Levobupivacaine differentially suppresses platelet aggregation by modulating calcium release in a dose-dependent manner.

OBJECTIVE: Levobupivacaine, an amide local anesthetic widely used in regional anesthesia, is reported in recent studies that it is a potent inhibitor of platelet functions. However, the concentrations of levobupivacaine were limitedly estimated in these reports. Additionally, the mechanisms by which it affects platelet function and blood coagulation is still not entirely known. The purpose of this study was to further investigate its effects on platelet function and the possible signaling mechanisms under various concentrations of levobupivacaine. METHODS: Blood samples collected from healthy volunteers were separated into whole blood, platelet-rich-plasma and washed platelets. The effect of levobupivacaine on platelet aggregation was studied using platelet function analyzer (PFA-100) and platelet aggregometer. Agonist-induced platelet adenosine triphosphate (ATP) release, cytosolic calcium mobilization, thromboxane B2 (TxB2) secretion and platelet P-selectin translocation under various concentrations of levobupivacaine were investigated. RESULTS: Our results indicated that levobupivacaine possessed negative effect on platelet aggregation. The closure times of (PFA-100) were lengthened and the agonist-induced platelet aggregation was significantly attenuated by levobupivacaine even at a low dose (50 µgml(-1)). Pretreatment with levobupivacaine produced significant changes in agonist-induced platelet P-selectin translocation, ATP release, thromboxane A2 (TxA2) production, and calcium mobilization in a dose-dependent manner. The p38 mitogen-activated protein kinases (MAPK), protein kinase C (PKC) δ subtype, cytosolic phospholipase A2 (cPLA2), and protein kinase B (PKB or Akt) were involved in collagen-induced platelet signaling, which would be responsible for antiplatelet effects of levobupivacaine. CONCLUSION: We explored possible targets of levobupivacaine on platelets aggregation signaling mechanisms. Our data revealed that p38 MAPK, PKC δ subtype, cPLA2, and Akt were pathways involved in collagen-induced platelet signaling, which might be responsible for antiplatelet effects of levobupivacaine. Our study did provide direct evidence bolstering the critical mechanisms of levobupivacaine within different contexts. Additionally, levobupivacaine imposed a negative effect on platelet aggregation through multiple signaling pathways.

Secondary metabolites from the root of Ehretia longiflora and their biological activities.

Bioassay-guided fractionation of the methanolic extract of the root of Ehretia longiflora (Boraginaceae) afforded eight compounds, ehretiquinone (1), ehretiolide (2), ehreticoumarin (3), ehretilactone A (4), ehretilactone B (5), ehretiamide (6), ehretine (7), and ehretiate (8), together with 12 known compounds (9-20). The relative configuration of 1 was determined by single crystal X-ray diffraction. Among the isolates, 1 and prenylhydroquinone (14) showed antitubercular activity against Mycobacterium tuberculosis strain H37Rv with MIC values of 25.0 and 26.2 µg/mL, respectively. Moreover, 1 exhibited inhibitory effects on N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide production, with IC50 value of 0.36±0.03µM.

Antiplatelet effect by p-cresol, a uremic and environmental toxicant, is related to inhibition of reactive oxygen species, ERK/p38 signaling and thromboxane A2 production.

P-cresol is a well-known uremic toxin and environmental toxicant that may affect platelet functions. In this study, p-cresol (1-5 µM) inhibited the arachidonic acid (AA)-induced platelet aggregation, with 47% and 82% of inhibition at concentrations of 2 and 5 µM, respectively. Under similar experimental condition, p-cresol showed little effect on the U46619-induced platelet aggregation. p-cresol (<500 µM) revealed no discernable cytotoxicity to platelets as analyzed by quantification of lactate dehydrogenase release. Antiplatelet effect of p-cresol was related to inhibition of thromboxane A(2) (TXA(2)) and prostaglandin D(2) (PGD(2)) formation. P-cresol (2-100 µM) partly inhibited the AA-induced reactive oxygen species (ROS) production as well as the extracellular signal-regulated kinase (ERK1/2) and p38 phosphorylation in platelets. P-cresol further inhibited the AA-induced aggregation of rabbit platelet-rich plasma (PRP) with an IC50 of 2 µM and aggregation of human PRP (IC50 = 13.6 µM). Intravenous administration of p-cresol (250-1000 nmole) into mice effectively suppressed the ex vivo platelet aggregation, whereas showed little effect on the value of RBC, hemoglobin (HGB), hematocrit, MCV, MCH, MCHC, platelets and lymphocyte counts. These results indicate that in acute p-cresol-poisoning and long-term exposure to cresol as in severe uremic patients, p-cresol may potentially inhibit blood clot formation and lead to hemorrhagic disorders via inhibition of platelet aggregation, ROS production, ERK/p38 activation and TXA(2) production.

Secondary metabolites from the leaves of Neolitsea hiiranensis and the anti-inflammatory activity of some of them.

Seven sesquiterpenoids, hiiranlactones A-D (1-4), (-)-ent-6α-methoxyeudesm-4(15)-en-1ß-ol (5), (+)-villosine (6), hiiranepoxide (7), and one triterpenoid, hiiranterpenone (8), together with 22 known compounds, were isolated from the leaves of Neolitsea hiiranensis (Lauraceae). Their structures were elucidated by spectroscopic analysis and single crystal X-ray diffraction. Among the isolates, hiiranlactone B (2) and hiiranlactone D (4) exhibited inhibitory activity against fMLP-induced superoxide production by human neutrophils with IC(50) values of 21.86±3.97 and 25.78±4.77µM, respectively.

5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2) suppresses fMLP-mediated respiratory burst in human neutrophils by inhibiting phosphatidylinositol 3-kinase activity.

Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2), a lignan extracted from the fruit of Melicope Semecarprifolia, on fMLP-induced respiratory burst in human neutrophils and suggests a possible therapeutic approach to ameliorate disease associated with neutrophil hyperactivation. MSF-2 inhibited fMLP-induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflecting inhibition of phosphatidylinositol 3-kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion production in neutrophils. MSF-2 suppresses PI3K activation and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production, and consequently inhibits downstream activation of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC-dependent elevation of intracellular calcium. MSF-2 reduces fMLP-mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC-dependent manner. However, MSF-2 is not a competitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, MSF-2 prevents fMLP-induced neutrophil infiltration and inflammation in mice. In conclusion, MSF-2 opposes fMLP-mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2.

Anti-inflammatory Biphenyls and Dibenzofurans from Rhaphiolepis indica.

Bioassay-guided fractionation of the methanolic extract of the roots of Rhaphiolepis indica var. tashiroi afforded four new dibenzofurans, 2-hydroxy-3,4,6-trimethoxydibenzofuran (1), 2-hydroxy-3,4,9-trimethoxydibenzofuran (2), 2-hydroxy-3,4,6,9-tetramethoxydibenzofuran (3), and 1,2-methylenedioxy-3,4,6-trimethoxydibenzofuran (4), two new biphenyls, 3-hydroxy-2',5-dimethoxybiphenyl (5) and 2',3-dihydroxy-5-methoxybiphenyl (6), and 3-hydroxy-5-methoxybiphenyl (7). Among the isolates, 3, 5, and 6 exhibited inhibitory effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with in vitro IC50 values < 8.36 µM.

Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions.

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca(2+)-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca(2+)-dependent binding that became partially Ca(2+) independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca(2+)-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5mM Ca(2+). We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.

Platelet function analyzer (PFA-100) offers higher sensitivity and specificity than thromboelastography (TEG) in detection of platelet dysfunction.

BACKGROUND: The development of advanced surgical procedures requires novel and early diagnostic techniques. One of the prime difficulties that need to be overcome is an abnormality in the coagulation system. The blood clot formation and fibrinolysis processes are very complicated and important perioperatively. However, most of the major surgeries, such as liver transplantation, use thromboelastography (TEG) for detection of coagulation abnormalities, even though TEG is not actually an ideal option. Therefore, we compared the sensitivity and specificity of the platelet function analyzer (PFA-100) and thromboelastogram (TEG) in predicting platelet dysfunction and bleeding risk. METHODS: Human blood samples were drawn from healthy volunteers for this study. Levobupivacaine and CGS21680 have antiplatelet effects which were used as the detection target. The platelet counts before comparison, platelet aggregation, the closure time of PFA-100, and the parameters of TEG were examined for data analysis. RESULTS: Platelet aggregations were suppressed by all levobupivacaine doses (10 microg/mL, 50 micropg/mL, 200 microg/mL) and CGS21680 (100 nM, 500 nM, 1 microM) in a dose-dependent manner. Levobupivacaine and CGS21680 at maximal test doses produced no significant alteration in any parameter in the TEG assay. In the samples measured with PFA-100, both levobupivacaine and CGS21680 at maximal test doses significantly prolonged the closure time in the PFA-100 assay. CONCLUSION: We conclude that PFA-100 offers a higher sensitivity and specificity than TEG in detection of platelet dysfunction.

Hydroxyethyl starch interferes with human blood ex vivo coagulation, platelet function and sedimentation.

BACKGROUND: Hydroxyethyl starch (HES) solutions are widely used for intravascular volume expansion. In Taiwan, the medium molecular weight of HES 200/0.5 and HES 130/0.4 solutions are most commonly used. It has been demonstrated that HES may affect coagulation and platelet function significantly. However, the differential effects of each medium molecular weight HES on platelets remain poorly reported. Therefore, we studied the influence of the two HES solutions on platelet function in vitro by mixing whole blood with different proportions of HES 130 kD, HES 200 kD, and saline to determine the differences. METHODS: Human blood samples for platelet function analyzer (PFA), aggregometry and blood/HES mixed test were drawn from the antecubital vein and put into test tubes containing 3.2% trisodium citrate (blood:citrate, 9:1). The specimens were divided into four groups, designated as whole blood, 10%, 20%, and 30% dilution with normal saline (N/S), HES130 or HES200 solution. The platelet function of each sample was measured by both PFA and platelet aggregometry. RESULTS: The results showed that the PFA-100 closure times CEPI-CT and CADP-CT were significantly prolonged in the samples diluted with normal saline, HES130 and HES200 than in the controls. The ADP triggered whole blood aggregometry showed that attenuated impedance was observed in samples of 20% diluted with HES130 and HES200 groups. The blood/HES mixed sedimentation test showed significantly increased proportion of the upper liquid layer in the HES200 group than in other groups. CONCLUSION: Our data demonstrated that HES200 and HES130 possess noticeably inhibitory effects on platelet function, especially when the HES replaced proportion was more than 20%. HES200 has a greater effect on blood cells and plasma separation than does HES130.

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase.

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca(2+) release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(2+) mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.

A new dibenzofuran and further constituents from the stems of Pourthiaea lucida with inhibitory activity on superoxide generation by neutrophils.

A new dibenzofuran, lucidafuran (1), was isolated from the stems of Pourthiaea lucida, together with eight known compounds. The structure of this new compound was determined through NMR and mass-spectrometric analyses. Among the isolated compounds, lucidafuran (1) and aucuparin (3) exhibited potent inhibitory activity against fMLP-induced superoxide (O(*-)(2)) production by human neutrophils with IC(50) values of 18.7+/-4.4 and 17.0+/-6.8 microM, resp.

Thrombin regulates matrix metalloproteinase-9 expression in human monocytes.

We investigated whether thrombin, the final activator of coagulation cascade, regulates expression of matrix metalloproteinases (MMP)-9 in human monocytes. We show that thrombin stimulation induced MMP-9 secretion of monocytes dose- and time-dependently as revealed by gelatin zymography. Real-time RT-PCR and Western blot analysis demonstrated that thrombin up-regulated mRNA and protein levels of MMP-9. Pre-incubation with anti-protease-activated receptor (PAR)-1 or anti-PAR-3 antibody partially inhibited the thrombin-induced MMP-9 secretion. Simultaneous incubation with both showed synergistic effect, indicating the involvement of both receptors in this thrombin effect. BAPTA, a Ca(2+) chelator, abolished the thrombin-induced MMP-9 secretion, indicating the requirement of Ca(2+) mobilization in this process. Inhibition of thrombin-induced MMP-9 secretion by either MEK inhibitor or p38 kinase inhibitor revealed that the thrombin effect was mediated by both ERK1/2 and p38 pathways. The activation of NFkappaB by thrombin as demonstrated by electromobility shift assay was also shown to be critical to the thrombin-induced MMP-9 up-regulation.

Hemeoxygenase-1 upregulation is critical for sirtinol-mediated attenuation of lung injury after trauma-hemorrhage in a rodent model.

BACKGROUND: Hemeoxygenase-1 induction in response to adverse circulatory conditions is protective. Our recent study has shown that administration of sirtinol attenuates hepatic injury in male Sprague-Dawley rats after trauma-hemorrhage; however, the mechanism by which sirtinol produces the salutary effects remains unknown. We hypothesized that sirtinol administration in male Sprague-Dawley rats after trauma-hemorrhage decreases cytokine production and protects against lung injury through a hemeoxygenase-1 related pathway. METHODS: Male Sprague-Dawley rats (n = 8 per group) underwent trauma-hemorrhage (mean arterial blood pressure 40 mm Hg for 90 min, then resuscitation). A single dose of sirtinol (1 mg/kg of body weight) with or without a hemeoxygenase enzyme inhibitor (chromium-mesoporphyrin) or vehicle was administered IV during resuscitation. Twenty-four hours thereafter, myeloperoxidase activity (a marker of neutrophil sequestration) and tumor necrosis factor alpha, interleukin-6, and interleukin-10 levels in the lung, protein concentrations in bronchoalveolar lavage fluid and tissue histology were measured. Lung hemeoxygenase-1 protein level was also determined. RESULTS: In the sirtinol-treated rats subjected to trauma-hemorrhage, there were significant improvements in lung myeloperoxidase activity (4.68 +/- 0.31 vs 9.36 +/- 1.03 U/mg protein, P < 0.05), tumor necrosis factor alpha levels (710.7 +/- 28 vs 1288 +/- 40.69 pg/mg protein, P < 0.05), interleukin-6 levels (343.6 +/- 18.41 vs 592.7 +/- 22.3 pg/mg protein, P < 0.05), and protein concentrations (303.8 +/- 24.54 vs 569.6 +/- 34.82 microg/mL, P < 0.05) and lesser damage in histology. There was no statistically significant difference in interleukin-10 levels in the lung between sirtinol-treated trauma-hemorrhaged rats and vehicle-treated trauma-hemorrhaged rats (842.5 +/- 54.18 vs 756.2 +/- 41.34 pg/mg protein, respectively). Lung hemeoxygenase-1 protein levels were increased in rats receiving sirtinol treatment as compared with vehicle-treated trauma-hemorrhaged rats (5.18 +/- 0.25 vs 2.70 +/- 0.16, P < 0.05). Administration of the hemeoxygenase inhibitor chromium-mesoporphyrin prevented the sirtinol-induced attenuation of shock-induced lung damage. CONCLUSION: The salutary effects of sirtinol administration on attenuation of lung inflammation after trauma-hemorrhage are mediated via upregulation of hemeoxygenase-1 expression.

Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca++ release.

Splitomicin is derived from beta-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 microg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 microM) or ADP (10 microM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 microM) was combined with PGE1 (0.5 microM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, splitomicin (300 microM) attenuated intracellular Ca(++) mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(++) mobilization, TXB2 formation and ATP release.

A benzodiazepines derived compound, 4-(3-chlorophenyl)-1,3-dihydronaphtho [2,3-b][1,4]diazepin-2-one (ND700C), inhibits fMLP-induced superoxide anion release by activating protein phosphatase 2A in human neutrophils.

UNLABELLED: We studied the mechanism underlying the inhibitory effect of a benzodiazepines derivative, 4-(3-chlorophenyl)-1,3-dihydronaphtho [2,3-b][1,4]diazepin-2-one (ND700C), on superoxide anion production induced by formly-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. ND700C inhibited the fMLP-induced superoxide anion production and cathepsin G release in a concentration-dependent manner with respective IC50 values of 5.0+/-0.5 and 8.7+/-0.8muM. In addition, ND700C was found to suppress fMLP-induced intracellular calcium mobilization and the phosphorylation of ERK and Akt. In another study, ND700C was observed to cause a rapid increase in intracellular cAMP level by up to threefold. Furthermore, when H89 was used to inhibit cAMP-dependent protein kinase A (PKA), we discovered that ND700C's suppressive effects on calcium mobilization, phosphorylation, and superoxide anion production were abrogated. ND700C demonstrated additive effect on the PGE1-induced increase in cAMP. However, this additive effect was not demonstrated with the IBMX-induced rise in cAMP. Our results indicated that ND700C did not directly inhibit the activity of phosphodiesterase 4. In another set of experiments, calyculin A and okadaic acid (both protein phosphatase 2A inhibitors) were found to reverse ND700C's positive effect on cAMP level. This observation suggested the involvement of protein phosphatase 2A in ND700C's cAMP-elevating mechanism. We found that the activity of protein phosphatase 2A was activated by ND700C. Furthermore, protein phosphatase 2A was co-immunoprecipitated with phosphodiesterase 4 after ND700C treatment in human neutrophils. CONCLUSION: ND700C inhibited fMLP-induced superoxide anion production through a PKA-dependent pathway. ND700C increased cAMP by activating protein phosphatase 2A, which subsequently inhibited phosphodiesterase 4.

(2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils.

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.

New phenylpropenoids, bis(1-phenylethyl)phenols, bisquinolinone alkaloid, and anti-inflammatory constituents from Zanthoxylum integrifoliolum.

Five new compounds, including two new phenylpropenoids, (R,E)-1-[4-(3-hydroxyprop-1-enyl)phenoxy]-3-methylbutane-2,3-diol (1) and 4-hydroxy-3-(3-methyl-2-butenyl)cinnamyl alcohol (2), two new bis(1-phenylethyl)phenols, 2,6-bis(1-phenylethyl)phenol (3) and 2,4-bis(1-phenylethyl)phenol (4), and a new bisquinolinone alkaloid, 18-demethylparaensidimerin C (5), together with 17 known compounds have been isolated from the stem wood of Zanthoxylum integrifoliolum. The structures of these new compounds were determined through spectral analyses including extensive 2D nuclear magnetic resonance data. Among the isolates, N-methylflindersine (7), (-)-simulanol (10), and evofolin-C (16) exhibited potent inhibition against N-formylmethionylleucylphenylalanine-induced superoxide production with IC(50) values less than 12 microM.

Synthesis, antiproliferative, and antiplatelet activities of oxime- and amide-containing quinolin-2(1H)-one derivatives.

Certain oxime- and amide-containing quinolin-2(1H)-one derivatives were synthesized and evaluated for their antiproliferative and antiplatelet activities. These compounds were synthesized via alkylation of hydroxyl precursors followed by the reaction with NH(2)OH or NaN(3) (Schmidt reaction). The preliminary assays indicated that amide derivatives are either weakly active or inactive while the oxime counterparts exhibited potent inhibitory activities against platelet aggregation induced by collagen, AA (arachidonic acid), and U46619 (the stable thromboxan A(2) receptor agonist). Among them, (Z)-6-[2-(4-methoxyphenyl)-2-hydroxyiminoethoxy]quinolin-2(1H)-one (7c) was the most active against AA induced platelet aggregation with an IC(50) of 0.58microM and was inactive against cell proliferation. For the inhibition of U46619 induced aggregation, 7a and 8a-c exhibited very potent activities with IC(50) values in a range between 0.54 and 0.74microM. For the antiproliferative evaluation, N-(biphenyl-4-yl)-2-(2-oxo-1,2-dihydroquinolin-7-yloxy)acetamide (11d) was the most potent with GI(50) values of <10, 10.8, and <10microM against the growth of MT-2, NCI-H661, and NPC-Tw01, respectively, and possessed only a weak antiplatelet activity. Further evaluation of 11d as a potential anticancer agent is on-going.

Modulation of human monocyte-derived dendritic cells maturation by a soluble guanylate cyclase activator, YC-1, in a cyclic nucleotide independent manner.

This study evaluated how YC-1, a guanylate cyclase activator, affects the maturation of human monocyte-derived dendritic cells. Maturation markers and intracellular signaling pathways were evaluated. YC-1 inhibited the lipopolysaccharide up-regulation of mature markers, including CD40, CD80 or CD86 in a concentration-dependent manner with IC(50) values of 4.6+/-0.4, 4.9+/-0.6 or 4.5+/-0.5 microM, respectively. YC-1, at a higher concentration, inhibited lipopolysaccharide-induced HLADR expression. These effects of YC-1 were not reversed by ODQ (10 microM), which is a soluble guanylate cyclase inhibitor, nor by KT5823 (1 microM), which is a PKG inhibitor. Additionally, YC-1 did not increase levels of cyclic nucleotides in dendritic cells, supporting the claim that YC-1 affects dendritic cells maturation in a cGMP-independent manner. YC-1, in a cGMP-independent manner, inhibited lipopolysaccharide-induced Akt activation, IkappaBalpha degradation and NF-kappaB translocation, all of which are associated with co-stimulatory molecules expression. YC-1 inhibited the capacity of dendritic cell to activate allogenic T cells with an IC(50) value of 1.2+/-0.3 microM. YC-1-treated dendritic cells have mature phenotypes that exhibit up-regulated CCR7, enhanced IL-10 release and low phagocytosis activity in the presence of lipopolysaccharide. In conclusion, YC-1 inhibited the lipopolysaccharide-induced co-stimulatory molecular expression of dendritic cells by inhibiting Akt activation, IkappaBalpha degradation and NF-kappaB translocation. These inhibitory effects on co-stimulatory molecules suppressed the capacity of dendritic cells to activate allogenic T cells. Additionally, YC-1 treated dendritic cells exhibit the up-regulation of CCR7, enhanced IL-10 release and the down-regulation of phagocytosis in the presence of lipopolysaccharide. Accordingly, YC-1 might be a useful tool for evaluation of dendritic cells on autoimmune or allergic disease.

Lignans, an amide and anti-platelet activities from Piper philippinum.

Investigation of the stem extract of Piper philippinum led to isolation of eight compounds, piperphilippinins I-VI (1-6), philippinamide (7), and (+)-bornyl caffeate (8), together with 26 known compounds. Among the isolates, (-)-3',4'-O,O-demethylenehinokinin (10) and 3,4-methylenedioxycinnamaldehyde (23) showed anti-platelet activities in vitro.