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Objective: To investigate the safety and effectiveness of direct oral anticoagulants (DOAC) versus vitamin K antagonists (VKA) after recent stroke in patients with atrial fibrillation (AF) aged ≥85 years. Methods: Individual patient data analysis from seven prospective stroke cohorts. We compared DOAC versus VKA treatment among patients with AF and recent stroke (

Background: Leukoaraiosis (LA) is a common radiological finding in elderly, frequently associated with several clinical disorders, including unexplained dizziness. The pathogenesis of LA is multifactorial, with a dysfunction of cerebral microcirculation resulting in chronic hypoperfusion and tissue loss, with oxidative stress involved in this cascade. Objective: The aim of this study was to analyse some oxidative stress biomarkers in a cohort of LA patients. Method: Fifty-five consecutive patients (33 males, median age 75 years) with LA were recruited. In a subgroup of 33 patients with LA and unexplained dizziness, we have then performed an open study to evaluate if 60-day supplementation with a polyphenol compound may modify these biomarkers and influence quality of life, analysed with the Dizziness Handicap Inventory (DHI) scale. Results: At baseline, blood oxidative stress parameters values were outside normal ranges and compared to matched healthy controls. After the two months supplementation, we observed a significant decrement of advanced oxidation protein products values and a significant improvement of DHI. Conclusion: Oxidative stress biomarkers may be useful to detect redox imbalance in LA and to provide non-invasive tools to monitor disease status and response to therapy.

Spontaneous dissection of cervical arteries (sCAD) is a major cause of ischemic stroke in young patients, with an incidence varying from 1.7 to 3/100,000/year for extracranial internal carotid artery (ICAD) and 1 to 1.9/100,000/year for extracranial vertebral artery (VAD). Reliable epidemiological data on stroke incidence related to sCAD are scarce in Italy. This study aims to evaluate the incidence, clinical features, and outcome of cerebrovascular events related to sCAD and spontaneous intracranial arteries dissections (sIAD) in the city of Pisa (Italy). We retrospectively analyzed consecutive patients admitted between December 1997 and June 2015 with a diagnosis of stroke, TIA, or Bernard-Horner syndrome due to acute cervical or intracranial artery dissection. Considering that our hospital collects presumptively all patients hospitalized with sCAD coming from the referral geographical area, data may provide a good approximation to real incidence of sCAD in our population. Clinical and radiological features, acute treatment and outcome were collected. Seventy-seven cases were included (mean age 48.1±10.4 years, range 23-77,72.7% males), 66 residents in the district of Pisa. Crude incidence rate of cerebrovascular events due to intra or extracranial dissection was 1.88/100,000/year. The incidence of ICAD was 0.80/100,000/year and 0.43/100,000/year for VAD. Stroke occurred in 76.6% of patients. VAD was more prone to cause ischemic stroke and present with cervical pain or focal signs (p

A great deal of interest exists in gene transfer and manipulation techniques for the predictable modification of plant genomes, in particular those of economically or nutritionally important plants. Such combinations, successful so far in prokaryotic systems, might improve lipofection as a tool for genetic engineering of plants as well. Electroporation has become a popular tool for introducing exogenous DNA into plant protoplasts. A classical example of the combination of the advantages of both liposome technology and electroporation has been described by P. Machy et al. Besides the wide utilization of electroporation for transient gene expression experiments, permanent transformation and viral RNA-mediated infection of plant protoplasts, the technique has been increasingly used for antisense RNA-mediated inhibition of gene expression. A wide array of transformation techniques is available, but there is to date no universal, ideal, generally applicable method for gene transfer to plants.

Background and Purpose-Bridging therapy with low-molecular-weight heparin reportedly leads to a worse outcome for acute cardioembolic stroke patients because of a higher incidence of intracerebral bleeding. However, this practice is common in clinical settings. This observational study aimed to compare (1) the clinical profiles of patients receiving and not receiving bridging therapy, (2) overall group outcomes, and (3) outcomes according to the type of anticoagulant prescribed. Methods-We analyzed data of patients from the prospective RAF and RAF-NOACs studies. The primary outcome was defined as the composite of ischemic stroke, transient ischemic attack, systemic embolism, symptomatic cerebral bleeding, and major extracerebral bleeding observed at 90 days after the acute stroke. Results-Of 1810 patients who initiated oral anticoagulant therapy, 371 (20%) underwent bridging therapy with full-dose low-molecular-weight heparin. Older age and the presence of leukoaraiosis were inversely correlated with the use of bridging therapy. Forty-two bridged patients (11.3%) reached the combined outcome versus 72 (5.0%) of the nonbridged patients (P=0.0001). At multivariable analysis, bridging therapy was associated with the composite end point (odds ratio, 2.3; 95% CI, 1.4-3.7; P

Background: The relationship between different patterns of atrial fibrillation and early recurrence after an acute ischaemic stroke is unclear. Purpose: In a prospective cohort study, we evaluated the rates of early ischaemic recurrence after an acute ischaemic stroke in patients with paroxysmal atrial fibrillation or sustained atrial fibrillation which included persistent and permanent atrial fibrillation. Methods: In patients with acute ischaemic stroke, atrial fibrillation was categorised as paroxysmal atrial fibrillation or sustained atrial fibrillation. Ischaemic recurrences were the composite of ischaemic stroke, transient ischaemic attack and symptomatic systemic embolism occurring within 90 days from acute index stroke. Results: A total of 2150 patients (1155 females, 53.7%) were enrolled: 930 (43.3%) had paroxysmal atrial fibrillation and 1220 (56.7%) sustained atrial fibrillation. During the 90-day follow-up, 111 ischaemic recurrences were observed in 107 patients: 31 in patients with paroxysmal atrial fibrillation (3.3%) and 76 with sustained atrial fibrillation (6.2%) (hazard ratio (HR) 1.86 (95% CI 1.24–2.81)). Patients with sustained atrial fibrillation were on average older, more likely to have diabetes mellitus, hypertension, history of stroke/ transient ischaemic attack, congestive heart failure, atrial enlargement, high baseline NIHSS-score and implanted pacemaker. After adjustment by Cox proportional hazard model, sustained atrial fibrillation was not associated with early ischaemic recurrences (adjusted HR 1.23 (95% CI 0.74–2.04)). Conclusions: After acute ischaemic stroke, patients with sustained atrial fibrillation had a higher rate of early ischaemic recurrence than patients with paroxysmal atrial fibrillation. After adjustment for relevant risk factors, sustained atrial fibrillation was not associated with a significantly higher risk of recurrence, thus suggesting that the risk profile associated with atrial fibrillation, rather than its pattern, is determinant for recurrence. © European Stroke Organisation 2018.

Background and Purpose-Despite treatment with oral anticoagulants, patients with nonvalvular atrial fibrillation (AF) may experience ischemic cerebrovascular events. The aims of this case-control study in patients with AF were to identify the pathogenesis of and the risk factors for cerebrovascular ischemic events occurring during non-Vitamin K antagonist oral anticoagulants (NOACs) therapy for stroke prevention. Methods-Cases were consecutive patients with AF who had acute cerebrovascular ischemic events during NOAC treatment. Controls were consecutive patients with AF who did not have cerebrovascular events during NOACs treatment. Results-Overall, 713 cases (641 ischemic strokes and 72 transient ischemic attacks; median age, 80.0 years; interquartile range, 12; median National Institutes of Health Stroke Scale on admission, 6.0; interquartile range, 10) and 700 controls (median age, 72.0 years; interquartile range, 8) were included in the study. Recurrent stroke was classified as cardioembolic in 455 cases (63.9%) according to the A-S-C-O-D (A, atherosclerosis; S, small vessel disease; C, cardiac pathology; O, other causes; D, dissection) classification. On multivariable analysis, off-label low dose of NOACs (odds ratio [OR], 3.18; 95% CI, 1.95-5.85), atrial enlargement (OR, 6.64; 95% CI, 4.63-9.52), hyperlipidemia (OR, 2.40; 95% CI, 1.83-3.16), and CHA2DS2-VASc score (OR, 1.72 for each point increase; 95% CI, 1.58-1.88) were associated with ischemic events. Among the CHA2DS2-VASc components, age was older and presence of diabetes mellitus, congestive heart failure, and history of stroke or transient ischemic attack more common in patients who had acute cerebrovascular ischemic events. Paroxysmal AF was inversely associated with ischemic events (OR, 0.45; 95% CI, 0.33-0.61). Conclusions-In patients with AF treated with NOACs who had a cerebrovascular event, mostly but not exclusively of cardioembolic pathogenesis, off-label low dose, atrial enlargement, hyperlipidemia, and high CHA2DS2-VASc score were associated with increased risk of cerebrovascular events. © 2019 American Heart Association, Inc.

Background—In patients with acute ischemic stroke and atrial fibrillation, early anticoagulation prevents ischemic recurrence but with the risk of hemorrhagic transformation (HT). The aims of this study were to evaluate in consecutive patients with acute stroke and atrial fibrillation (1) the incidence of early HT, (2) the time to initiation of anticoagulation in patients with HT, (3) the association of HT with ischemic recurrences, and (4) the association of HT with clinical outcome at 90 days. Methods and Results—HT was diagnosed by a second brain computed tomographic scan performed 24 to 72 hours after stroke onset. The incidence of ischemic recurrences as well as mortality or disability (modified Rankin Scale scores >2) were evaluated at 90 days. Ischemic recurrences were the composite of ischemic stroke, transient ischemic attack, or systemic embolism. Among the 2183 patients included in the study, 241 (11.0%) had HT. Patients with and without HT initiated anticoagulant therapy after a mean 23.3 and 11.6 days, respectively, from index stroke. At 90 days, 4.6% (95% confidence interval, 2.3-8.0) of the patients with HT had ischemic recurrences compared with 4.9% (95% confidence interval, 4.0-6.0) of those without HT; 53.1% of patients with HT were deceased or disabled compared with 35.8% of those without HT. On multivariable analysis, HT was associated with mortality or disability (odds ratio, 1.71; 95% confidence interval, 1.24-2.35). Conclusions—In patients with HT, anticoagulation was initiated about 12 days later than patients without HT. This delay was not associated with increased detection of ischemic recurrence. HT was associated with increased mortality or disability. © 2018 The Authors.

Background and Purposes - This study was designed to derive and validate a score to predict early ischemic events and major bleedings after an acute ischemic stroke in patients with atrial fibrillation. Methods - The derivation cohort consisted of 854 patients with acute ischemic stroke and atrial fibrillation included in prospective series between January 2012 and March 2014. Older age (hazard ratio 1.06 for each additional year; 95% confidence interval, 1.00-1.11) and severe atrial enlargement (hazard ratio, 2.05; 95% confidence interval, 1.08-2.87) were predictors for ischemic outcome events (stroke, transient ischemic attack, and systemic embolism) at 90 days from acute stroke. Small lesions (≤1.5 cm) were inversely correlated with both major bleeding (hazard ratio, 0.39; P=0.03) and ischemic outcome events (hazard ratio, 0.55; 95% confidence interval, 0.30-1.00). We assigned to age ≥80 years 2 points and between 70 and 79 years 1 point; ischemic index lesion >1.5 cm, 1 point; severe atrial enlargement, 1 point (ALESSA score). A logistic regression with the receiver-operating characteristic graph procedure (C statistic) showed an area under the curve of 0.697 (0.632-0.763; P=0.0001) for ischemic outcome events and 0.585 (0.493-0.678; P=0.10) for major bleedings. Results - The validation cohort consisted of 994 patients included in prospective series between April 2014 and June 2016. Logistic regression with the receiver-operating characteristic graph procedure showed an area under the curve of 0.646 (0.529-0.763; P=0.009) for ischemic outcome events and 0.407 (0.275-0.540; P=0.14) for hemorrhagic outcome events. Conclusions - In acute stroke patients with atrial fibrillation, high ALESSA scores were associated with a high risk of ischemic events but not of major bleedings. © 2017 American Heart Association, Inc.

Background--The optimal timing to administer non-vitamin K oral anticoagulants (NOACs) in patients with acute ischemic stroke and atrial fibrillation is unclear. This prospective observational multicenter study evaluated the rates of early recurrence and major bleeding (within90 days)and their timing in patients with acute ischemic stroke and atrial fibrillation who received NOACs for secondary prevention. Methods and Results--Recurrence was defined as the composite of ischemic stroke, transient ischemic attack, and symptomatic systemic embolism, and major bleeding was defined as symptomatic cerebral and major extracranial bleeding. For the analysis, 1127 patients were eligible: 381 (33.8%) were treated with dabigatran, 366 (32.5%) with rivaroxaban, and 380 (33.7%) with apixaban. Patients who received dabigatran were younger and had lower admission National Institutes of Health Stroke Scale score and less commonly had a CHA2DS2-VASc score > 4 and less reduced renal function. Thirty-two patients (2.8%) had early recurrence, and 27 (2.4%) had major bleeding. The rates of early recurrence and major bleeding were, respectively, 1.8% and 0.5% in patients receiving dabigatran, 1.6% and 2.5% in those receiving rivaroxaban, and 4.0% and 2.9% in those receiving apixaban. Patients who initiated NOACs within 2 days after acute stroke had a composite rate of recurrence and major bleeding of 12.4%; composite rates were 2.1% for those who initiated NOACs between 3 and 14 days and 9.1% for those who initiated > 14 days after acute stroke. Conclusions--In patients with acute ischemic stroke and atrial fibrillation, treatment with NOACs was associated with a combined 5% rate of ischemic embolic recurrence and severe bleeding within 90 days. © 2017 The Authors.

Cb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to protein-coding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion in Cb2, exon-1B/exon-2 Cb2 messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2 transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2 complementary DNA and a myeloid cell line carrying a virus insertion in Cb2 (ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. Exposure of Cb2-expressing cells to different cannabinoids showed that the true ligand for Cb2 is 2-arachidonoylglycerol (2-AG), which may act as chemoattractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between 2-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb2 receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed 2-AG-induced migration of B220-, CD19-, immunoglobulin M-, and immunoglobulin D-expressing B lymphocytes. Our study demonstrates that a major function of Cb2 receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration.

This paper describes a program load voltage generator for flash memories. It is based on an adaptive feedback loop which senses the current delivered to the memory cells during programming and adjusts the output voltage accordingly to compensate for the voltage drop caused by the programming current across the bit-line select transistors. The proposed circuit (silicon area=0.065 mm/sup 2/) was integrated in a 0.8-/spl mu/m CMOS 4 Mb flash memory device (0.6 /spl mu/m in the matrix). Experimental evaluations showed that very effective compensation is achieved, with bit-line voltage kept at the desired value during the whole programming operation. A spread as small as 70 mV was measured between the single-bit and 16-b programming cases.

Nearly 30 years after the discovery of the psychoactive ingredient of Cannabis sativa, Δ9-tetrahydrocannabinol (THC) in 1964, the endogenous counterparts of THC were discovered and were collectively termed endocannabinoids (eCBs). To date, among several members of the eCB family N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are widely recognized as the most bioactive substances, able to bind to and activate G protein-coupled type-1 (CB1) and type-2 (CB2) cannabinoid receptors, as well as other non-CB1/non-CB2 receptor targets. In addition, an ensemble of proteins that synthesize, transport and degrade eCBs have been identified in the last 20 years, altogether forming the so-called eCB system. eCBs control basic biological processes spanning from cell choice between survival and death, progenitor/stem cell proliferation and differentiation, and neurotransmission to reproductive events, energy balance and immune response. Unsurprisingly, in the last two decades eCBs have emerged as key regulators of human pathophysiology at multiple levels. Here, a modern view of the biochemistry and pharmacology of the eCB system will be presented, in order to put in a better perspective the impact of eCB signaling on human health and disease, both centrally and peripherally. In particular, it will be shown that eCBs regulate bone elongation and remodeling, as well as inflammation and adaptive/innate immunity. Moreover, unprecedented results on AEA and 2-AG alterations in human patients with systemic lupus erythematosus will be presented, that may help to better understand the role of lipid signaling in this disease. Disclosure of Interest None declared

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of Δ9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm's endocannabinoid system. Long-term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users. HU210, a selective agonist for CB1 and CB2 cannabinoid receptors impairs spermatogenesis and sperm motility and deregulates the endocannabinoid system.

Type-1 cannabinoid receptor (CB(1)) is the most abundant G-protein-coupled receptor (GPCR) in the brain. CB(1) and its endogenous agonists, the so-called 'endocannabinoids (eCBs)', belong to an ancient neurosignalling system that plays important functions in neurodegenerative and neuroinflammatory disorders like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. For this reason, research on the therapeutic potential of drugs modulating the endogenous tone of eCBs is very intense. Several GPCRs reside within subdomains of the plasma membranes that contain high concentrations of cholesterol: the lipid rafts. Here, the hypothesis that changes in membrane fluidity alter function of the endocannabinoid system, as well as progression of particular neurodegenerative diseases, is described. To this end, the impact of membrane cholesterol on membrane properties and hence on neurodegenerative diseases, as well as on CB(1) signalling in vitro and on CB(1) -dependent neurotransmission within the striatum, is discussed. Overall, present evidence points to the membrane environment as a critical regulator of signal transduction triggered by CB(1) , and calls for further studies aimed at better clarifying the contribution of membrane lipids to eCBs signalling. The results of these investigations might be exploited also for the development of novel therapeutics able to combat disorders associated with abnormal activity of CB(1).

The presence of pyrroloquinoline quinone (PQQ) as the organic cofactor of Dactylium dendroides galactose oxidase and lentil (Lens culinaris) seedling amine oxidase, purported PQQ-containing oxidoreductases (Van der Meer, R. A., Jongejan, J. A., and Duine, J. A. (1989) J. Biol. Chem. 264, 7792-7794; Citro, G., Verdina, A., Galati, R., Floris, G., Sabatini, S., and Finazzi-Argo', A. (1989) FEBS Lett. 247, 201-204), was reinvestigated using the nitro blue tetrazolium redoxcycling method (Paz, M. A., Gallop, P. M., Torrelio, B. M., and Fluckiger, R. (1988) Biochem. Biophys. Res. Commun. 154, 1330-1337; Paz, M. A., Fluckiger, R., Boak, A., Kagan, H. M., and Gallop, P. M. (1991) J. Biol. Chem. 266, 689-692) and the enzyme-linked immunosorbent assay with polyclonal antibodies against PQQ. The possible quinoprotein nature of the laccases from Polyporus versicolor and Rhus vernicifera was also investigated because of the similarities in spectroscopic and kinetic features of these enzymes and the laccase from Phlebia radiata, reported to be a PQQ protein (Karhunen, E., Niku-Paavola, M.-L., Viikari, L., Haltia, T., Van der Meer, R. A., and Duine, J. A. (1990) FEBS Lett. 267, 6-8). The presence of a quinonoid cofactor in lentil seedling amine oxidase is confirmed, whereas galactose oxidase and both laccases do not display any quinoprotein nature.

In the present study, we used electrophysiological, biochemical, and confocal microscopy techniques, to investigate the functional role of transient receptor potential vanilloid type 1 (TRPV1) and cannabinoid type 1 receptors (CB1-R) in the substantia nigra pars compacta (SNpc) and their stimulation by the endocannabinoid N-arachidonoyl-dopamine (NADA). Liquid chromatography-mass spectrometry analyses revealed that a NADA-like compound is produced in substantia nigra slices, in conditions of hyperactivity. Moreover, the functional role of both TRPV1 and CB1-R in modulating synaptic transmission in this area was suggested by confocal microscopy data, showing TRPV1 and CB1-R immunoreactivity in punctate structures, probably representing synaptic contacts on cell bodies of the SNpc. In patch-clamp recordings from dopamine (DA) neurons of the SNpc, we found that NADA increases or reduces glutamatergic transmission onto DA neurons by activating TRPV1 and CB1 receptors, respectively, whereas it decreases GABAergic transmission via CB1 stimulation. Facilitation of glutamate release through TRPV1 was blocked in the presence of a selective blocker of the putative endocannabinoid membrane transporter (EMT), indicating that NADA needs to be taken up by cells to interact with this receptor. In line with these data, biochemical results demonstrated that NADA selectively acted at CB1-R when its re-uptake was blocked. Altogether these data demonstrate a significant role exerted by the endocannabinoid/endovanilloid NADA in the regulation of synaptic transmission to DA neurons of the SNpc. Moreover, they highlight a key function of the EMT transporter in promoting the stimulation of TRPV1 or CB1-R, thus favoring facilitation or inhibition of glutamate synaptic release.

Marijuana and cannabinoids have been shown to exert profound effects on hypothalamic regulatory functions and reproduction in both experimental animals and humans. Here we review the role of (endo)cannabinoids in the regulation of appetite and food intake. There is converging evidence that the hypothalamic endocannabinoid system changes after leptin treatment. Cannabinoid administration decreases heat production by altering hypothalamic neurotransmitter production. Experimental and human data have also shown that the endocannabinoid system is involved in the regulation of reproductive function at both central and peripheral levels. We discuss also the role of fatty acid amide hydrolase (FAAH) in gestation, and in particular the regulation of the activity of FAAH by progesterone and leptin. We show that endocannabinoids inhibit the release of leukaemia inhibitory factor (LIF) from peripheral T lymphocytes. Taken together, endocannabinoids not only help to maintain neuroendocrine homeostasis, but also take part in immunological changes occurring during early pregnancy.

In this study, we extended our previous observations on the effects of altered gravity on the "lipoxygenase pathway", by ascertain the possible role of the force of gravity in modulating the activity and expression of PHS in human erythro-leukemia K562 cells.

The mechanisms underlying cell sensitivity to the gravistimulus are still largely unknown. Recently, the oxidation of membrane lipids and the alteration of polyamine content have been shown to be involved in several cellular processes, from cell growth and differentiation, to aging and resistance to (a)biotic stress. Such an involvement was mediated by the modification of the activity and expression of lipoxygenase (LOX; E.C. 1.13.11.12) and diamine oxidase (DAO; E.C. 1.4.3.6), the enzymes which control membrane properties (LOX) and the catabolism of polyamines (DAO), respectively. In this study, the possible effects of altered gravity on membrane lipid peroxidation and polyamine metabolism were investigated, by subjecting human erythroleukemia K562 cell cultures to simulated hypogravity (by clinorotation) or hypergravity (by centrifugation).

The endocannabinoid 2-arachidonoylglycerol (2-Delta(4)Ach-Gro) activates human platelets in platelet-rich plasma at physiological concentrations. The activation was inhibited by selective antagonists of CB(1) and CB(2) cannabinoid receptors, but not by acetylsalicylic acid. Human platelets can metabolize 2-Delta(4)Ach-Gro by internalization through a high affinity transporter (K(m) = 300 +/- 30 nM, V(max) = 10 +/- 1 pmol.min(-1).mg protein(-1)), followed by hydrolysis by a fatty acid amide hydrolase (K(m) = 8 +/- 1 microM, V(max) = 400 +/- 50 pmol.min(-1).mg protein(-1)). The anandamide transport inhibitor AM404, and anandamide itself, were ineffective on 2-Delta(4)Ach-Gro uptake by platelets, whereas anandamide competitively inhibited 2-Delta(4)Ach-Gro hydrolysis (inhibition constant = 10 +/- 1 microM). Platelet activation by 2-Delta(4)Ach-Gro was paralleled by an increase of intracellular calcium and inositol-1,4,5-trisphosphate, and by a decrease of cyclic AMP. Moreover, treatment of preloaded platelet-rich plasma with 2-Delta(4)Ach-Gro induced an approximately threefold increase in [(3)H]2-Delta(4)Ach-Gro release, according to a CB receptor-dependent mechanism. On the other hand, ADP and collagen counteracted the activation of platelets by 2-Delta(4)Ach-Gro, whereas 5-hydroxytryptamine (serotonin) enhanced and extended its effects. Remarkably, ADP and collagen also reduced [(3)H]2-Delta(4)Ach-Gro release from 2-Delta(4)Ach-Gro-activated platelets, whereas 5-hydroxytryptamine further increased it. These findings suggest a so far unnoticed interplay between the peripheral endocannabinoid system and physiological platelet agonists.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.

Oxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2-induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2-induced apoptosis. On the other hand, lentil lipoxygenase products 9- and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.

The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitor Nomega-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.

Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.

The effect of ozone stress on polyamine metabolism and membrane lipid peroxidation in lentil seedlings through the amine oxidase and lipoxygenase activity and expression has been investigated. Ozone is shown to control the expression of these enzymes at the transcriptional level, down-regulating the amine oxidase gene and up-regulating the lipoxygenase gene. The decrease of amine oxidase activity correlated with the increase of putrescine concentration in the ozone-treated plantlets, whereas the increase of lipoxygenase activity was paralleled by enhanced membrane lipid peroxidation. Finally, polyamines are shown to inhibit lipoxygenase activity in lentils.

Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.

The modulation of the activity and expression of soybean lipoxygenases 1 (LOX-1) [Vliegenthart, J.F.G. and Veldink, G.A. (1982) in: Free Radicals in Biology (Pryor, W.A., Ed.) pp. 29–64, Academic Press, New York] and 2 (LOX-2) by water deficit (osmotic stress) has been investigated, by following gene expression at the transcriptional and translational levels. Osmotic stress enhanced the transcription of the genes of both isoenzymes, leading to increased levels of the corresponding mRNAs, protein contents and specific activities. Abscisic acid (ABA) did not mediate enhancement of LOX expression, but caused a decrease of LOX-2 activity and was ineffective on LOX-1. Water deficit also caused oxidative modification of soybean membrane pool lipids [Schmidt, W.E. and Ebel, J. (1987) Proc. Natl. Acad. Sci. USA 84, 4117–4121], attributable to the increase of conjugated hydroperoxides in the esterified fatty acids of the lipid bilayer.

The ability of soybean lipoxygenases-1 and -2 to oxygenate biomembranes isolated from soybean seedlings has been investigated. Constituents of the lipid bilayer were analyzed by means of reversed phase and chiral phase high performance liquid chromatography, gas chromatography/mass spectrometry, high performance thin layer chromatography and uv spectroscopy. Evidence is presented that soybean lipoxygenase-2, at variance with the type-1 enzyme, oxygenates the esterified unsaturated fatty acid moieties in biomembranes, whereas membrane-embedded free unsaturated fatty acid moieties were not a suitable substrate for either isoenzyme. The oxygenation products derived from the biomembranes were the 9- and 13-hydroperoxides of linoleic acid residues, in a molar ratio of 1.0 to 1.7, and the 9- and 13-hydroperoxides of alpha-linolenic acid residues, in a molar ratio of 1.0 to 0.1. The R/S ratios of 13-hydroperoxy-9Z,11E-octadecadienoic acid and 9-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid were found to be 0.5 and 25.0, respectively. These stereospecificity values were much higher than those of hydroperoxides isolated after incubation of lipoxygenase-2 with non-membraneous fatty acids or their methyl esters. The hydroperoxy fatty acids produced were distributed in neutral lipids and phospholipids isolated from soybean membranes, the former being oxidized to a larger extent. Furthermore, both intracellular and plasma membranes were substrates for the enzymic oxygenation, with a preference for those of chloroplasts followed by those of Golgi apparatus, endoplasmic reticulum, plasma membrane and mitochondria. These data point towards a different action of the two lipoxygenases in soybean cells. We suggest that the type-2 enzyme plays a role in the in vivo remodelling of biomembranes. The physiological relevance of these findings is discussed.

The effects of thermal injury (cold and heat shock) and ozone treatment on lipoxygenases 1 (LOX-1) and 2 (LOX-2) of soybean seedlings have been investigated. Cold stress led to a decrease of the specific activities of both isoenzymes, attributable at least in part to a down-regulation of gene expression at the translational level. Both heat shock and ozone treatment enhanced lipoxygenases-specific activities, acting at the level of transcription of the genes. It is proposed that LOX-1 and LOX-2 are involved in the thermotolerance of soybeans and in the precocious aging induced by ozone.

The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporated monoclonal antibody. Moreover, the inhibition of lipoxygenase activity was well correlated with the increase of protoplast viability.

The presence of pyrroloquinoline quinone (PQQ) as the organic cofactor of Dactylium dendroides galactose oxidase and lentil (Lens culinaris) seedling amine oxidase, purported PQQ-containing oxidoreductases (Van der Meer, R. A., Jongejan, J. A., and Duine, J. A. (1989) J. Biol. Chem. 264, 7792-7794; Citro, G., Verdina, A., Galati, R., Floris, G., Sabatini, S., and Finazzi-Argo', A. (1989) FEBS Lett. 247, 201-204), was reinvestigated using the nitro blue tetrazolium redoxcycling method (Paz, M. A., Gallop, P. M., Torrelio, B. M., and Flückiger, R. (1988) Biochem. Biophys. Res. Commun. 154, 1330-1337; Paz, M. A., Flückiger, R., Boak, A., Kagan, H. M., and Gallop, P. M. (1991) J. Biol. Chem. 266, 689-692) and the enzyme-linked immunosorbent assay with polyclonal antibodies against PQQ. The possible quinoprotein nature of the laccases from Polyporus versicolor and Rhus vernicifera was also investigated because of the similarities in spectroscopic and kinetic features of these enzymes and the laccase from Phlebia radiata, reported to be a PQQ protein (Karhunen, E., Niku-Paavola, M.-L., Viikari, L., Haltia, T., Van der Meer, R. A., and Duine, J. A. (1990) FEBS Lett. 267, 6-8). The presence of a quinonoid cofactor in lentil seedling amine oxidase is confirmed, whereas galactose oxidase and both laccases do not display any quinoprotein nature.

The effects of different light irradiations and anoxia treatment on the activity and expression of the lipoxygenases 1 (LOX-1) and 2 (LOX-2) of soybean seedlings have been investigated. A phytochrome-mediated decrease of the specific activities of both isoenzymes was found which was due to the regulation of the gene expression at the transcriptional level. The anoxia treatment of the seedlings led to a decrease of both lipoxygenase specific activities as well, but this effect was attributable to the reduction of the amount of the two isoenzymes, without changes in the steady-state levels of specific mRNAs. Therefore, light and anoxia are both regulators of soybean lipoxygenases, but their mechanism of action at the molecular level is different.

Soybean lipoxygenase-1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase-1 contains pyrroloquinoline quinone as the organic cofactor [1]. Because spectroscopie data were found to be inconsistent [2] with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase-1 is not a quinoprotein.

The activity of beta-lactamases from Citrobacter diversus ULA-27 on ceftriaxone, a widely recognized third-generation cephalosporin, has been examined and compared to the activity of various other beta-lactamases from different sources. Ceftriaxone (Roche S.p.A. Milan) was found to be resistant to hydrolysis by beta-lactamases from Enterobacter cloacae and Bacillus cereus, but susceptible to beta-lactamases from Mycobacterium fortuitum strain Cow 18 and, mostly, to beta-lactamases from various strains of Citrobacter diversus. Derivatives with substituents in the 3-position of ceftriaxone, namely cefotaxime (Roussel Maestretti S.p.A., Milan) and ceftizoxime (Farmitalia Carlo Erba, Milan), were much less susceptible to hydrolysis by C. diversus ULA-27 enzymes (22 and 6% of ceftriaxone hydrolysis, respectively), the hydrolysis rate being paralleled by differences in MIC values. Ceftriaxone inhibited the activity of E. cloacae beta-lactamases toward cefazolin as substrate, but the inhibition was totally abolished by preincubation of ceftriaxone with the enzyme before addition of the substrate. Overall, the data point to a relevance of C. diversus ULA-27 beta-lactamases in the mechanism of resistance of this strain to the various third-generation cephalosporins.