Polycyclic aromatic hydrocarbons present in cigarette smoke cause endothelial cell apoptosis by a phospholipase A2-dependent mechanism. (65/915)

Smoking is a major risk factor for endothelial cell injury and subsequent coronary artery disease. Epidemiological studies implicate the phospholipase A2/arachidonic acid cascade in the mechanism by which smoking causes heart disease. However, specific components of cigarette smoke that activate this pathway have not been identified. The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons contained in cigarette smoke on phospholipase A2 (PLA2) activity and apoptosis of human coronary artery endothelial cells. 1-methylanthracene (1-MA), phenanthrene (PA), and benzo(a)pyrene (B(a)P) caused significant release of 3H-arachidonate from endothelial cells. 1-MA and PA, but not B(a)P, also caused significant release of 3H-linoleic acid. Release of fatty acids from membrane phospholipids preceded the onset of apoptosis. 3H-arachidonate release and apoptosis induced by 1-MA, B(a)P, and PA were inhibited by methylarachidonoyl-fluorophosphonate, an inhibitor of Groups IV and VI PLA2s. Bromoenol lactone, an inhibitor of Group VI enzymes, inhibited both 3H-arachidonate release and apoptosis induced by 1-MA and PA, but not B(a)P. MJ33, an inhibitor of the acidic calcium-independent PLA2, attenuated 3H-arachidonate release and apoptosis by PA, but not 1-MA or B(a)P. The presence of Groups IV and VI and the acidic iPLA2 in endothelial cells was demonstrated by reverse transcriptase-polymerase chain reaction and Western analysis. These data suggest that 1-MA, B(a)P and PA induce apoptosis of endothelial cells by a mechanism that involves activation of these three distinct isoforms of PLA2.  (+info)

In vitro reversal of chloroquine resistance in Plasmodium falciparum with dihydroethanoanthracene derivatives. (66/915)

The effects of combining four dihydroethanoanthracenic (DEA) derivatives and chloroquine were assessed in vitro against Plasmodium falciparum chloroquine resistant parasites W2, Palo Alto, FCR3, and Bres1. Like verapamil or promethazine, the four dihydroethanoanthracenic derivatives tested can be added to the growing list of agents that show capability in enhancing the activity of chloroquine against resistant parasites. The structurally related tricyclic antihistaminic compounds examined in this study exerted different intrinsic antimalarial activity, but the same chloroquine-potentiating activity as verapamil or promethazine. They may act both on the rate of chloroquine accumulation and on its access to ferriprotoporphyrin IX. The reversal mechanism would be assumed to result from competition between DEA derivatives and chloroquine for efflux translocation sites, thus causing an increase in steady-state accumulation of chloroquine and a return to susceptibility. Restoration of therapeutic efficacy of chloroquine against resistant parasites by the administration of an additional drug available at relatively low cost may be a more effective strategy than the introduction of another antimalarial drug at the national level.  (+info)

A gene cluster from a marine Streptomyces encoding the biosynthesis of the aromatic spiroketal polyketide griseorhodin A. (67/915)

The telomerase inhibitor griseorhodin A is probably the most heavily oxidized bacterial polyketide known and features a unique epoxyspiroketal moiety crucial for its activity. To gain insight into which tailoring enzymes generate this pharmacophore, we have cloned and fully sequenced the griseorhodin biosynthesis gene cluster. Among other unusual features, this aromatic polyketide synthase (PKS) system encodes an unprecedented number of functionally diverse oxidoreductases, which are involved in the oxidative modification of a polyaromatic tridecaketide precursor by cleavage of three carbon-carbon bonds. The cluster was highly unstable on a variety of shuttle plasmids but could finally be functionally expressed in its entirety in Streptomyces lividans using a novel integrative cosmid vector. The availability of the tailoring system now opens up the possibility of engineering nonnatural biosynthetic pathways yielding novel pharmacologically active analogs with a similar pharmacophore.  (+info)

c-Jun N-terminal kinase activation mediates downregulation of connexin43 in cardiomyocytes. (68/915)

Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP-mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.  (+info)

Expression, purification, and characterization of AknX anthrone oxygenase, which is involved in aklavinone biosynthesis in Streptomyces galilaeus. (69/915)

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.  (+info)

Salicylic acid reverses phorbol 12-myristate-13-acetate (PMA)- and tumor necrosis factor alpha (TNFalpha)-induced insulin receptor substrate 1 (IRS1) serine 307 phosphorylation and insulin resistance in human embryonic kidney 293 (HEK293) cells. (70/915)

Salicylates, including aspirin, have been shown to improve insulin sensitivity both in human and animal models. Although it has been suggested that salicylates sensitize insulin action by inhibiting IkappaB kinase beta (IKKbeta), the detailed mechanisms remain unclear. Protein kinase C isoforms and tumor necrosis factor alpha (TNFalpha) signaling pathways are well described mediators of insulin resistance; they are implicated in the activation of IKKbeta and the subsequent inhibition of proximal insulin signaling via insulin receptor substrate 1 (IRS1) and Akt. This study investigated the effect of salicylic acid on phorbol 12-myristate 13-acetate (PMA)- and TNFalpha-induced insulin resistance in a human embryonic kidney 293 (HEK293) cell line stably expressing recombinant human IRS1. The results showed that both PMA and TNFalpha inhibited insulin-induced Akt phosphorylation and promoted IRS1 phosphorylation on Ser-307. Salicylic acid pretreatment completely reversed the effects of PMA and TNFalpha on both Akt and IRS1. Whereas PMA activated protein kinase C isoforms and IKKbeta, TNFalpha activated neither. On the other hand, both PMA and TNFalpha activated the c-Jun N-terminal kinase (JNK), which has been reported to directly phosphorylate IRS1 Ser-307. SP600125, a JNK inhibitor, prevented PMA and TNFalpha-induced IRS1 Ser-307 phosphorylation. Finally, salicylic acid inhibited JNK activation induced by both PMA and TNFalpha. Taken together, these observations suggest that salicylic acid can reverse the inhibitory effects of TNFalpha on insulin signaling via an IKKbeta-independent mechanism(s), potentially involving the inhibition of JNK activation. The role of JNK in salicylic acid-mediated insulin sensitization, however, requires further validation because the JNK inhibitor SP600125 appears to have other nonspecific activity in addition to inhibiting JNK activity.  (+info)

The p65/RelA subunit of NF-kappaB suppresses the sustained, antiapoptotic activity of Jun kinase induced by tumor necrosis factor. (71/915)

Tumor necrosis factor (TNF) signaling through the TNF receptors involves the recruitment of key signaling factors, leading to the activation of both the transcription factor NF-kappaB and the stress-activated Jun kinase (JNK). In most cells, TNF signaling leads to a rapid and transient increase in JNK activity. However, we show that TNF treatment leads to the sustained activation of JNK in cells that are null for the p65/RelA subunit of NF-kappaB as well as in cells expressing the super-repressor form of IkappaB. In addition, the data indicate that the ability of p65/RelA to regulate gene expression is required to suppress the persistent activation of JNK. Interestingly, this suppression occurs upstream of JNK, within the signal transduction cascade leading to JNK activation, without affecting the stress-activated kinase p38. Since NF-kappaB has previously been shown to be involved in the suppression of TNF-induced apoptosis, we were interested in determining the role of deregulated JNK activity, induced by the loss of NF-kappaB, in controlling the cell death response. Through the use of different approaches for inhibition of JNK, we show that the suppression of JNK activity in cells that lack active NF-kappaB enhances the apoptotic response to TNF. These data suggest that the activity of JNK in cells blocked for NF-kappaB function provides an antiapoptotic signal and explains, at least partly, why a significant number of NF-kappaB null cells remain viable following TNF treatment.  (+info)

Mitogen-activated protein kinase routes as targets in the action of diaza-anthracene compounds with a potent growth-inhibitory effect on cancer cells. (72/915)

1,8-Diaza-anthracene-tetraones are novel intermediates in the synthesis of the antifolate antibiotic diazaquinomycin A that was found before to have potent antitumor activity. Three of them (CV65, CV66, and CV70) were found to inhibit growth of a panel of several human tumor cell lines. The IC50s ranged from 0.05 to 1.5 microM and are comparable with that of doxorubicin. Among the three drugs, CV70 showed the highest cytotoxic activity. The growth-inhibitory action of these compounds was unrelated to the p53 status of the cells. At micromolar concentrations, all three compounds induced apoptosis, CV70 being the most proapoptotic. The incubation of HeLa cells with CV65, CV66, and CV70, at concentrations between 10 and 20 microM, inhibited the activation of c-Jun NH2-terminal kinase by various stimuli and prevented growth factor-induced extracellular signal-regulated kinase (ERK) 5 activation. At least one drug, CV65, also inhibited p38. This was surprising because proapoptotic antitumor drugs activate stress signaling pathways. Activation of ERK1/ 2 by growth factors or phorbol esters was unaffected by preincubation of cells with CV compounds. In vitro, CV compounds inhibit the enzyme quinone reductase but not c-Jun NH2-terminal kinase or ERK5. Because doxorubicin also inhibits quinone reductase, we conclude that the inhibitory effect of CV compounds on stress signaling kinases is not a direct effect on the kinases and is likely attributable to upstream elements of the activation cascades.  (+info)