Four randomized, placebo-controlled, crossover studies were conducted among 282 healthy subjects to investigate whether an interaction exists between clopidogrel (300-mg loading dose/75-mg/day maintenance dose) and the proton-pump inhibitor (PPI) omeprazole (80 mg) when they are administered simultaneously (study 1); whether the interaction, if any, can be mitigated by administering clopidogrel and omeprazole 12 h apart (study 2) or by increasing clopidogrel to 600-mg loading/150-mg/day maintenance dosing (study 3); and whether the interaction applies equally to the PPI pantoprazole (80 mg) (study 4). Relative to levels after administration of clopidogrel alone in studies 1,2,3, and 4, coadministration of PPI decreased the AUC(0-24) of the clopidogrel active metabolite H4 by 40, 47, 41, and 14% (P ≤ 0.002), respectively; increased maximal platelet aggregation (MPA) induced by 5 micromol/l adenosine diphosphate (ADP) by 8.0, 5.6, 8.1, and 4.3% (P ≤ 0.014), respectively; and increased the vasodilator-stimulated phosphoprotein phosphorylation-platelet reactivity index (VASP-PRI) by 20.7, 27.1, 19.0 (P < 0.0001), and 3.9% (P = 0.3319), respectively. The results suggest that a metabolic drug-drug interaction exists between clopidogrel and omeprazole but not between clopidogrel and pantoprazole.

Sera from 33 patients with cystic fibrosis and two pediatric patients being treated for chronic pulmonary infections not related to cystic fibrosis and six sera or serum pools from uninfected individuals were tested with a microtiter radioimmunoassay for reactivity against exotoxin A and two proteases from Pseudomonas aeruginosa. Exotoxin A was purified from a low-protease strain of P. aeruginosa and shown to have adenosine diphosphate-ribose transferase activity and mouse lethality. Proteases were purified from an isolate of P. aeruginosa from a patient with cystic fibrosis and had proteolytic activity against elastin and collagen in an assay employing dimethylated protein substrates. The antibody responses of the patients detected using 125I-labeled antibody to human immunoglobulin were correlated with clinical evaluations expressed as a composite score based on pulmonary findings, case histories, growth and nutrition, and chest X rays. Values in the radioimmunoassay for patients' sera were compared with those of a control serum pool and expressed as the ratio of counts per minute (cpm) in patient serum to the cpm in the control pool. Inverse correlations were found between these ratios for each of the pseudomonas exoproducts and clinical scores; highest ratios occurred in patients showing the lowest clinical scores. These results confirm that proteases and exotoxin A of P. aeruginosa are produced in cystic fibrosis pulmonary infections due to P. aeruginosa and suggest that they may serve as significant virulence factors in these chronic infectious states.

OBJECTIVE

This study investigated the effects of physical training on skeletal muscle metabolism in patients with chronic heart failure.

BACKGROUND

Skeletal muscle metabolic abnormalities in patients with chronic heart failure have been associated with exercise intolerance. Muscle deconditioning is a possible mechanism for the intrinsic skeletal muscle metabolic changes seen in chronic heart failure.

METHODS

We used phosphorus-31 nuclear magnetic resonance spectroscopy to study muscle metabolism during exercise in 12 patients with stable ischemic chronic heart failure undergoing 8 weeks of home-based bicycle exercise training in a randomized crossover controlled trial. Changes in muscle pH and concentrations of phosphocreatine and adenosine diphosphate (ADP) were measured in phosphorus-31 spectra of calf muscle obtained at rest, throughout incremental work load plantar flexion until exhaustion and during recovery from exercise. Results were compared with those in 15 age-matched control subjects who performed a single study only.

RESULTS

Before training, phosphocreatine depletion, muscle acidification and the increase in ADP during the 1st 4 min of plantar flexion exercise were all increased (p < 0.04) compared with values in control subjects. Training produced an increase (p < 0.002) in incremental plantar flexion exercise tolerance. After training, phosphocreatine depletion and the increase in ADP during exercise were reduced significantly (p < 0.003) at all matched submaximal work loads and at peak exercise, although there was no significant change in the response of muscle pH to exercise. After training, changes in ADP were not significantly different from those in control subjects, although phosphocreatine depletion was still greater (p < 0.05) in trained patients than in control subjects. The phosphocreatine recovery half-time was significantly (p < 0.05) shorter after training, although there was no significant change in the half-time of adenosine diphosphate recovery. In untrained subjects, the initial rate of phosphocreatine resynthesis after exercise (a measure of the rate of oxidative adenosine triphosphate [ATP] synthesis) and the inferred maximal rate of mitochondrial ATP synthesis were reduced compared with rates in control subjects (p < 0.003) and both were significantly increased (p < 0.05) by training, so that they were not significantly different from values in control subjects.

CONCLUSIONS

The reduction in phosphocreatine depletion and in the increase in ADP during exercise, and the enhanced rate of phosphocreatine resynthesis in recovery (which is independent of muscle mass) indicate that a substantial correction of the impaired oxidative capacity of skeletal muscle in chronic heart failure can be achieved by exercise training.

A number of enzymes presumably implicated in starch synthesis were assayed at various stages of endosperm development ranging from 8 days to 28 days after pollination. Activity for invertase, hexokinase, the glucose phosphate isomerases, the phosphoglucomutases, phosphorylase I, uridine diphosphate glucose pyrophosphorylase, and the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase was present at the earliest stage of development (8 days) studied. Activity was detectable for phosphorylase III, the soluble adenosine diphosphate glucose-starch glucosyltransferase, adenosine diphosphate glucose pyrophosphorylase, and sucrose-uridine diphosphate glucosyltransferase at 12 days. For phosphorylase II and cytidine diphosphate glucose pyrophosphorylase, activity was first detectable at the 14- and 16-day stages, respectively. Rapid increases in starch content are observed prior to detectable activity for adenosine diphosphate glucose pyrophosphorylase, the soluble adenosine diphosphate glucose-starch glucosyltransferase and phosphorylases II and III. For all enzymes, except invertase, activity per endosperm rises to a peak at 22 or 28 days. Greatest activity for invertase is found at 12 days with a steady decline thereafter. The pattern of invertase activity in comparison with that of sucrose-uridine diphosphate glucosyltransferase supports previous suggestions, that the latter plays a key role in the conversion of sucrose to starch. In addition to phosphorylases I, II, and III, multiple forms of glucosephosphate isomerase and phosphoglucomutase were detected.

The ADP-ribosyltransferase (ADPRT) gene encodes a zinc-finger DNA-binding protein, poly(ADP-ribose) polymerase-1 (PARP-1), that modifies various nuclear proteins by poly(ADP-ribosyl)ation and functions as a key enzyme in the base excision repair pathway. We have conducted two studies to test whether an amino acid substitution variant, ADPRT V762A (T2444C), is associated with prostate cancer (CaP) risk and decreased enzyme function. The first study used genomic DNA samples from an ongoing, clinic-based case-control study (488 cases and 524 controls) to show that a higher percentage of the CaP cases carried the ADPRT 762 AA genotype than controls (4% versus 2%). In Caucasians, the AA genotype was significantly associated with increased CaP risk [odds ratio (OR), 2.65; 95% confidence interval (CI), 1.08-6.49], and the VA genotype was associated with a slight but not significantly increased CaP risk (OR, 1.18; 95% CI, 0.85-1.64) using VV as the referent group after adjustment for age, benign prostatic hyperplasia, and family history. Furthermore, this association was stronger in younger (<65) men (OR, 4.77; 95% CI, 1.01-22.44) than older >> or =65) men (OR, 1.78; 95% CI, 0.55-5.82). The second study used freshly isolated peripheral lymphocytes from 354 cancer-free subjects to demonstrate that the ADPRT 762 A allele contributed to significantly lower adenosine diphosphate ribosyl transferase (ADPRT)/PARP-1 activities in response to H2O2 in a gene dosage-dependent manner (P < 0.0001, test for linear trend). The PARP-1 activities (mean +/- SD dpm/10(6) cells) were 18,554 +/- 9,070 (n=257), 14,847 +/- 7,082 (n=86), and 12,155 +/- 6,334 (n=11) for VV, VA, and AA genotypes, respectively. This study is the first to provide evidence that the ADPRT V762A-genetic variant contributes to CaP susceptibility and altered ADPRT/PARP-1 enzyme function in response to oxidative damage.

The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein. This gene is frequently activated in human cancer. As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied. In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism. Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle. Although analysis of RAS biochemistry in S. cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism. In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells. However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism. The identification of other GTP-binding proteins in S. cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.

A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.

Hsp70 of the mitochondrial matrix (mtHsp70) provides a critical driving force for the import of proteins into mitochondria. Tim44, a peripheral inner-membrane protein, tethers it to the import channel. Here, regulated interactions were found to maximize occupancy of the active, adenosine 5'-triphosphate (ATP)-bound mtHsp70 at the channel through its intrinsic high affinity for Tim44, as well as through release of adenosine diphosphate (ADP)-bound mtHsp70 from Tim44 by the cofactor Mge1. A model peptide substrate rapidly released mtHsp70 from Tim44, even in the absence of ATP hydrolysis. In vivo, the analogous interaction of translocating polypeptide would release mtHsp70 from the channel. Consistent with the ratchet model of translocation, subsequent hydrolysis of ATP would trap the polypeptide, driving import by preventing its movement back toward the cytosol.

The mitochondria isolated from dark-grown mung bean hypocotyls oxidize succinate, l-malate, and externally added reduced nicotine adenine dinucleotide (NADH) with good respiratory control. While the pattern of respiration resembles that of animal mitochondria, there are 4 basic differences between the respiratory properties of mung bean and animal mitochondria: A) the ability to oxidize NADH, B) the pattern of succinate and malate oxidation, C) the rate of oxygen uptake, and D) the adenosine-5'-diphosphate to oxygen ratios.The apparent ;Km' for malate of mung bean mitochondria is about one order higher than that expected from malic dehydrogenase in animal mitochondria, whereas the affinity for phosphate is about 5 times higher with plant mitochondria than rat-liver mitochondria. While the half-maximal stimulation of respiration by adenosine-5'-diphosphate is practically identical to that of animal mitochondria, higher concentrations of adenosine-5'-diphosphate cause some decrease in its stimulating action.

High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo. In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis and bioenergetics are presented. In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR. In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR. In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 NMR while the pH difference across the membrane was measured simultaneously.

Congenital hyperinsulinism is a condition of dysregulated insulin secretion often caused by inactivating mutations of the ATP-sensitive K+ (KATP) channel in the pancreatic beta cell. Though most disease-causing mutations of the 2 genes encoding KATP subunits, ABCC8 (SUR1) and KCNJ11 (Kir6.2), are recessively inherited, some cases of dominantly inherited inactivating mutations have been reported. To better understand the differences between dominantly and recessively inherited inactivating KATP mutations, we have identified and characterized 16 families with 14 different dominantly inherited KATP mutations, including a total of 33 affected individuals. The 16 probands presented with hypoglycemia at ages from birth to 3.3 years, and 15 of 16 were well controlled on diazoxide, a KATP channel agonist. Of 29 adults with mutations, 14 were asymptomatic. In contrast to a previous report of increased diabetes risk in dominant KATP hyperinsulinism, only 4 of 29 adults had diabetes. Unlike recessive mutations, dominantly inherited KATP mutant subunits trafficked normally to the plasma membrane when expressed in COSm6 cells. Dominant mutations also resulted in different channel-gating defects, as dominant ABCC8 mutations diminished channel responses to magnesium adenosine diphosphate or diazoxide, while dominant KCNJ11 mutations impaired channel opening, even in the absence of nucleotides. These data highlight distinctive features of dominant KATP hyperinsulinism relative to the more common and more severe recessive form, including retention of normal subunit trafficking, impaired channel activity, and a milder hypoglycemia phenotype that may escape detection in infancy and is often responsive to diazoxide medical therapy, without the need for surgical pancreatectomy.

The aim of this work was to investigate the possible effects of resveratrol on the mitochondrial respiratory chain in rat brains. Isolation of mitochondria was performed at 4 degrees C using differential centrifugation. Mitochondrial respiration rate (0.4 mg of protein/ml) was determined by measuring mitochondrial oxygen consumption with a Clark electrode at 37 degrees C. Respiratory control ratio (RCR) was evaluated as the state 3/state 4 ratio of oxidative phosphorylation with substrates adenosine 5'-diphosphate (ADP) and malate plus glutamate, respectively in the presence and in the absence of resveratrol. The rate of oxygen consumption by the different complexes was checked using rotenone (2 microM), malonate (10 mM), antimycin A (1 microM), potassium cyanide (KCN) (0.3 mM) and oligomycin (10 microM) to inhibit complexes II, III, IV, V and I, respectively. Moreover, enzyme activity determinations were checked as follows: the activities of complexes II-III were measured as the rate of cytochrome c reduction at 550 nm (37 degrees C) successively triggered either by succinate (complexes II and III) or by decylubiquinol (DUQH2) (complex III), in the presence and in the absence of resveratrol. Adenosine 5'-triphosphate (ATP) synthase activity was checked as ATP hydrolysis (ATPase) at 37 degrees C for 10 min from purified mitochondria on Percoll gradient. The inorganic phosphate (Pi) concentration was measured by the Fiske and Subbarow method. When complexes I to V were activated by glutamate plus malate, resveratrol (10(-11) - 10(-4) M) significantly decreased RC (p < 0.001) following a biphasic curve with two EC50 values, 0.162 +/- 0.072 microM and 24.5 +/- 4.0 microM, representing about 56% of total oxygen consumption inhibition. We also observed a concentration-dependent effect on state 3 with two EC50 values, 2.28 +/- 0.87 nM and 27 +/- 5 microM respectively. On the other hand, resveratrol inhibited state 4 following a concentration-dependent curve with an EC50 of 37 +/- 11 microM. When complex IV operated alone, resveratrol (100 microM) did not modify oxygen consumption compared with control, indicating that this molecule did not inhibit complex IV. Thus resveratrol inhibits the mitochondrial respiratory chain through complexes I to III. In order to confirm these data, we measured the enzymatic activity of ubiquinol cytochrome c reductase alone and in the presence of resveratrol. In the presence of disrupted mitochondria, after freeze thawing cycles (three times), resveratrol inhibited about 20% of complex III activity. These results suggest that resveratrol and DUQH2 could be competitive on complex III. Resveratrol significantly inhibited ATPase activity (p < 0.001) following a biphasic curve with two EC50 values, 0.39 +/- 0.15 nM and 23.1 +/- 6.4 microM, both representing about 80% of oligomycin-dependent ATPase total activity. Resveratrol was effective as a protecting agent on the three models of oxidation. On lipid peroxidation of brain synaptosomes induced by the Fenton reaction, it was three times more potent than DUQH2. Its effectiveness in reducing 1,1-diphenyl-2-picryl hydrazyl radical (DPPH degrees) showed a stoichiometry of two, indicating that two hydrogen atoms of resveratrol were abstracted by the process. Resveratrol was also able to scavenge the superoxide anion (O2 degrees) generated from rat forebrain mitochondria in a concentration dependent manner. In conclusion, resveratrol can decrease complex III activity by competition with coenzyme Q. This property is especially interesting as this complex is the site where reactive oxygen substances (ROS) are generated. By decreasing the activity of complex III, resveratrol cannot only oppose the production of ROS but can also scavenge them.

BACKGROUND

High residual platelet reactivity (HRPR) in patients receiving clopidogrel has been associated with high risk of ischemic events after percutaneous coronary intervention (PCI).

OBJECTIVE

To test the hypothesis that HRPR after clopidogrel loading is an independent prognostic marker of risk of long-term thrombotic events in patients with acute coronary syndromes (ACS) undergoing an invasive procedure and antithrombotic treatment adjusted according to the results of platelet function tests.

METHODS

Prospective, observational, referral center cohort study of 1789 consecutive patients with ACS undergoing PCI from April 2005 to April 2009 at the Division of Cardiology of Careggi Hospital, Florence, Italy, in whom platelet reactivity was prospectively assessed by light transmittance aggregometry.

METHODS

All patients received 325 mg of aspirin and a loading dose of 600 mg of clopidogrel followed by a maintenance dosage of 325 mg/d of aspirin and 75 mg/d of clopidogrel for at least 6 months. Patients with HRPR as assessed by adenosine diphosphate test (≥70% platelet aggregation) received an increased dose of clopidogrel (150-300 mg/d) or switched to ticlopidine (500-1000 mg/d) under adenosine diphosphate test guidance.

METHODS

The primary end point was a composite of cardiac death, myocardial infarction, any urgent coronary revascularization, and stroke at 2-year follow-up. Secondary end points were stent thrombosis and each component of the primary end point.

RESULTS

The primary end point event rate was 14.6% (36/247) in patients with HRPR and 8.7% (132/1525) in patients with low residual platelet reactivity (absolute risk increase, 5.9%; 95% CI, 1.6%-11.1%; P = .003). Stent thrombosis was higher in the HRPR group compared with the low residual platelet reactivity group (6.1% [15/247] vs 2.9% [44/1525]; absolute risk increase, 3.2%; 95% CI, 0.4%-6.7%; P = .01). By multivariable analysis, HRPR was independently associated with the primary end point (hazard ratio, 1.49; 95% CI, 1.08-2.05; P = .02) and with cardiac mortality (hazard ratio, 1.81; 95% CI, 1.18-2.76; P = .006).

CONCLUSIONS

Among patients receiving platelet reactivity-guided antithrombotic medication after PCI, HRPR status was significantly associated with increased risk of ischemic events at short- and long-term follow-up.

BACKGROUND

clinicaltrials.gov Identifier: NCT01231035.

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.

In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis ML3 rapidly accumulated thiomethyl-beta-D-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the beta-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (approximately 39 mM; 2-PG), but an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the beta-galactoside and the total available concentration of the two intermediates; TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; fluoride inhibition of 2-phospho-D-glycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'-triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis.

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.

The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

Neutrophil granulocytes traffic into sites of organ injury in which they may not only participate in tissue repair and pathogen clearance but may also contribute to collateral cell damage through the release of noxious mediators. The dynamics and mechanisms of neutrophil migration in the extravascular space toward loci of tissue damage are not well understood. Here, we have used intravital multi-photon microscopy to dissect the behavior of neutrophils in response to tissue injury in the dermis of mice. We found that, following confined physical injury, initially rare scouting neutrophils migrated in a directional manner toward the damage focus. This was followed by the attraction of waves of additional neutrophils, and finally stabilization of the neutrophil cluster around the injury. Although neutrophil migration in the steady state and during the scouting phase depended on pertussis toxin-sensitive signals, the amplification phase was sensitive to interference with the cyclic adenosine diphosphate ribose pathway. We finally demonstrated that neutrophil scouts also transit through the non-inflamed dermis, suggesting immunosurveillance function by these cells. Together, our data unravel a three-step cascade of events that mediates the specific accumulation of neutrophils at sites of sterile tissue injury in the interstitial space.

BACKGROUND

Recent randomized trials suggest that women may not accrue the same cardioprotective benefits as men do from low-dose aspirin therapy used in primary prevention. Failure of aspirin to suppress platelet aggregation in women is one hypothesized mechanism.

OBJECTIVE

To examine differential platelet reactivity to low-dose aspirin therapy by sex.

METHODS

A clinical trial of aspirin at 81 mg/d for 14 days was conducted in 571 men and 711 women. Baseline and post-aspirin therapy measures included platelet aggregation to arachidonic acid, adenosine diphosphate, epinephrine, and platelet function analyzer closure time.

METHODS

Sex differences in cyclooxygenase 1 (COX-1) direct and indirect platelet activation pathways before and after administration of aspirin.

RESULTS

In 10 of the 12 platelet agonist exposures, women's platelets were significantly more reactive at baseline. However, after aspirin therapy, the percent aggregation to arachidonic acid (the direct COX-1 pathway) decreased more in women than in men (P<.001) and demonstrated near total suppression of residual platelet reactivity in both men and women. In COX-1 indirect pathways, women experienced the same or more platelet inhibition than men in 8 of the 9 assays yet retained modestly greater platelet reactivity after aspirin therapy. In multivariable analysis, female sex significantly predicted aggregation to 2 muM and 10 muM of adenosine diphosphate (P = .02 and <.001, respectively) and collagen at 5 mug/mL (P<.001) independent of risk factors, age, race, menopausal status, and hormone therapy.

CONCLUSIONS

Women experienced the same or greater decreases in platelet reactivity after aspirin therapy, retaining modestly more platelet reactivity compared with men. However, most women achieved total suppression of aggregation in the direct COX-1 pathway, the putative mechanism for aspirin's cardioprotection.

The crystal structures of the catalytic fragment of chicken poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] with and without a nicotinamide-analogue inhibitor have been elucidated. Because this enzyme is involved in the regulation of DNA repair, its inhibitors are of interest for cancer therapy. The inhibitor shows the nicotinamide site and also suggests the adenosine site. The enzyme is structurally related to bacterial ADP-ribosylating toxins but contains an additional alpha-helical domain that is suggested to relay the activation signal issued on binding to damaged DNA.

CD73/ecto-5'-nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti-tumor immunity has not been studied. Here, we show that under physiological conditions CD73-deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor-infiltrating leukocytes there were fewer Tregs and mannose receptor-positive macrophages, and increased IFN-γ and NOS2 mRNA production in CD73-deficient mice. Treatment of tumor-bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor-positive macrophages in the WT C57BL/6 mice but not in the CD73-deficient mice. Pharmacological inhibition of CD73 with α,β-methylene-adenosine-5'-diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti-tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

OBJECTIVE

This study sought to assess the influence of type 2 diabetes mellitus (T2DM) and the impact of hypoglycemic treatment (insulin vs. noninsulin) on platelet function profiles in patients treated with dual oral antiplatelet therapy.

BACKGROUND

Insulin inhibits platelet aggregation by suppressing the P2Y12 pathway. However, T2DM patients have a loss of responsiveness to insulin that leads to upregulation of the P2Y12 pathway, increased platelet reactivity, and reduced responsiveness to antiplatelet agents. Patients with insulin-treated diabetes mellitus (ITDM) have a more advanced disease status and higher atherothrombotic risk compared with non-ITDM (NITDM). However, the impact of insulin therapy on platelet dysfunction in patients treated with P2Y12 antagonists is unknown.

METHODS

A total of 201 T2DM and 65 nondiabetic patients with coronary artery disease in a steady phase of aspirin and clopidogrel treatment were studied. Platelet aggregation was assessed using agonists specific (6 and 20 microM adenosine diphosphate [ADP]) and nonspecific (6 microg/ml collagen and 20 microM epinephrine) for the P2Y12 pathway. High shear-induced platelet reactivity was assessed by means of the PFA-100 system (Dade-Behring International, Miami, Florida).

RESULTS

The T2DM patients had platelet aggregation and shear-induced platelet function significantly increased compared with nondiabetic patients using all assays. Platelet aggregation was increased in ITDM (n = 68) compared with NITDM (n = 133) patients after P2Y12-specific stimuli. Insulin treatment was the strongest predictor of ADP-induced aggregation. Platelet function profiles were similar between ITDM and NITDM using assays nonspecific to the P2Y12 pathway. Platelet dysfunction was independent of glycemic control and inflammatory status.

CONCLUSIONS

The P2Y12-dependent and -independent pathways of platelet reactivity are altered in T2DM compared with nondiabetic patients, and ITDM have greater ADP-induced platelet aggregation compared with NITDM.