid-phase endocytosis and concentrated in vesicular-like structures. At a ten-fold higher concentration (1 mg/ml), a plasmalemmal anionic carrier system allowed LY uptake and its accumulation in the central vacuole by a vacuolar anionic transporter. Chloroquine, cytochalasin B, monensin, and phorbol-12-myristate-13-acetate (PMA) hindered LY endocytosis. Brefeldin A did not modify LY uptake. The probenecidsensitive carrier uptake machinery showed sensitivity to chloroquine and PMA. Therefore the probenecid-sensitive transport mechanism seems to be complex and involve both acidification of a compartment and protein kinase C activity.Abbreviations CH carbohydrazide - DMSO dimethylsulfoxide - LY Lucifer Yellow - MES 2-[N-morpholino]-ethanesulfonic acid - MS Murashige and Skoog's medium - PMA phorbol-12-myristate-13-acetate - NAA naphthalene acetic acid     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets

Effects of 1-(5-isoquinolinesulfonyl)-2-methylpeperazine (H-7), a potent inhibitor of protein kinase C in vitro (1), were investigated with regard to stimulus-induced protein phosphorylation of rabbit platelets. While H-7 inhibited the protein kinase C-mediated phosphorylation in 12-0-tetradecanoylphorbol-13-acetate (TPA)-stimulated platelets, this compound did not block the Ca2+-calmodulin-dependent phosphorylation in Ca2+ ionophore A23187-stimulated cells. This selective inhibitor of protein kinase C, in intact cells, will facilitate studies on the biological functions of protein kinase C.     

Bombesin-like peptides stimulate gastrin release from isolated rat G-cells

Bombesin-like peptides as well as receptor-independent activators were tested for their effect on gastrin release from acutely dispersed rat gastric G-cells. The amphibian peptide bombesin as well as its mammalian analogues neuromedin B and neuromedin C stimulated gastrin release. Maximal responses were achieved with 10(-9) M bombesin (191.0 +/- 16.8% of basal release), 10(-8) M neuromedin C(205.9 +/- 17.6%) and 10(-7) M neuromedin B (162.2 +/- 10.4%), respectively. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and the synthetic diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) are receptor-independent activators of the protein kinase C. Both TPA (10(-6) M) and OAG (10(-5) M) stimulated gastrin release to 214.0 +/- 29.3% and 198.2 +/- 20.8% of basal, respectively. Calcium ionophore A23187 (10(-5) M) was the most effective stimulant tested (364.7 +/- 39.6%). Its effect was reversed by the calmodulin antagonist W 7 (10(-6)-10(-5) M). Finally, forskolin (10(-5) M), a direct activator of cAMP-formation, as well as the cAMP-analogue dbcAMP (10(-3) M) induced gastrin release. IN conclusion, neuromedin B is less potent and less effective than neuromedin C and bombesin in stimulating rat gastric G-cells. In addition, gastrin release is activated by calcium- and phospholipid-dependent as well as by cAMP-induced cellular signal transduction mechanisms.     

Effect of Age on Brain Cortical Protein Kinase C and Its Mediation of 5-Hydroxytryptamine Release

The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5-hydroxytryptamine (5-HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12-myristate 13-acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer-344 rats. Parietal cortical brain slices from 6-, 12-, and 24-month-old animals were preloaded with [3H]5-HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5-HT release induced by either K+ or A23187 was found to be reduced in 12- and 24-month-old as compared to 6-month-old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6- and 12-month-old animals but reduced [3H]5-HT release in slices from 24-month-old animals. These responses were prevented by the putative PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age-related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC-mediated functions that are altered during senescence.     

Activation of adipocyte adenylate cyclase by protein kinase C

Adenylate cyclase activity in purified rat adipocyte membranes is stimulated by the calcium- and phospholipid-dependent enzyme protein kinase C. Over the concentration range of 100-1000 milliunits/ml, both highly purified (approximately 3000 units/mg of protein) protein kinase C from rat brain and partially purified (14 units/mg of protein) protein kinase C from guinea pig pancreas stimulate cyclase activity. The actions of both protein kinase C preparations on adenylate cyclase activity are dependent on added calcium, which is effective at concentrations less than 10 microM. Exogenous phospholipids are not required for stimulation of adenylate cyclase by protein kinase C; but, under typical cyclase assay conditions, the adipocyte membranes satisfy the lipid requirement for protein kinase C phosphorylation of histone. The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate enhances the kinase action on cyclase, and the phorbol ester is effective at concentrations equimolar with the kinase (less than 10 nM). With the brain protein kinase C, 12-O-tetradecanoylphorbol-13-acetate effects are especially evident at limiting calcium concentrations. Inhibitors of protein kinase C activity, such as chlorpromazine, palmitoylcarnitine, and polymyxin B, inhibit selectively that adenylate cyclase activity which is stimulated by protein kinase C plus calcium. It is concluded that protein kinase C acts directly on the adipocyte adenylate cyclase system.     

Dehydroepiandrosterone sulfate (DHEA-S) and 3', 5'-cyclic adenosine monophosphate (cAMP) production in a cultured human adrenocortical carcinoma cell line (SW-13)

Very little has been known of the biochemical function of a human adrenocortical carcinoma cell line, SW-13. In this study, the production of several adrenal steroids and 3', 5'-cyclic adenosine monophosphate (cAMP) were investigated in this cell line. The cells were incubated in L-15 medium containing 0.1% bovine serum albumin with several reagents in an atmosphere of 5% CO2 and 95% air for 2 hours at 37 degrees C. Aldosterone (Ald), corticosterone (B), cortisol (F), dehydroepiandrosterone sulfate (DHEA-S) and cAMP were simultaneously assayed by specific radioimmunoassays in the medium and cells. Significant increases in cAMP production were observed by cholera toxin (10 ng/ml) and forskolin (10 nM), both direct stimulators of adenylate cyclase, in the cAMP concentration without an increase in the steroids. The DHEA-S concentration in the medium was significantly increased by angiotensin-II (10(-7)M), noradrenalin (3 X 10(-5) M), adrenalin (3 X 10(-5) M) or alpha-melanocyte-stimulating hormone (alpha-MSH, 10(-7) M), none of which was associated with cAMP production. Neither adrenocorticotropin (10(-10) M) nor human chorionic gonadotropin (500 mIU/ml) stimulated the release of the steroids or cAMP production. A calcium ionophore, A23187 (10(-7) M), and 12-O-tetradecanoylphorbol-13-acetate (10(-8) M), a direct stimulator of protein kinase C, stimulated the release of DHEA-S, but not those of Ald, B and F. The results suggest that SW-13 retains functioning adenylate cyclase which, however, is not linked with steroidogenesis and that DHEA-S is produced probably by the mechanisms which involve protein kinase C system or calcium ion. This report provides the first demonstration of cAMP and DHEA-S production in SW-13 and suggests that this cell line is potentially useful for investigating the mechanisms of steroidogenesis in the human adrenal cortex.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

A rapid,nongenomic action of glucocorticoids in rat B103 neuroblastoma cells

We report here a new example in which glucocorticoids (GCs) acted in a rapid, nongenomic way. In rat B103 neuroblastoma cells, 5-hydroxytryptamine (5-HT) was found to evoke an immediate rise in intracellular free calcium concentration ([Ca(2+)](i)). Pre-incubation of B103 cells for 5 min with corticosterone (B) or bovine serum albumin-conjugated corticosterone (B-BSA) concentration-dependently (10(-4)-10(-8) M) inhibited the peak increments in [Ca(2+)](i). Cortisol and dexamethasone had a similar effect, while deoxycorticosterone and cholesterol were ineffective. This rapid inhibitory effect of corticosterone could be mimicked by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and abolished completely by PKC inhibitors Ro31-8220 or GF-109203X. Neither pertussis toxin (PTX) nor nuclear GC receptor (GR) antagonist RU38486 influenced the rapid action of B. Our results suggest that GCs can modulate the 5-HT-induced Ca(2+) response in B103 cells in a membrane-initiated, nongenomic, and PKC-dependent manner.     

TPA(12-O-tetradecanoyl-phorbol-13-acetate) as a carcinogen for mouse skin

Virchows Archiv B Cell Pathology - In order to study a possible dose/response relationship in the tumorigenic effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), groups of hairless mice were...