Pertussis toxin (PT) and adenylate cyclase toxin (AT) are AB enterotoxins produced by Bordetella pertussis. PT is a powerful mucosal adjuvant whose cellular target and mechanism of action are unknown; however, emerging evidence suggests that dendritic cells (DC) may be a principal adjuvant target of PT. Here, we investigate the mechanism underlying the effects of these toxins on human monocyte-derived DC (MDDC) in vitro. We found that the effects of PT and AT on MDDC, including maturation, are mediated by cyclic adenosine monophosphate (cAMP). In this regard, adenosine 5'-diphosphate-ribosylation-defective derivatives of PT failed to induce maturation of MDDC, whereas dibutyryl-cAMP (d-cAMP) and Forskolin mimic the maturation of MDDC and dominant inhibition of cytokine production induced by these toxins. Also, cAMP-dependent kinase inhibitors blocked the ability of PT, AT, d-cAMP, and Forskolin to activate MDDC. Taken together, these results show that the effects of PT and AT on MDDC are mediated strictly by cAMP.

BACKGROUND

Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg-deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF(-/-)).

RESULTS

Here we studied Fg/VWF-independent platelet aggregation in vitro and found no aggregation in citrated platelet-rich plasma of Fg/VWF(-/-) mice. Surprisingly, in Fg/VWF(-/-) plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF(-/-) platelets but not of beta(3)-integrin-deficient (beta(3) (-/-)) platelets. In addition, beta(3) (-/-) platelets did not significantly incorporate into thrombi in Fg/VWF(-/-) mice. This Fg/VWF-independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF-NH(2)) induced aggregation of gel-filtered Fg/VWF(-/-) platelets in 1 mm Ca(2+) PIPES buffer. Notably, aggregation in PIPES buffer was only 50-60% of that observed in Fg/VWF(-/-) plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF(-/-) mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca(2+)in vitro.

CONCLUSIONS

beta(3) integrin, thrombin, and Ca(2+) play critical roles in this Fg/VWF-independent aggregation, and both plasma and platelet granule proteins contribute to this process.

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.

It has been shown that lipid peroxidation is associated with hepatic fibrosis and stellate cell activation. Sho-saiko-to (TJ-9) is an herbal medicine, which is commonly used to treat chronic hepatitis in Japan, although the mechanism by which TJ-9 protects against hepatic fibrosis is not known. As a result, we assayed the preventive and therapeutic effects of TJ-9 on experimental hepatic fibrosis, induced in rats by dimethylnitrosamine (DMN) or pig serum (PS), and on rat stellate cells and hepatocytes in primary culture, and assessed the antioxidative activities and the active components of TJ-9. Male Wistar rats were given a single intraperitoneal injection of 40 mg/kg DMN or 0.5 mL PS twice weekly for 10 weeks. In each model, rats were fed a basal diet throughout, or the same diet, which also contained 1.5% TJ-9, for 2 weeks before treatment or for the last 2 weeks of treatment. TJ-9 suppressed the induction of hepatic fibrosis, increased hepatic retinoids, and reduced the hepatic levels of collagen and malondialdehyde (MDA), a production of lipid peroxidation. Immunohistochemical examination showed that TJ-9 reduced the deposition of type I collagen and the number of alpha-smooth muscle actin (alpha-SMA) positive-stellate cells in the liver and inhibited, not only lipid peroxidation in cultured rat hepatocytes that were undergoing oxidative stress, but also the production of type I collagen, alpha-SMA expression, cell proliferation, and oxidative burst in cultured rat stellate cells. In addition, TJ-9 inhibited Fe2+/adenosine 5'-diphosphate-induced lipid peroxidation in rat liver mitochondria in a dose-dependent manner and showed radical scavenging activity. Among the active components of TJ-9, baicalin and baicalein were found to be mainly responsible for the antioxidative activity. These findings suggest that Sho-saiko-to (TJ-9) functions as a potent antifibrosuppressant by inhibition of lipid peroxidation in hepatocytes and stellate cells in vivo.

The platelet glycoproteins (GPs) Ib, integrin alpha(2)beta(1), and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s(-1)), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb alpha (12G1 Fab(2)) and alpha(2)beta(1) (6F1 mAb or GFOGER-GPP peptide). GPVI was found to be crucial for aggregate formation, Ca(2+) signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting alpha(2)beta(1) revealed its involvement in regulating Ca(2+) signaling, PS exposure, and aggregate size. Both GPIb alpha and alpha(2)beta(1) contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb alpha or alpha(2)beta(1). In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with alpha(2)beta(1) mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.

Adenosine diphosphate-ribosylation is a post-translational modification mediated by intracellular and membrane-associated extracellular enzymes and many bacterial toxins. The intracellular enzymes modify their substrates either by poly-ADP-ribosylation, exemplified by ARTD1/PARP1, or by mono-ADP-ribosylation. The latter has been discovered only recently, and little is known about its physiological relevance. The founding member of mono-ADP-ribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitin-interaction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-κB signalling. We therefore studied the role of ARTD10 in this pathway. ARTD10 inhibits the activation of NF-κB and downstream target genes in response to interleukin-1β and tumour necrosis factor-α, dependent on catalytic activity and poly-ubiquitin binding of ARTD10. Mechanistically ARTD10 interferes with poly-ubiquitination of NEMO, which interacts with and is a substrate of ARTD10. Our findings identify a novel regulator of NF-κB signalling and provide evidence for cross-talk between K63-linked poly-ubiquitination and mono-ADP-ribosylation.

The effect of synthetic 8-arginine vasopressin (vasopressin) was studied in isolated canine basilar, left circumflex coronary, and femoral arteries of the dog. Vascular rings with and without endothelium were suspended for isometric tension recording in physiological salt solution. The removal of the endothelium was confirmed by the absence of relaxations induced by either thrombin (basilar arteries) or acetylcholine (coronary and femoral arteries). In the basilar artery, vasopressin induced concentration-dependent inhibition of myogenic tone. In basilar and coronary arteries, the hormone caused concentration-dependent relaxations during contractions evoked by prostaglandin F2 alpha. In femoral arteries, vasopressin caused contraction. After removal of the endothelium, the inhibitory responses to vasopressin were abolished in basilar arteries and significantly reduced in left circumflex coronary arteries. The contractions of femoral arteries were not affected by endothelium removal. The V1-vasopressinergic antagonist d(CH2)5Tyr(Me)AVP prevented the inhibitory response to vasopressin, but did not alter endothelium-dependent relaxations of basilar arteries caused by adenosine diphosphate. These results demonstrate that the endothelial cells mediate relaxation induced by vasopressin via specific V1-vasopressinergic receptors.

The dehydrogenation reaction of cholest-7-en-3beta-ol (I) to cholesta-5,7-dien-3beta-ol (II) in the presence of NADH was studied in rat liver microsomes and in microsomal acetone powder preparations, using [3alpha-3H]cholest-7-en-3beta-ol. It was found that the reaction was inhibited by menadione, adenosine diphosphate, potassium ferricyanide, and cytochrome c while p-cresol had no effect. These results indicated the participation of a microsomal electron transport system in the dehydrogenation of cholest-7-en-3beta-ol. The conversion of cholest-7-en-3beta-ol to cholesta-5,7-dien-3beta-ol was also observed in the absence of NADH when ascorbic acid was included in the incubation mixture. However, the ascorbic acid-catalyzed dehydrogenation was not inhibited by potassium ferricyanide. Immunological evidence that microsomal cytochrome b5 is involved in the dehydrogenation of (I) to (II) was obtained. Antibodies specific for rat liver microsomal cytochrome b5 were elicited in rabbits. The anticytochrome b5 immunoglobulin fraction inhibited rat liver microsomal NADH-cytochrome c reductase but not NADPH-cytochrome c reductase. Also, the extent of reduction of cytochrome b5 was not affected by the antibodies. The conversion of (I) to (II) by rat liver microsomes was inhibited (73%) by anticytochrome b5 immunoglobulin at a ratio of microsomal protein:immunoglobulin of 1:5.6. These results are consistent with the participation of microsomal cytochrome b5 in the introduction of the C-5 double bond in cholesterol biosynthesis. A close analogy of the microsomal dehydrogenation of fatty acids and of cholest-7-en-3beta-ol is apparent and this suggests a possible similarity in the mechanisms of the two reactions.

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.

The aggregating effects of adenosine diphosphate, thrombin, 5-hydroxytryptamine, tryptamine, adrenaline and noradrenaline, and tri-ethyl tin have been carefully compared. The first three compounds in some circumstances produce remarkably similar effects although there are important differences. The kinetics of aggregation induced by adrenaline (and noradrenaline) are quite different and the tri-ethyl tin effects are different again. Anti-serotonins specifically inhibit 5-hydroxytryptamine and the anti-adrenaline drug phentolamine specifically inhibits the effects of the catecholamines. Experiments presented suggest but do not prove that aggregation produced by all these compounds is accompanied by the liberation of diphosphate from the platelets and that platelet triphosphate may be converted to diphosphate. How these different compounds all produce this effect is discussed. Either the presence of diphosphate or the action of a triphosphatase might be the immediate cause of aggregation if there is a single final common cause. The anti-adrenaline phentolamine prolongs the bleeding time, so adrenaline or noradrenaline may be involved in platelet phenomena in haemostasis.

Group N streptococci, which have the lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) and phospho-beta-d-galactosidase (beta-Pgal), grew rapidly on lactose and converted more than 90% of the sugar to l-lactate. In contrast, Streptococcus lactis 7962, which does not have a beta-Pgal, grew slowly on lactose and converted only 15% of the sugar to l-lactate. With glucose and galactose, this strain had growth rates and fermentation patterns similar to those of other S. lactis strains, suggesting that the rapid and homolactic fermentation of lactose that is characteristic of group N streptococci is dependent upon a functional PEP-dependent PTS and the presence of beta-Pgal. Seventeen strains of group N streptococci were examined for the activator specificities of pyruvate kinase and lactate dehydrogenase. The properties of each enzyme from all the strains, including S. lactis 7962, were similar. Pyruvate kinase had a broad activator specificity, whereas activation of lactate dehydrogenase was specific for ketohexose diphosphate. All intermediates of lactose metabolism from the hexose phosphates to the triose phosphates activated pyruvate kinase. No activation was obtained with adenosine 5'-monophosphate. K and Mg were required for pyruvate kinase activity but could be replaced by NH(4) and Mn, respectively. Lactate dehydrogenase was activated equally by fructose-1,6-diphosphate and tagatose-1,6-diphosphate, the activation characteristics being pH dependent. The roles of pyruvate kinase and lactate dehydrogenase in the regulation of lactose fermentation by group N streptococci are discussed.

Kinesin is a 'motor' molecule, consisting of two head domains, an alpha-helical coiled coil rod, and a tail part that binds to its cargo. When expressed in a bacterial system, the head domain is functional, and can bind to microtubules with the stoichiometry of one head per tubulin dimer. Kinesin moves along microtubules by means of a cyclic process of nucleotide binding, hydrolysis and product release. We have used negative-stain electron microscopy and image analysis to study the structures of microtubules and tubulin sheets decorated with the motor domain (head) of kinesin in three states: in the presence of an unhydrolysable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP); without nucleotides; and with adenosine 5'-diphosphate (ADP). A single kinesin head bound to a microtubule has a pear-shaped structure, with the broader end towards the 'plus' end of the microtubule under all conditions; the reverse motor, ncd, is similarly oriented. Three-dimensional maps reveal that kinesin heads have a spike that is assumed to form the attachment to the tail of a complete kinesin molecule. This spike is perpendicular to the microtubule axis in the presence of ADP, but points towards the plus end (approximately 45 degrees) in the presence of AMP-PNP or absence of nucleotides. Our results provide direct evidence for a conformational change of the kinesin motor domain during the ATPase cycle.

Platelet spreading on immobilized fibrinogen (Fg) involves progression through a number of morphologic stages that, although distinctive, are not well understood mechanistically. Here we demonstrate that an association between GPIIb/IIIa and calcium- and integrin-binding protein (CIB) is required for the process of platelet spreading. Upon platelet adhesion to immobilized Fg, CIB localizes to the transiently formed filopodia and then redistributes diffusely along the membrane periphery of spread platelets. Immunoprecipitation analyses indicate that CIB and glycoprotein IIb/IIIa (GPIIb/IIIa) interact with each other as platelets adhere to immobilized Fg, and together they associate with the platelet cytoskeleton. Introduction of anti-CIB antibody or GPIIb cytoplasmic peptide into platelets blocks lamellipodia but not filopodia formation. GPIIb peptide-induced inhibition of platelet spreading is recovered by the incorporation of recombinant CIB protein, suggesting that interaction between CIB and GPIIb/IIIa is required for progression from filopodial to spread morphologies. Further, anti-CIB- or GPIIb peptide-induced inhibition of platelet spreading can be overcome by the addition of exogenous adenosine diphosphate (ADP). These data suggest that formation of the CIB-GPIIb/IIIa complex may be necessary for initiation of downstream signaling events, such as ADP secretion, that lead to platelet spreading.

It has been suggested that ATP might play a role in synaptic transmission at developing vertebrate neuromuscular junctions. To increase our understanding of the events underlying synapse formation, we have used intracellular recording and patch clamp recording to examine the response of chick myoblasts and myotubes to to ATP and other nucleotides, ATP, applied at micromolar concentrations, has a potent depolarizing action on chick myoblasts and myotubes. The ATP depolarization declines during prolonged application of ATP and shows no recovery for at least 20 min after the removal of ATP. The physiological event that underlies the ATP response has a reversal potential near O mV and is due to a conductance increase. However, contrary to our expectation, in a series of nearly 200 cell-attached and outside-out patch recordings, we did not detect single-channel currents that were related to ATP. The myotube ATP receptor is pharmacologically distinct from putative ATP receptors in other systems. It is not activated by ADP, AMP, or adenosine. Furthermore, the nonhydrolyzable ATP analogs, AMP-PNP, alpha,beta-meATP, and beta,gamma-meATP (respectively, 5-adenylylimido diphosphate; alpha,beta-methylene adenosine 5'-triphosphate; and beta,gamma-methylene adenosine 5'-triphosphate), which are potent ATP agonists in other systems, have no depolarizing action on myotubes. The ATP receptor is also distinct from the nicotinic ACh receptor since responses to ATP are unaffected by the nicotinic antagonists d-tubocurarine and alpha-bungarotoxin. We therefore applied alpha-bungarotoxin to nerve-muscle co-cultures in the hope of uncovering an additional component of the postsynaptic potential, which might represent a synaptic action of ATP. Under these experimental conditions no evidence indicative of a postsynaptic action of ATP released from nerve terminals was observed.

The contractile and relaxant effects of the different P2 receptors were characterized in the rat isolated mesenteric artery by use of extracellular nucleotides, including the stable pyrimidines uridine 5'-O-thiodiphosphate (UDPbetaS) and uridine 5'-O-3-thiotriphosphate (UTPgammaS). The selective P2X receptor agonist, alphabeta-methylene-adenosine triphosphate (alphabeta-MeATP) stimulated a potent (pEC(50)=6.0) but relatively weak contraction (E:(max)=57% of 60 mM K(+)). The contractile concentration-response curve of adenosine triphosphate (ATP) was biphasic when added in single concentrations. The first part of the response could be desensitized by alphabeta-MeATP, indicating involvement of P2X receptors, while the second part might be mediated by P2Y receptors. The contractile P2Y receptors were further characterized after P2X receptor desensitization with 10 microM alphabeta-MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and ATP stimulated contraction only in high concentrations (1 - 10 mM). The selective P2Y(6) agonist, UDPbetaS, and the P2Y(2)/P2Y(4)-receptor agonists UTPgammaS and adenosine 5'-O-3-thiotriphosphate (ATPgammaS) were considerably more potent and efficacious (E:(max) approximately 250% of 60 mM K(+)). Adenosine 5'-O-thiodiphosphate (ADPbetaS) was inactive, excluding contractile P2Y(1) receptors. After precontraction with 1 microM noradrenaline, UTP, ADP and ATP induced relaxations with similar potencies (pEC(50) approximately 5.0). UTPgammaS, ADPbetaS and ATPgammaS were approximately one log unit more potent indicating the presence of endothelial P2Y(1) and P2Y(2)/P2Y(4) receptors. The P2Y(6) receptor agonist, UDPbetaS, had no effect. UDPbetaS and UTPgammaS are useful tools when studying P2 receptors in tissue preparations with ectonucleotidase activity. Contractile responses can be elicited by stimulation of P2Y(6) and, slightly less potently, P2Y(2)/P2Y(4) receptors. The P2X response was relatively weak, and there was no P2Y(1) response. Stimulation of P2Y(1) and P2Y(2)/P2Y(4) receptors elicited relaxation, while P2Y(6) did not contribute.

The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.

1. Pertussis toxin inactivates Gi-protein, which mediates the inhibitory effects of receptors on adenylate cyclase. The effects of the toxin on endothelium-dependent and independent relaxations were determined in porcine coronary arteries. 2. Arterial rings (with and without endothelium) were suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution (maintained at 37 degrees C, gassed with 95% O2 and 5% CO2). 3. Incubation of the tissues with pertussis toxin (100 ng/ml for 60 min) virtually abolished the endothelium-dependent relaxations produced by the alpha 2-adrenergic agonist, UK 14304, and by 5-hydroxytryptamine. Endothelium-dependent relaxations to thrombin and to aggregating platelets were markedly reduced, whereas those produced by bradykinin were only minimally affected. Endothelium-dependent responses produced by the calcium ionophore (A23187) and by adenosine diphosphate were not altered by pertussis toxin. 4. Pertussis toxin did not affect the direct, endothelium-independent relaxations produced by nitric oxide, or by adenosine diphosphate. 5. These experiments demonstrate that pertussis toxin interferes with the release of endothelium-derived relaxing factor(s) evoked by certain, but not all, endothelial activators. The release of endothelium-derived relaxing factor(s) may occur through different pathways involving Gi-protein-dependent and independent mechanisms.

BACKGROUND

Energy drink consumption has been anecdotally linked with sudden cardiac death and, more recently, myocardial infarction. As myocardial infarction is strongly associated with both platelet and endothelial dysfunction, we tested the hypothesis that energy drink consumption alters platelet and endothelial function.

METHODS

Fifty healthy volunteers (34 male, aged 22+/-2 years) participated in the study. Platelet aggregation and endothelial function were tested before, and 1 hour after, the consumption of 250 mL (1 can) of a sugar-free energy drink. Platelet function was assessed by adenosine diphosphate-induced (1 micromol/L) optical aggregometry in platelet-rich plasma. Endothelial function was assessed via changes in peripheral arterial tonometry and expressed as the reactive hyperemia index (RHI).

RESULTS

Compared with baseline values, there was a significant increase in platelet aggregation following energy drink consumption, while no change was observed with control (13.7+/-3.7% vs 0.3+/-0.8% aggregation, respectively, P <.01). Similarly, RHI decreased following energy drink consumption (-0.33+/-0.13 vs 0.07+/-0.12 RHI [control], P <.05). Mean arterial pressure significantly increased following energy drink consumption, compared with control (P <.05). Heart rate was unaffected by energy drink consumption.

CONCLUSIONS

Energy drink consumption acutely increases platelet aggregation and decreases endothelial function in healthy young adults.

A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.

A new enzyme, 5-oxoprolinase, was found in rat kidney and in several other tissues; it catalyzes the conversion of 5-oxo-L-proline (L-5-oxo-pyrrolidine-2-carboxylic acid, L-2-pyrrolidone-5-carboxylic acid, L-pyroglutamic acid) to L-glutamic acid, with concomitant stoichiometric cleavage of ATP to ADP and orthophosphate. The reaction catalyzed by 5-oxoprolinase, in which 5-oxoproline formed from gamma-glutamyl amino acids by the action of gamma-glutamylcyclotransferase is converted to glutamate, appears to function in the gamma-glutamyl cycle. 5-Oxoprolinase requires Mg(++) (or Mn(++)) and K(+) (or NH(4) (+)) for activity. The equilibrium is markedly in favor of glutamate formation at pH 7.8.

Salmonella typhimurium LT2 rapidly accumulates high levels of a family of five adenylylated nucleotides following exposure to a bacteriostatic quinone, 6-amino-7-chloro-5,8-dioxoquinoline. These compounds have been analyzed using our recently described two-dimensional thin layer chromatographic method. The five dinucleotides, which cannot be detected in exponentially growing cells, have been identified as diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), ApppGpp (guanosine 3'-diphosphate-5'-adenosine-5'-(P1,P3-triphosphate)), AppppG (adenosine 5'-guanosine-5'-(P1,P4-tetraphosphate)), ApppG (adenosine 5'-guanosine-5'-(P1,P3-triphosphate)), and ApppA (diadenosine 5',5"'-P1,P3-triphosphate). AppppA has been previously detected in vitro as an enzymatic product of aminoacyl-tRNA synthetases and in vivo at submicromolar levels in eucaryotic cells. The induced intracellular concentration of AppppA and the other adenylylated nucleotides in S. typhimurium is approximately 100-fold higher than that found in eucaryotic cells. We propose that these dinucleotides are alarmones, regulatory molecules signaling a particular metabolic stress.