OBJECTIVE

To investigate an association of responsiveness to clopidogrel loading dose with genotypes of cytochrome P450 (CYP) 2C19, other CYP isozymes and nongenetic factors in patients with coronary artery disease.

METHODS

Genotyping for CYP2C19 (*2, *3 and *17), CYP3A4*1B and CYP3A5*3 variants was performed in patients (n = 237) who underwent percutaneous coronary intervention. Adenosine diphosphate-induced platelet aggregation was determined after first administration of 600 mg clopidogrel.

RESULTS

CYP2C19*2 carriers showed significantly increased residual platelet aggregation (RPA) (OR: 4.6; 95% CI: 2.5-8.7; p < 0.0001) compared with noncarriers. All other polymorphisms had no influence on RPA. For the development of a risk score for better prediction of RPA, CYP2C19*2 genotype and previously identified nongenetic risk factors (age >65 years, Type 2 diabetes mellitus, decreased left ventricular function, renal failure and acute coronary syndrome) were analyzed. Multivariable logistic regression analysis showed a significant correlation of the nongenetic factors (chi (2) = 5.32; p = 0.021) and CYP2C19*2 (chi (2) = 21.31; p < 0.0001) with high RPA, and an even higher association for the combination of both (chi (2) = 25.85; p < 0.0001).

CONCLUSIONS

Prediction of responsiveness after clopidogrel loading dose may substantially be improved by adding CYP2C19*2 genotype to nongenetic risk factors.

Human platelets have been separated into two extreme density populations by centrifugation in specific density media. A large-heavy platelet population with specific gravity>> 1.055 and a light-small population with specific gravity < 1.046 were obtained, each representing approximately 15-20% of the total population volume. The average volume per platelet of the separated large-heavy and light-small platelet populations was 12 and 5 mu(3) respectively. When data are expressed per milliliter platelets or per gram wet weight, the large-heavy platelet population had a 2-fold greater glycogen content, 1.3-fold greater orthophosphate content, 1.3-fold greater total adenine nucleotide content, 4.2-fold greater rate of glycogenolysis, 2.6-fold greater rate of glycolysis, 2.9-fold greater rate of protein synthesis, and 5.7-fold greater rate of glycogen synthesis. Significant differences were not obtained with respect to total lipid content or total lipid synthesis. The large-heavy platelet had a 2.5-fold greater resistance to osmotic shock as measured by adenosine triphosphate (ATP) or adenosine diphosphate (ADP) release. These data, as well as diisopropyl fluorophosphate (DFP(32)) survival curves in rabbits, indicate that large-heavy platelets have a greater metabolic potential and suggest that they may be the young platelets which progress with age to light-small platelets with a diminished metabolic potential.

To test for the role of the P2Y(2) receptor (P2Y(2)-R) in the regulation of nucleotide-promoted Ca(2+) signaling in the lung, we generated P2Y(2)-R-deficient (P2Y(2)-R(-/-)) mice and measured intracellular Ca(2+)(i) responses (DeltaCa(2+)(i)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y(2)-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced DeltaCa(2+)(i) was as follows: UTP>>/= ATP>>) ADP>> UDP. The responses induced by these agonists were completely absent in the P2Y(2)-R(-/-) fibroblasts. Inositol phosphate responses paralleled those of DeltaCa(2+)(i) in both groups. ATP and UTP also induced Ca(2+)(i) responses in wild type airway epithelial cells. In the P2Y(2)-R(-/-) airway epithelial cells, UTP was ineffective. A small fraction (25%) of the ATP response persisted. Adenosine and alpha,beta-methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca(2+), indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca(2+)(i) response in P2Y(2)-R(-/-) cells, which was inhibited by the P2Y(1) receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y(2)-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y(1) receptor, and that the P2Y(2)-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca(2+) mobilization in mouse lung fibroblasts.

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

Dendritic cells (DCs) are considered the principal initiators of immune response because of their ability to migrate into peripheral tissues and lymphoid organs, process antigens, and activate naive T cells. There is evidence that extracellular nucleotides regulate certain functions of DCs via G-protein-coupled P2Y receptors (P2YR) and ion-channel-gated P2X receptors (P2XR). Here we investigated the chemotactic activity and analyzed the migration-associated intracellular signaling events such as actin reorganization and Ca(++) transients induced by common P2R agonists such as adenosine 5'-triphosphate (ATP) and 2-methylthioadenosine triphosphate, the P2YR agonists UTP and adenosine 5'-diphosphate (ADP), or the P2XR agonists alphabeta-methylenadenosine-5'-triphosphate and 2',3'-(4-benzoyl)benzoyl-ATP. The common P2R agonists and the selective P2YR agonists turned out to be potent chemotactic stimuli for immature DCs, but not for mature DCs. In contrast, P2XR agonists had only marginal chemotactic activity in both DC types. Chemotaxis was paralleled by a rise in the intracellular Ca(++) concentration and by actin polymerization. Studies with pertussis toxin implicated that intracellular signaling events such as actin polymerization, mobilization of intracellular Ca(++), and migration induced by nucleotides was mediated via G(i/o) protein-coupled P2YR. Moreover, functional studies revealed selective down-regulation of this G(i/o) protein-coupled chemotactic P2YR responsiveness during maturation, although immature and mature DCs expressed similar amounts of mRNA for the P2R subtypes (P2Y(2)R, P2Y(4)R, P2Y(5)R, P2Y(7)R, P2Y(11)R and P2X(1)R, P2X(4)R, P2X(7)R), and no major differences in respect to the mRNA expression of these receptors could be observed by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR). In summary, our data describe a differential chemotactic response of immature and mature DCs to nucleotides, and lend further support to the hypothesis that P2R are a novel class of immunomodulatory plasma membrane receptors suitable for pharmacological intervention.

To define the molecular pathways involved in radiation-induced apoptosis and the role of the mitochondria, 32D cl 3 hematopoietic cells and subclones overexpressing either the human manganese superoxide dismutase (SOD2) transgene (1F2 and 2C6) or BCL2L1 (also known as Bcl-xl) transgene (32D-Bcl-xl) were compared for their response to radiation at the subcellular level, comparing nuclear to mitochondrial localized pathways. All cell lines showed complete detectable DNA repair by 30 min after irradiation, and clearly delayed migration of BAX and active stress-activated protein (SAP) kinases MAPK1 (also known as p38) and MAPK8 (also known as JNK1) to the mitochondria at 3 h. Radioresistant clonal lines 1F2, 2C6 and 32D-Bcl-xl showed significant decreases in mitochondrial membrane permeability, cytochrome C release, caspase 3 and poly(adenosine diphosphate-ribose) polymerase (PARP) activation at 6-12 h, and in apoptosis at 24 h. Since the nuclear-to-cytoplasm events preceding the release of cytochrome C were similar in all cell lines, and increased expression of either the SOD2 or the BCL2L1 transgene provided radiation protection, we conclude that events at the level of the mitochondria are critically involved in radiation-induced apoptosis.

Nickel, cadmium, cobalt, and arsenic compounds are well-known carcinogens to humans and experimental animals. Even though their DNA-damaging potentials are rather weak, they interfere with the nucleotide and base excision repair at low, noncytotoxic concentrations. For example, both water-soluble Ni(II) and particulate black NiO greatly reduced the repair of DNA adducts induced by benzo[a]pyrene, an important environmental pollutant. Furthermore, Ni(II), As(III), and Co(II) interfered with cell cycle progression and cell cycle control in response to ultraviolet C radiation. As potential molecular targets, interactions with so-called zinc finger proteins involved in DNA repair and/or DNA damage signaling were investigated. We observed an inactivation of the bacterial formamidopyrimidine-DNA glycosylase (Fpg), the mammalian xeroderma pigmentosum group A protein (XPA), and the poly(adenosine diphosphate-ribose)polymerase (PARP). Although all proteins were inhibited by Cd(II) and Cu(II), XPA and PARP but not Fpg were inhibited by Co(II) and Ni(II). As(III) deserves special attention, as it inactivated only PARP, but did so at very low concentrations starting from 10 nM. Because DNA is permanently damaged by endogenous and environmental factors, functioning processing of DNA lesions is an important prerequisite for maintaining genomic integrity; its inactivation by metal compounds may therefore constitute an important mechanism of metal-related carcinogenicity.

The polycomb group (PcG) proteins are epigenetic regulators of gene expression that enhance cell survival. This regulation is achieved via action of two multiprotein PcG complexes--PRC2 (EED) and PRC1 [B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1)]. These complexes modulate gene expression by increasing histone methylation and reducing acetylation--leading to a closed chromatin conformation. Activity of these proteins is associated with increased cell proliferation and survival. We show increased expression of key PcG proteins in immortalized keratinocytes and skin cancer cell lines. We examine the role of two key PcG proteins, Bmi-1 and enhancer of zeste homolog 2 (Ezh2), and the impact of the active agent in green tea, (-)-epigallocatechin-3-gallate (EGCG), on the function of these regulators. EGCG treatment of SCC-13 cells reduces Bmi-1 and Ezh2 level and this is associated with reduced cell survival. The reduction in survival is associated with a global reduction in histone H3 lysine 27 trimethylation, a hallmark of PRC2 complex action. This change in PcG protein expression is associated with reduced expression of key proteins that enhance progression through the cell cycle [cyclin-dependent kinase (cdk)1, cdk2, cdk4, cyclin D1, cyclin E, cyclin A and cyclin B1] and increased expression of proteins that inhibit cell cycle progression (p21 and p27). Apoptosis is also enhanced, as evidenced by increased caspase 9, 8 and 3 cleavage and increased poly(adenosine diphosphate ribose) polymerase cleavage. EGCG treatment also increases Bax and suppresses Bcl-xL expression. Vector-mediated enhanced Bmi-1 expression reverses these EGCG-dependent changes. These findings suggest that green tea polyphenols reduce skin tumor cell survival by influencing PcG-mediated epigenetic regulatory mechanisms.

Accumulation of reactive oxygen species has been implicated in various diseases and aging. However, the precise physiological effects of accumulating oxidants are still largely undefined. Here, we applied a short-term peroxide stress treatment to young Caenorhabditis elegans and measured behavioral, physiological, and cellular consequences. We discovered that exposure to peroxide stress causes a number of immediate changes, including loss in mobility, decreased growth rate, and decreased cellular adenosine triphosphate levels. Many of these alterations, which are highly reminiscent of changes in aging animals, are reversible, suggesting the presence of effective antioxidant systems in young C. elegans. One of these antioxidant systems involves the highly abundant protein peroxiredoxin 2 (PRDX-2), whose gene deletion causes phenotypes symptomatic of chronic peroxide stress and shortens lifespan. Applying the quantitative redox proteomic technique OxICAT to oxidatively stressed wild-type and prdx-2 deletion worms, we identified oxidation-sensitive cysteines in 40 different proteins, including proteins involved in mobility and feeding (e.g., MYO-2 and LET-75), protein translation and homeostasis (e.g., elongation factor 1 [EFT-1] and heat shock protein 1), and adenosine triphosphate regeneration (e.g., nucleoside diphosphate kinase). The oxidative modification of some of these redox-sensitive cysteines may contribute to the physiological and behavioral changes observed in oxidatively stressed animals.

In recent years, there have been multiple breakthroughs in our understanding of lung cancer biology. Despite significant advances in molecular targeted therapies, DNA-damaging cytotoxic therapies will remain the mainstay of lung cancer management for the near future. Similar to the concept of personalized targeted therapies, there is mounting evidence that perturbations in DNA repair pathways are common in lung cancers, altering the resistance of the affected tumors to many chemotherapeutics as well as radiation. Defects in DNA repair may be due to a multitude of mechanisms including gene mutations, epigenetic events, and alterations in signal transduction pathways such as epidermal growth factor receptor and phosphoinositide 3-kinase/AKT. Functional biomarkers that assess the subcellular localization of central repair proteins in response to DNA damage may prove useful for individualization of cytotoxic therapies including poly(adenosine diphosphate-ribose) polymerase inhibitors. A better mechanistic understanding of cellular sensitivity and resistance to DNA damaging agents should facilitate the development of novel, individualized treatment approaches. Absolute resistance to radiation therapy, however, does not exist. To some extent, radiation therapy will always have to remain unselective and indiscriminant to eradicate persistent, drug-resistant tumor stem cell pools.

Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

A new antithrombotic drug, cilostazol (6-[4-(1-cyclohexyl-1 H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone, OPC-13013) was studied for its inhibitory effect on platelet aggregation in vitro in various experimental animals and man and in dogs ex vivo, for its effect to disperse platelet aggregates in vitro in rabbits and man and for its antithrombotic effect in vivo using its effect to prevent death due to the formation of pulmonary thrombi in mice. Cilostazol produced a potent inhibition of platelet aggregation both in vitro and ex vivo and a dispersion of platelet aggregates in vitro. The mode of action of cilostazol was different from that of acetylsalicylic acid (ASA) in that cilostazol inhibits not only secondary platelet aggregation but also primary platelet aggregation induced by aggregating agents such as adenosine diphosphate (ADP). The drug potently prevented death due to pulmonary thrombosis by platelet aggregates in mice in vivo. Unlike ASA which prevented only death due to collagen-induced platelet aggregation, cilostazol prevented both collagen- and ADP-induced platelet aggregation. These results suggest that cilostazol is a promising antithrombotic drug.

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.

The low rate of endogenous respiration exhibited by the blue-green algae Anacystis nidulans and Phormidium luridum was not increased by the addition of respiratory substrates. However, endogenous respiration was inhibited by low concentrations of cyanide and by high carbon monoxide tensions. In addition, the uncouplers dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone both stimulated the respiratory rate. The transition of cells from the aerobic steady state to anaerobiosis was accompanied by a decrease in the concentration of cellular nicotinamide adenine dinucleotide phosphate (NADP(+)) and adenosine triphosphate (ATP), whereas the concentration of nicotinamide adenine dinucleotide (NAD(+)) was unchanged. Concomitant with the metabolite decreases were stoichiometric increases io reduced NADP(+) (NADPH), adenosine diphosphate, and adenosine monophosphate. A decrease in ATP was also observed after the addition of uncouplers. These data are interpreted as evidence for the association of oxidative phosphorylation with the oxidation of NADP(+)-linked substrates in these algae. Membrane fragments isolated from the algal cells oxidized succinate, malate, ferrocytochrome c, ascorbate-tetramethyl-p-phenylenediamine, and reduced 2,6-dichlorophenol indophenol but did not oxidize NADPH or reduced NAD(+) in a cyanide-sensitive system. Oxidative phosphorylation has not yet been demonstrated in these fragments, but a dark ATP-P(i) exchange, distinct from the lighttriggered exchange associated with photosynthesis, is readily observed. This exchange was inhibited by phloridzin, Atabrine, and uncouplers in concentrations which suggest that the mechanism of oxidative phosphorylation in blue-green algae is different from that found in other bacteria and in mitochondria. These results led to the conclusion that the biochemical basis for obligate autotrophy in these organisms does not lie in the metabolic events associated with terminal electron transport and energy conservation.

Purified endothelial cells isolated from guinea pig hearts by enzymatic perfusion were grown in monolayer culture and used to test the ability of a variety of vasoactive agents to stimulate ATP release from these cells. Stimulation of endothelial cells with the peptide agonist bradykinin (1 nmol/L), acetylcholine (1 mumol/L), serotonin (1 mumol/L), or adenosine 5'-diphosphate (10 mumol/L) resulted in the rapid appearance of ATP in the incubation medium determined with the firefly luciferase assay for ATP. Addition of antagonists for muscarinic (atropine, 0.1 mumol/L) and purinergic (suramin, 100 mumol/L; reactive blue-2, 100 mumol/L) receptors suggested that ATP release from these cells was receptor-mediated. Bradykinin-induced release of ATP was rapid (peak < 30 seconds at 3 nmol/L bradykinin), dose-dependent (EC50, 0.18 nmol/L), and diminished with repeated administration of agonist. Desensitization to bradykinin also affected the ability of acetylcholine to induce release and was reversible when cells were returned to growth conditions for short periods. Measurement of released adenyl purines as their fluorescent N6-ethenopurine derivatives by high-performance liquid chromatography revealed the origin of the purine released to be ATP and confirmed its rapid dephosphorylation. Addition of the purine nucleotide analogues 2-methylthio-ATP (2-methyl-S-ATP), ADP, and beta gamma-methylene ATP to endothelial cell cultures resulted in a dose-dependent increase in the appearance of ATP measured in the medium bathing the cells at 30 seconds, suggesting the presence of ATP-induced ATP release.(ABSTRACT TRUNCATED AT 250 WORDS)

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.

Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P(2) accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

BACKGROUND

Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) is essential in cellular processing of DNA damage via the base excision repair pathway (BER). The PARP inhibition can be directly cytotoxic to tumour cells and augments the anti-tumour effects of DNA-damaging agents. This study evaluated the optimally tolerated dose of olaparib (4-(3--4-fluorophenyl) methyl-1(2H)-one; AZD2281, KU0059436), a potent PARP inhibitor, with dacarbazine and assessed safety, toxicity, clinical pharmacokinetics and efficacy of combination treatment.

METHODS

Patients with advanced cancer received olaparib (20-200 mg PO) on days 1-7 with dacarbazine (600-800 mg m(-2) IV) on day 1 (cycle 2, day 2) of a 21-day cycle. An expansion cohort of chemonaive melanoma patients was treated at an optimally tolerated dose. The BER enzyme, methylpurine-DNA glycosylase and its substrate 7-methylguanine were quantified in peripheral blood mononuclear cells.

RESULTS

The optimal combination to proceed to phase II was defined as 100 mg bd olaparib with 600 mg m(-2) dacarbazine. Dose-limiting toxicities were neutropaenia and thrombocytopaenia. There were two partial responses, both in patients with melanoma.

CONCLUSIONS

This study defined a tolerable dose of olaparib in combination with dacarbazine, but there were no responses in chemonaive melanoma patients, demonstrating no clinical advantage over single-agent dacarbazine at these doses.

Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.

OBJECTIVE

We studied the effects of sildenafil, a phosphodiesterase 5 inhibitor, on coronary and peripheral vascular function, platelet activation, and myocardial ischemia.

BACKGROUND

Nitric oxide vasodilates and inhibits platelet activation by generating cyclic guanosine 5'-monophosphate, which is metabolized by phosphodiesterase type 5.

METHODS

The effect of oral sildenafil on resting coronary vascular tone, endothelium-dependent and -independent function and platelet activation was measured in 24 patients. An additional 24 patients with coronary artery disease (CAD) and ischemia during exercise, and 12 control subjects received either 100 mg of sildenafil, 10 mg of isosorbide dinitrate (ISDN) or placebo during exercise on three separate days in a randomized, double-blind manner. Flow-mediated dilation of the brachial artery was measured, and CAD patients underwent treadmill exercise testing.

RESULTS

Sildenafil (100 mg) vasodilated epicardial coronary arteries (+6.9 +/- 1.3%, p < 0.0001). Coronary epicardial and microvascular responses with acetylcholine and cold-pressor testing improved, with a greater enhancement in patients with CAD and endothelial dysfunction. Verapamil responses were unchanged. Both resting and adenosine diphosphate-stimulated platelet IIb/IIIa receptor activation was inhibited by sildenafil (p < 0.05). Brachial arteries dilated in response to sildenafil in controls. Peak flow-mediated dilation was similar, but the duration of hyperemia was prolonged after sildenafil administration (p < 0.001). Compared with placebo, ISDN improved myocardial ischemia during exercise (p < 0.05), whereas the effect of sildenafil was intermediate between the two.

CONCLUSIONS

Sildenafil dilates epicardial coronary arteries, improves endothelial dysfunction and inhibits platelet activation in patients with CAD. It has an intermediate effect on myocardial ischemia compared with ISDN and placebo.

Cyclophosphamide (CP) and ifosfamide (IF) are widely used antineoplastic agents, but their side-effect of hemorrhagic cystitis (HC) is still encountered as an important problem. Acrolein is the main molecule responsible of this side-effect and mesna (2-mercaptoethane sulfonate) is the commonly used preventive agent. Mesna binds acrolein and prevent its direct contact with uroepithelium. Current knowledge provides information about the pathophysiological mechanism of HC: several transcription factors and cytokines, free radicals and non-radical reactive molecules, as well as poly(adenosine diphosphate-ribose) polymerase (PARP) activation are now known to take part in its pathogenesis. There is no doubt that HC is an inflammatory process, including when caused by CP. Thus, many cytokines such as tumor necrosis factor (TNF) and the interleukin (IL) family and transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) also play a role in its pathogenesis. When these molecular factors are taken into account, pathogenesis of CP-induced bladder toxicity can be summarized in three steps: (1) acrolein rapidly enters into the uroepithelial cells; (2) it then activates intracellular reactive oxygen species and nitric oxide production (directly or through NF-kappaB and AP-1) leading to peroxynitrite production; (3) finally, the increased peroxynitrite level damages lipids (lipid peroxidation), proteins (protein oxidation) and DNA (strand breaks) leading to activation of PARP, a DNA repair enzyme. DNA damage causes PARP overactivation, resulting in the depletion of oxidized nicotinamide-adenine dinucleotide and adenosine triphosphate, and consequently in necrotic cell death. For more effective prevention against HC, all pathophysiological mechanisms must be taken into consideration.

A new in vitro system for the detection of platelet dysfunction, PFA-100, has been developed. It provides a quantitative measure of platelet function in anticoagulated whole blood. The system comprises a microprocessor-controlled instrument and a disposable test cartridge containing a biologically active membrane. The instrument aspirates a blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture cut into the membrane. The membrane is coated with collagen and epinephrine or adenosine 5'-diphosphate. The presence of these biochemical stimuli, and the high shear rates generated under the standardized flow conditions, result in platelet attachment, activation, and aggregation, slowly building a stable platelet plug at the aperture. The time required to obtain full occlusion of the aperture is reported as the "closure time." We have found that impairment of von Willebrand factor, or inhibition of platelet receptors glycoprotein Ib or IIb/IIIa with monoclonal antibodies or peptides, resulted in abnormal closure times. An antifibrinogen antibody, in contrast, failed to show any effect. The test appears to be sensitive to platelet adherence and aggregation abnormalities. The PFA-100 system has potential applications in routine evaluation of platelet function in the clinical setting because of its accuracy, ease of operation, and rapid turnaround of results.

1. Properties of the pinacidil-sensitive K+ channel in the smooth muscle of the rabbit portal vein were investigated using cell-attached and inside- and outside-out patch clamp techniques. 2. In the cell-attached patch configuration, a K+ channel with a unitary conductance of 150 pS could be recorded when physiological salt solution (PSS) was in the pipette and high-K+ solution was in the bath. Tetraethylammonium (TEA; less than 1 mM) and charybdotoxin (CTX; greater than 50 nM) inhibited the 150 pS K+ channel from the outside of the membrane. This channel was activated by an increase in the concentrations of intracellular Ca2+ but not by pinacidil (less than or equal to 500 microM). 3. In the cell-attached patch configuration, bath application of pinacidil (greater than 3 microM) activated a K+ channel (ATP-sensitive K+ channel) with a unitary conductance of 15 pS and the enhancing action of pinacidil was blocked by glibenclamide. However, in the cell-free patch configuration, pinacidil (100 microM) failed to open the 15 pS K+ channel. With pinacidil in the pipette, the 15 pS K+ channel was completely inactivated within 5 s of the excision of the membrane. Opening of the 15 pS K+ channel also disappeared after saponin treatment (50 micrograms/ml). 4. In the cell-free patch configuration, application of guanosine 5'-diphosphate (GDP; greater than 100 microM) re-activated the inactivated 15 pS K+ channel only when pinacidil was present either in the pipette or bath. GDP increased the mean open time and open probability of the 15 pS K+ channel in a concentration-dependent manner. Simultaneous application of MgCl2 (less than or equal to 1 mM) with GDP did not modify the GDP-induced activation. Neither GDP nor GTP (1 mM) had any effect on the 150 pS K+ channel. 5. Guanosine 5'-triphosphate (GTP; 1 mM) activated the 15 pS K+ channel to a lesser extent that did GDP. Other guanine nucleotides (guanosine 5'-monophosphate, GMP, 1 mM; guanosine 5'-O-(3-thiotriphosphate), GTP gamma S, 100 microM; and guanosine 5'-O-(2-thiodiphosphate), GDP beta S, 1 mM) failed to activate the 15 pS K+ channel. However, GDP beta S, but not GMP or GTP gamma S, inhibited this channel when it was activated by 1 mM-GDP. 6. In the presence of pinacidil, adenosine 5'-triphosphate (ATP; greater than or equal to 10 microM) inhibited the ATP-sensitive K+ channel when it was activated by 1 mM-GDP.(ABSTRACT TRUNCATED AT 400 WORDS)

In the energy transduction of muscle contraction, it is important to know the nature and extent of conformational changes of the head portion of the myosin molecules. In the presence of magnesium adenosine triphosphate (MgATP), fairly large conformational changes of the myosin head [subfragment-1 (S1)] in solution were observed by small-angle x-ray scattering with the use of synchrotron radiation as an intense and stable x-ray source. The presence of MgATP reduced the radius of gyration of the molecule by about 3 angstrom units and the maximum chord length by about 10 angstroms, showing that the shape of S1 becomes more compact or round during hydrolysis of MgATP. Comparison with various nucleotide-bound S1 complexes that correspond to the known intermediate states during ATP hydrolysis indicates that the shape of S1 in a key intermediate state, S1-bound adenosine diphosphate (ADP) and phosphate [S1**.ADP.P(i)], differs significantly from the shape in the other intermediate states of the S1 adenosine triphosphatase cycle as well as that of nucleotide-free S1.