The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.

BACKGROUND

Clopidogrel is activated by CYP2C19, which also metabolizes proton pump inhibitors (PPI). As proton pump inhibitors are metabolized to varying degrees by CYP2C19, we hypothesized that the reported negative omeprazole-clopidogrel drug interaction may not be a class effect.

METHODS

Responsiveness to clopidogrel was assessed by the vasodilator-stimulated phosphoprotein phosphorylation (VASP) assay and aggregometry (Multiplate Analyzer) in 300 patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI).

RESULTS

The mean platelet reactivity index (PRI, assessed by the VASP assay) was nearly the same in patients with (n = 226; PRI = 51%) or without PPI treatment (n = 74; PRI = 49%; P = .724). Likewise, the adenosine diphosphate-induced platelet aggregation did not differ significantly between patients with or without PPI treatment (45 vs. 41 U; P = .619). Similarly, there was no difference in the PRI or the adenosine diphosphate-induced platelet aggregation between patients with pantoprazole (n = 152; PRI = 50%; aggregation = 47 U), esomeprazole (n = 74; PRI = 54%; aggregation = 42 U), or without PPI (n = 74; PRI = 49%; aggregation = 41 U; P = .382).

CONCLUSIONS

In contrast to the reported negative omeprazole-clopidogrel drug interaction, the intake of pantoprazole or esomeprazole is not associated with impaired response to clopidogrel.

The transporter associated with antigen processing (TAP) is an ABC transporter formed of two subunits, TAP1 and TAP2, each of which has an N-terminal membrane-spanning domain and a C-terminal ABC ATPase domain. We report the structure of the C-terminal ABC ATPase domain of TAP1 (cTAP1) bound to ADP. cTAP1 forms an L-shaped molecule with two domains, a RecA-like domain and a small alpha-helical domain. The diphosphate group of ADP interacts with the P-loop as expected. Residues thought to be involved in gamma-phosphate binding and hydrolysis show flexibility in the ADP-bound state as evidenced by their high B-factors. Comparisons of cTAP1 with other ABC ATPases from the ABC transporter family as well as ABC ATPases involved in DNA maintenance and repair reveal key regions and residues specific to each family. Three ATPase subfamilies are identified which have distinct adenosine recognition motifs, as well as distinct subdomains that may be specific to the different functions of each subfamily. Differences between TAP1 and TAP2 in the nucleotide-binding site may be related to the observed asymmetry during peptide transport.

OBJECTIVE

P2Y(12) receptor antagonism and platelet inhibition by prasugrel vs. clopidogrel were investigated in patients with stable coronary artery disease.

RESULTS

One hundred and ten aspirin treated subjects were randomized to double-blind treatment with clopidogrel (n = 55) 600 mg loading dose (LD) and 75 mg maintenance dose (MD) or prasugrel (n = 55) 60 mg LD and 10 mg MD for 28 days. Concentrations of prasugrel and clopidogrel active metabolites were determined. Platelet aggregation to 20 microM adenosine diphosphate, measured by light transmission aggregometry, was reported as maximal platelet aggregation (MPA). P2Y(12) function was assessed by the vasodilator-stimulated phosphoprotein assay and reported as platelet reactivity index (PRI). The same pharmacodynamic measurements were performed after ex vivo addition of clopidogrel's active metabolite. At 2 h post-LD, mean MPA was 31 vs. 55%, and mean PRI 8.3 vs. 55.9% for prasugrel and clopidogrel, respectively (P < 0.001). During MD on day 14 and 28, mean MPA was 42 vs. 54% and mean PRI was 25 vs. 51%, respectively (P < 0.001). Peak level of the active metabolite and P2Y(12) inhibition occurred earlier and was greater with prasugrel (P < 0.001). Mean area under the time-concentration curve (AUC; microM.h) of the respective active metabolite was higher with prasugrel vs. clopidogrel post-LD (1.11 vs. 0.24) and post-MD (0.16 vs. 0.062). Ex vivo addition of clopidogrel's active metabolite further reduced PRI in all patients whose platelets were not already maximally inhibited.

CONCLUSIONS

In aspirin-treated subjects with coronary artery disease, prasugrel 60/10 mg provides faster onset and greater inhibition of P2Y(12) receptor-mediated platelet aggregation than clopidogrel 600/75 mg, because of greater and more efficient generation of the active metabolite.

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best-characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDH-mediated responses). As regards the latter, hydrogen peroxide (H2 O2 ) now appears to play a dominant role. Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi (e.g. responses to α2 -adrenergic agonists, serotonin, and thrombin) and pertussis toxin-insensitive Gq (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low-density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H2 O2 ), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factors. Recent evidence confirms that most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium-dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin-1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.

BACKGROUND

For patients undergoing percutaneous coronary intervention, the administration of a clopidogrel loading dose ranging from 300 to 600 mg is currently recommended. It is unknown, though, whether loading doses higher than 600 mg exert additional suppression of platelet function.

RESULTS

Sixty patients with suspected or documented coronary artery disease admitted to our hospital for coronary angiography were included in this trial. They were allocated to 1 of 3 clopidogrel loading doses (300, 600, or 900 mg) in a double-blinded, randomized manner. Plasma concentrations of the active thiol metabolite, unchanged clopidogrel, and the inactive carboxyl metabolite of clopidogrel were determined before and serially after drug administration. Optical aggregometry was performed before and 4 hours after administration of clopidogrel. Loading with 600 mg resulted in higher plasma concentrations of the active metabolite, clopidogrel, and the carboxyl metabolite compared with loading with 300 mg (P< or =0.03) and lower values for adenosine diphosphate-induced (5 and 20 micromol/L) platelet aggregation 4 hours after drug administration (P=0.01 and 0.004). With administration of 900 mg, no further increase in plasma concentrations of active metabolite and clopidogrel (P> or =0.38) and no further suppression of adenosine diphosphate-induced (5 and 20 micromol/L) platelet aggregation 4 hours after drug administration was achieved when compared with administration of 600 mg (P=0.59 and 0.39).

CONCLUSIONS

Single doses of clopidogrel higher than 600 mg are not associated with an additional significant suppression of platelet function because of limited clopidogrel absorption.

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20-100 nM) released prostacyclin (9.6 +/- 1.5 nM per 10(6) cells; mean +/- s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 +/- 0.6 nM per 10(6) cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500-2000 nM) also released both prostacyclin and PGE2 from the cells. This release was long-lasting and not reproducible. Bk (20-100 nM) and A23187 (30-300 nM) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half-life less than 7s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5-Hydroxytryptamine 0.2, noradrenaline 1.0, platelet-activating factor (Paf-acether) 1.0, formylmethionyl-leucyl-phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 microM did not release either prostanoids or EDRF from the column.

The maize mutant shrunken-2 synthesizes only 25 to 30 percent as much starch as normal maize; it completely lacks adenosine diphosphate glucose pyrophosphorylase activity in both endosperm and embryo tissue. Identification of the mutant block indicates that the greater portion of starch in the endosperm of normal maize is synthesized by way of enzyme systems that utilize adenosine diphosphate glucose as a substrate, and that the latter is formed chiefly by adenosine diphosphate glucose pyrophosphorylase.

OBJECTIVE

We investigated whether patients who suffered subacute stent thrombosis (SAT) have higher post-treatment reactivity than those who do not encounter stent thrombosis.

BACKGROUND

High post-treatment platelet reactivity has been reported after coronary stenting after clopidogrel therapy and may be an important factor in the occurrence of SAT.

METHODS

We identified patients with SAT treated at two tertiary care centers over a 1.5-year period. Light transmittance aggregation induced by adenosine diphosphate (ADP) and arachidonic acid, total and activated glycoprotein (GP) IIb/IIIa after stimulation with ADP, and vasodilator-stimulated phosphoprotein phosphorylation levels to measure P2Y12 receptor inhibition were determined (n = 20) and compared with an age-matched group of patients without SAT (n = 100). High post-treatment platelet reactivity was defined as >75th percentile ADP-induced aggregation in the group without SAT.

RESULTS

The SAT patients had higher mean platelet reactivity than those without SAT by all measurements (p < 0.05): 49 +/- 4% versus 33 +/- 2% for 5 micromol/l ADP-induced aggregation and 65 +/- 3% versus 51 +/- 2% for 20 micromol/l ADP-induced aggregation (p < 0.001), 69 +/- 5% versus 46 +/- 9% for P2Y12 reactivity ratio (p = 0.03), and 138 +/- 19 mean fluorescence intensity (MFI) versus 42 +/- 4 MFI for stimulated GP IIb/IIIa expression (p < 0.001). Of patients with SAT, 60% had high platelet reactivity.

CONCLUSIONS

High post-treatment platelet reactivity and incomplete P2Y12 receptor inhibition are risk factors for SAT. Measures to uniformly determine platelet reactivity after coronary stenting and treatment strategies to improve P2Y12 receptor inhibition in patients with high post-treatment platelet reactivity should be further investigated.

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-dependent relaxations involve both pertussis-toxin-sensitive G(i) (e.g., responses to serotonin, sphingosine 1-phosphate, alpha(2)-adrenergic agonists, and thrombin) and pertussis-toxin-insensitive G(q) (e.g., adenosine diphosphate and bradykinin) coupling proteins. eNOS undergoes a complex pattern of intracellular regulation, including post-translational modifications involving enzyme acylation and phosphorylation. eNOS is reversibly targeted to signal-transducing plasmalemmal caveolae where the enzyme interacts with a number of regulatory proteins, many of which are modified in cardiovascular disease states. The release of nitric oxide by the endothelial cell can be up- (e.g., by estrogens, exercise, and dietary factors) and down-regulated (e.g. oxidative stress, smoking, and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g., diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis-toxin-sensitive pathway for NO release which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction leading to atherosclerosis. The unraveling of the complex interaction of the pathways regulating the presence and the activity of eNOS will enhance the understanding of the perturbations in endothelium-dependent signaling that are seen in cardiovascular disease states, and may lead to the identification of novel targets for therapeutic intervention.

1. We carried out confocal Ca2+ imaging in myocytes permeabilized with saponin in 'internal' solutions containing: MgATP, EGTA and fluo-3 potassium salt. 2. Permeabilized myocytes exhibited spontaneous Ca2+ sparks and waves similar to those observed in intact myocytes loaded with fluo-3 AM. 3. In the presence of 'low' [EGTA] (0.05 mM), Ca2+ waves arose regularly, even at relatively low [Ca2+] (50-100 nM, free). Increasing [EGTA] resulted in decreased frequency and propagation velocity of Ca2+ waves. Propagating waves were completely abolished at [EGTA]>> 0.3 mM. 4. The frequency of sparks increased as a function of [Ca2+] (50-400 nM range) with no sign of a high affinity Ca2+-dependent inactivation process. 5. The rate of occurrence of Ca2+ sparks was increased by calmodulin and cyclic adenosine diphosphate-ribose (cADPR).

BACKGROUND

Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors, with novel and selective mechanisms of action, have moved from the laboratory to the clinic in just the last few years.

METHODS

We conducted an extensive review of PARP inhibitors using a Medline search. We also searched abstracts in databases of major international oncology meetings from the last 4 years.

RESULTS

To understand the mechanisms of action of PARP inhibitors requires a basic understanding of DNA repair mechanisms and the critical role of the PARP enzyme. We briefly review these DNA repair mechanisms, the concept of 'synthetic lethality', and how PARP inhibitors play a role to selectively disrupt DNA repair in cells with absent or dysfunctional BRCA genes. We review the preclinical data highlighting this unique and selective mechanism of action and we discuss early but highly promising clinical data and ongoing studies.

CONCLUSIONS

PARP inhibitors show promise as a powerful therapeutic tool, especially in the management of BRCA-associated breast and ovarian cancers but also in tumours where BRCA genes may be dysfunctional. Clinical studies are ongoing and many translational questions remain unanswered that will help clarify how to determine the best way to use PARP inhibitors.

OBJECTIVE

This study was designed to determine whether mitochondrial function in a systemic organ is acutely impaired in a resuscitated model of sepsis (endotoxemia, lipopolysaccharide) and the relationship, if any, between this impairment and the extent of mitochondrial ultrastructural damage that occurs.

METHODS

Perspective, controlled laboratory investigation.

METHODS

Animal laboratory in a university research institute.

METHODS

Adult male cats.

METHODS

A well-established feline model of acute endotoxemia was used wherein measures were taken to minimize tissue hypoxia. After lipopolysaccharide (3 mg/kg intravenously, n = 9) or isotonic saline vehicle (control, n = 5) administration, liver samples were obtained at 4 hrs posttreatment, and mitochondrial ultrastructure and respiratory function were assessed. Mitochondrial ultrastructural injury was graded on a scale of 0 (no injury) to 5 (severe injury), and mitochondrial respiration was evaluated by using standard techniques.

RESULTS

Significant mitochondrial injury was apparent by 4 hrs, but only in the lipopolysaccharide-treated group (2.5 +/- 0.2 vs. 1.3 +/- 0.2, p <.001) and despite maintenance of tissue oxygen availability. In addition, lipopolysaccharide treatment reduced the rate of state 3 (adenosine 5'-diphosphate-dependent) respiration, especially at complex IV (40% inhibition), and increased the rate of state 4 (adenosine 5'-diphosphate-independent) respiration, reflecting partial uncoupling of mitochondrial oxidative phosphorylation. Finally, a significant correlation was demonstrated between the severity of ultrastructural injury and the magnitude of mitochondrial respiratory dysfunction after lipopolysaccharide treatment and despite resuscitation efforts.

CONCLUSIONS

Mitochondrial function is significantly impaired during acute sepsis, and this impairment is strongly associated with the extent of mitochondrial ultrastructural abnormalities present in the tissues. These findings in conjunction with those previously shown suggest that mitochondrial functional impairment may contribute to the pathogenesis of altered oxygen metabolism in systemic organs during sepsis.

Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.

p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

Resveratrol, an edible polyphenolic stilbene, has been reported to possess substantial antileukemic activities in different leukemia cell lines. We investigated whether resveratrol is active against fresh acute myeloid leukemia (AML) cells and its mechanism of action. Because interleukin 1beta(IL-1beta) plays a key role in proliferation of AML cells, we first tested the effect of resveratrol on the AML cell lines OCIM2 and OCI/AML3, both of which produce IL-1beta and proliferate in response to it. Resveratrol inhibited proliferation of both cell lines in a dose-dependent fashion (5-75 microM) by arresting the cells at S phase, thus preventing their progression through the cell cycle; IL-1beta partially reversed this inhibitory effect. Resveratrol significantly reduced production of IL-1beta in OCIM2 cells. It also suppressed the IL-1beta-induced activation of transcription factor nuclear factor kappaB (NF-kappaB), which modulates an array of signals controlling cellular survival, proliferation, and cytokine production. Indeed, incubation of OCIM2 cells with resveratrol resulted in apoptotic cell death. Because caspase inhibitors Ac-DEVD-CHO or z-DEVD-FMK partially reversed the antiproliferative effect of resveratrol, we tested its effect on the caspase pathway and found that resveratrol induced the activation of the cysteine protease caspase 3 and subsequent cleavage of the DNA repair enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase. Finally, resveratrol suppressed colony-forming cell proliferation of fresh AML marrow cells from 5 patients with newly diagnosed AML in a dose-dependent fashion. Taken together, our data showing that resveratrol is an effective in vitro inhibitor of AML cells suggest that this compound may have a role in future therapies for AML.

Levels of phosphorylated adenosine nucleotides, including the universal energy carrier adenosine 5(')-triphosphate (ATP) and its metabolites adenosine 5(')-diphosphate (ADP) and adenosine 5(')-monophosphate (AMP), define the energy state in living cells and are dependent mainly on mitochondrial function. In this article, we describe a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues. We also describe a technique that uses the expression of recombinant targeted luciferase to report ATP content in different cell compartments. Finally, we describe an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a recently described family of ectonucleotidases that differentially hydrolyze the gamma and beta phosphate residues of extracellular nucleotides. Expression of this enzymatic activity has the potential to influence nucleotide P2 receptor signaling within the vasculature. We and others have documented that NTPDase1 (CD39, 78 kd) hydrolyzes both triphosphonucleosides and diphosphonucleosides and thereby terminates platelet aggregation responses to adenosine diphosphate (ADP). In contrast, we now show that NTPDase2 (CD39L1, 75 kd), a preferential nucleoside triphosphatase, activates platelet aggregation by converting adenosine triphosphate (ATP) to ADP, the specific agonist of P2Y(1) and P2Y(12) receptors. We developed specific antibodies to murine NTPDase1 and NTPDase2 and observed that both enzymes are present in the cardiac vasculature; NTPDase1 is expressed by endothelium, endocardium, and to a lesser extent by vascular smooth muscle, while NTPDase2 is associated with the adventitia of muscularized vessels, microvascular pericytes, and other cell populations in the subendocardial space. Moreover, NTPDase2 represents a novel marker for microvascular pericytes. Differential expression of NTPDases in the vasculature suggests spatial regulation of nucleotide-mediated signaling. In this context, NTPDase1 should abrogate platelet aggregation and recruitment in intact vessels by the conversion of ADP to adenosine monophosphate, while NTPDase2 expression would promote platelet microthrombus formation at sites of extravasation following vessel injury. Our data suggest that specific NTPDases, in tandem with ecto-5'-nucleotidase, not only terminate P2 receptor activation and trigger adenosine receptors but may also allow preferential activation of specific subsets of P2 receptors sensitive to ADP (e.g., P2Y(1), P2Y(3), P2Y(12)) and uridine diphosphate (P2Y(6)).

The Vibrio cholerae bacterium causes devastating diarrhea when it infects the human intestine. The key event is adenosine diphosphate (ADP)-ribosylation of the human signaling protein GSalpha, catalyzed by the cholera toxin A1 subunit (CTA1). This reaction is allosterically activated by human ADP-ribosylation factors (ARFs), a family of essential and ubiquitous G proteins. Crystal structures of a CTA1:ARF6-GTP (guanosine triphosphate) complex reveal that binding of the human activator elicits dramatic changes in CTA1 loop regions that allow nicotinamide adenine dinucleotide (NAD+) to bind to the active site. The extensive toxin:ARF-GTP interface surface mimics ARF-GTP recognition of normal cellular protein partners, which suggests that the toxin has evolved to exploit promiscuous binding properties of ARFs.

Light-regulated translation of chloroplast messenger RNAs (mRNAs) requires trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. Chloroplast polyadenylate-binding protein (cPABP) specifically binds to the 5'-UTR of the psbA mRNA and is essential for translation of this mRNA. A protein disulfide isomerase that is localized to the chloroplast and copurifies with cPABP was shown to modulate the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP through redox potential or adenosine 5'-diphosphate-dependent phosphorylation. This mechanism allows for a simple reversible switch regulating gene expression in the chloroplast.

Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor kappa B (NF-kappa B) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies.

Regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is unusual in that phosphorylated channels require cytosolic adenosine triphosphate (ATP) to open. The CFTR contains two regions predicted to be nucleotide-binding domains (NBDs); site-directed mutations in each NBD have now been shown to alter the relation between ATP concentration and channel activity, which indicates that ATP stimulates the channel by direct interaction with both NBDs. The two NBDs are not, however, functionally equivalent: adenosine diphosphate (ADP) competitively inhibited the channel by interacting with NBD2 but not by interacting with NBD1. Four cystic fibrosis-associated mutations in the NBDs reduced absolute chloride channel activity, and one mutation also decreased the potency with which ATP stimulates channel activity. Dysfunction of ATP-dependent stimulation through the NBDs may be the basis for defective CFTR chloride channel activity in some cystic fibrosis patients.

OBJECTIVE

This study sought to determine the prognostic implications of high platelet reactivity (HPR) assessed in type 2 diabetes mellitus (T2DM) patients while in their steady-state phase of dual antiplatelet therapy.

BACKGROUND

Type 2 diabetes mellitus patients have increased platelet reactivity compared with nondiabetic patients. Whether HPR assessed in T2DM while in their steady-state phase of dual antiplatelet therapy is associated with an increased risk of major adverse cardiovascular events (MACE) is unknown.

METHODS

Platelet function analyses, which included measures of platelet aggregation and activation, were performed in 173 T2DM patients with coronary artery disease on chronic treatment with aspirin and clopidogrel. The HPR was defined as the upper quartile of maximal platelet aggregation (Agg(max)) after 20 micromol/l adenosine diphosphate stimuli. Patients were followed up for 2 years and MACE were recorded.

RESULTS

A total of 41 MACE occurred in 34 patients (19.7%) during the 2-year follow-up. The MACE occurred in 15.2%, 12.2%, 12.2%, and 37.7% of patients from the lowest to upper quartile, respectively (p = 0.005). The HPR was the strongest independent predictor of MACE (hazard ratio 3.35, 95% confidence interval [CI] 1.68 to 6.66, p = 0.001). Receiver-operating characteristic analysis indicated that a cutoff value of 62% Agg(max) best predicted MACE (37.8% vs. 13.2%, odds ratio 3.96, 95% CI 1.8 to 8.7, p < 0.001). Patients with HPR had up-regulation of multiple platelet signaling pathways (p < 0.0001 for all assays), indicative of a global hyperreactive platelet status.

CONCLUSIONS

High platelet reactivity determined in T2DM patients with coronary artery disease while on chronic dual antiplatelet therapy is associated with a higher risk of long-term adverse cardiovascular events, suggesting the need for tailored antithrombotic drug regimens in these high-risk patients.