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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.

Transactivation-proficient (TA) p73 is a transcription factor belonging to the p53 family, which regulates a variety of biological processes, including neurogenesis, differentiation, apoptosis, and DNA damage checkpoint response. In the present study, we adopted multiple Omics approaches, based upon the simultaneous application of metabolomics, lipidomics, and proteomics, in order to dissect the intracellular pathways activated by p73. As cellular model, we utilized a clone of the human osteosarcoma SAOS-2 cell line that allows the expression of TAp73alpha in an inducible manner. We found that TAp73alpha promoted mitochondrial activity (accumulation of metabolic intermediates and up-regulation of proteins related to the Krebs cycle), boosted glutathione homeostasis, increased arginine-citrulline-NO metabolism, altered purine synthesis, and promoted the pentose phosphate pathway toward NADPH accumulation for reducing and biosynthetic purposes. Indeed, lipid metabolism was driven toward the accumulation and oxidation of long-chain fatty acids with pro-apoptotic potential. In parallel, the expression of TAp73alpha was accompanied by the dephosphorylation of key proteins of the mitotic spindle assembly checkpoint. In conclusion, the obtained results confirm existing evidence from transcriptomics analyses and suggest a role for TAp73alpha in the regulation of cellular metabolism, cell survival, and cell growth.

In this study, we analyzed the components of the endocannabinoid system (ECS) in R6/2 mice, a widely used model of Huntington's disease (HD). We measured the endogenous content of N-arachidonoylethanolamine and 2-arachidonoylglycerol and the activity of their biosynthetic enzymes (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol lipase, respectively) and hydrolytic enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively] and of their target receptors (type 1 cannabinoid receptor, type 2 cannabinoid receptor, and transient receptor potential vanilloid-1) in the brains of wild-type and R6/2 mice of different ages, as well as in the striatum and cortex of 12-week-old animals. In addition, we measured FAAH activity in lymphocytes of R6/2 mice. In the whole brains of 12-week-old R6/2 mice, we found reductions in N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D activity, diacylglycerol lipase activity and cannabinoid receptor binding, mostly associated with changes in the striatum but not in the cortex, as well as an increase in 2-arachidonoylglycerol content as compared with wild-type littermates, without any other change in ECS elements. Then, our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and noninduced conditions, we demonstrated a fully functional ECS. Overall, our data suggest that the ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high-throughput screening of FAAH-oriented drugs affecting HD progression.

OBJECTIVE The best treatment for moderately displaced radial head fractures (Mason type II) still remains controversial. In cases of isolated fractures, there is no evidence that a fragment displacement of ≥ 2 mm gives poor results in conservatively treated fractures. PATIENTS AND METHODS We retrospectively reviewed 52 patients (31M, 21F) affected by an isolated Mason type II fracture, treated with a long arm cast for two weeks between 2008 and 2013. All patients had practiced sports before being injured. They were all either bicyclists, or baseball, boxers, basketball, rugby, tennis or football players. The mean follow-up was 36 months. Elbow and forearm range of motion were measured. The Mayo Elbow Performance Score, the Broberg and Morrey rating system and the Disabilities of the Arm, Shoulder and Hand Score (DASH score) were analyzed. Follow-up radiographs were examined for evidence of consolidation, late displacement, early arthritis and non-unions. RESULTS Flexion was slightly impaired in the injured limb when compared to the uninjured limb (137°± 6° versus 139°±5°) as were extension (-3°±6° versus 1°±4°, p < 0.05), supination (86°±6° versus 88°±3°), pronation (87°±4° versus 88°±6°) and valgus deviation (10°±4° versus 8°±3°, p < 0.05). 40 patients had no elbow complaints; 9 patients experienced occasional pain, 2 a mild instability of the elbow, and 4 a mild loss of grip strength. The DASH score was excellent in 48 patients (92.31%). In only 6 cases (11.53%) degenerative changes were greater in formerly injured elbows than in uninjured elbows. All patients returned to their previous sports activities. CONCLUSIONS Isolated Mason type II fractures can have a good or excellent mid-term functional outcome even when treated conservatively.

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the "endocannabinoid system." Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an approximately 3 to approximately 5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.

Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-beta-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of 2-arachidonoylglycerol (2-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous 2-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated 2-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU210 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous 2-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.

The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(2) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB(2) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.

Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.

Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of ≈30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of ≈11 kcal/mol that accounted for ≈80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (ΔG ≈3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-β-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to ∼3-fold higher [35S]GTPγS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to ∼50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Anandamide (AEA) is the best-studied member of the endocannabinoids, a group of bioactive fatty acid amides and esters that activate cannabinoid receptors and have several roles in both the CNS and the periphery. The tone, signaling and activity of AEA in vivo are terminated by a specific fatty acid amide hydrolase (FAAH), whose inhibition has potential therapeutic applications in pain, neurodegenerative disorders, cancer and anxiety. In this article, we discuss the participation of AEA in hormone–cytokine networks that are essential for reproduction, and support the view that FAAH plays a key role in regulating this activity of AEA. We underline the fact that FAAH in maternal lymphocytes is a molecular integrator of signals that are crucial for human gestation, and that its downregulation to date is the only early marker of spontaneous abortion, both in vivo and in vitro . We propose that drugs that are able to enhance FAAH activity might become useful therapeutic tools for the management of human infertility.

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

Transglutaminases (TGases) are calcium-dependent enzymes that catalyse cross-linking between proteins by acyl transfer reaction; they are involved in many biological processes including coagulation, differentiation, and tissue repair. Transglutaminase 5 was originally cloned from keratinocytes, and a partial biochemical characterisation showed its involvement in skin differentiation, in parallel to TGase 1 and TGase 3. Here, we demonstrate, by electrospray tandem mass spectrometry that TGase 5 is acetylated at the N-terminal end. Moreover, in situ measurement of TGase activity shows that endogenous TGase 5 is active upon treatment with phorbol acetate, and the enzyme co-localises with vimentin intermediate filaments.

5-Lipoxygenase-mediated mitochondrial damage and apoptosis of mononuclear cells in ESRD patients. Background 5-Lipoxygenase activity is enhanced in peripheral blood mononuclear cells (PBMC) from end-stage renal disease (ESRD) patients on maintenance hemodialysis (HD), leading to lipoperoxidation and reactive oxygen species formation. These effects are prevented by vitamin E, which inhibits 5-lipoxygenase activity. The present study was designed to test the possibility that 5-lipoxygenase activation might cause mitochondrial damage and cytochrome c release, ultimately leading PBMC to apoptosis. Methods Apoptosis, mitochondrial uncoupling, and cytochrome c release were investigated in PBMC from 16 healthy volunteers and 16 ESRD patients on maintenance HD with cuprammonium rayon (CL-S) membranes in a two-step crossover study: after a four-week treatment with vitamin E-coated cuprammonium rayon (CL-E) membranes, and again after a four-week treatment with oral vitamin E. Results Compared to healthy controls, PBMC from ESRD patients showed a ∼threefold increase in mitochondrial uncoupling and cytochrome c release (within 4 and 8 hours, respectively), followed by a ∼threefold increase in apoptotic body formation (within 48 hours). Regardless of the administration route, vitamin E reduced mitochondrial uncoupling, cytochrome c release and apoptosis of mononuclear cells, as did the 5-lipoxygenase inhibitor eicosatetraynoic acid. Conversely, the cyclooxygenase inhibitor indomethacin was ineffective. Conclusions Reported data suggest that the 5-lipoxygenase branch of the arachidonate cascade is only responsible for mitochondrial disruption and apoptosis of PBMC of ESRD patients, and that vitamin E may be helpful in the control of oxidative stress-related disease in these subjects, independent of the administration route.

Lipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819-6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0-4.5 m). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable.

Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinson's disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty-acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate-mediated spontaneous excitatory post-synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6-OHDA-lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson's disease.

Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to approximately 300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (K(d)) of 1.95 +/- 0.14 nm and maximum binding (B(max)) of 392 +/- 8 fmol x mg protein(-1). Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (approximately 25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility.

An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of copper-containing amine oxidases (E.C. 1.4.3.6). The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K(M) and k(CAT) (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity. A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms. From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger. Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases. Some potential biotechnological applications are proposed. The cDNA Accession No. is AJ320485.

The human immunodeficiency virus type 1 (HIV-1) coat glycoprotein gp120 binds to its (co)receptors and orchestrates cell entry by the direct fusion of viral and target cell membranes. Here, we modulated membrane fluidity of human neuroblastoma CHP100 cells by modulating their cholesterol content, and investigated the ability of gp120 to induce cell death in comparison with the untreated cells. We show that in normal CHP100 cells gp120 induces necrosis by: (i) increased cyclooxygenase and 5-lipoxygenase activity, and metabolites generated thereof (prostaglandin E2 and leukotriene B4, respectively); (ii) increased membrane lipoperoxidation; and (iii) increased mitochondrial uncoupling. These events were triggered by a rapid increase in intracellular calcium, and in cholesterol-depleted cells engaged CXCR4 chemokine receptors. The intracellular calcium chelator EGTA-AM protected CHP100 cells almost completely against the toxic effects of gp120. However, gp120-induced necrosis and related biochemical changes were negligible in cholesterol-enriched, and significantly enhanced in cholesterol-depleted, CHP100 cells exposed to the viral glycoprotein under the same experimental conditions. Taken together, these results suggest that membrane fluidity may control the neurotoxic effects of HIV-1 glycoprotein gp120.

Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.

Endocannabinoids are an emerging class of lipid mediators, which include amides and esters of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors. Endotoxic shock is a potentially lethal failure of multiple organs that can be initiated by the inflammatory agent lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria. LPS has been recently shown to stimulate the production of AEA in rat macrophages, and of 2-AG in rat platelets. The mechanism responsible for this effect has not been elucidated. On the other hand, mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play an essential role in inflammation. As yet, little is known about endogenous modulators and mechanisms of mast cell activation. Here, we review recent literature on the role of endocannabinoids in endotoxic shock and inflammation, and report our recent research on the effects of LPS on the production of AEA and 2-AG in human lymphocytes, and on AEA degradation by a specific AEA membrane transporter (AMT) and an AEA-degrading enzyme (fatty acid amide hydrolase, FAAH). We also report the ability of the HMC-1 human mast cells to degrade AEA through a nitric oxide-sensitive AMT and a FAAH. The role of endocannabinoids in HMC-1 degranulation is discussed as well. Taken together, it can be suggested that human lymphocytes and mast cells take part in regulating the peripheral endocannabinoid system, which can affect some activities of these cells.

Human reproduction is a rather inefficient process, yet the molecular reasons for this inefficiency remain unknown. IVF and embryo transfer (IVF-embryo transfer) also results in a high frequency of implantation failures and early spontaneous abortions. Here we show that the anandamide (AEA)-degrading enzyme, fatty acid amide hydrolase (FAAH), had significantly lower activity (46 +/- 17 versus 161 +/- 74 pmol/min per mg protein) and protein content (0.10 +/- 0.03 versus 0.23 +/- 0.06 units) in lymphocytes of IVF-embryo transfer patients who failed to achieve an ongoing pregnancy than in those who become pregnant, and this was paralleled by a significant increase in blood AEA (4.0 +/- 2.2 pmol/ml and 0.9 +/- 1.0 pmol/ml respectively). The blood levels of the other endocannabinoid, 2-arachidonoylglycerol, or of the AEA congener, N-palmitoylethanolamine, which are metabolized by enzymes different from FAAH, was not different between the pregnant and nonpregnant women, nor was there any difference in the activity of the AEA membrane transporter or the amounts of cannabinoid receptors in lymphocytes. Taken together with the reported negative effects of AEA on embryo implantation, this study indicates that low FAAH activity and subsequent increased AEA levels in blood might be one of the causes of implantation failure or pregnancy loss, thereby leading to a better understanding of the pathophysiological and therapeutic implications of endocannabinoids in human fertility.

CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-month-old knockout versus wild-type mice, and even higher in 12-month-old animals. The molecular mass (≈ 67 kDa) and isoelectric point (≈ 7.6) of mouse brain FAAH were determined and the FAAH protein content was shown to parallel the enzyme activity. The kinetic constants of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amounts of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), 2-arachidonoylglycerol (5%), N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compounds were twice as abundant in the hippocampus as in the cortex. Minor differences were observed in AEA or 2-arachidonoylglycerol content in knockout versus wild-type mice, whereas the other compounds were lower in the hippocampus of knockout versus wild-type animals.

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.

Immunoelectron microscopy analysis of brain tissue sections and rat-specific sandwich ELISA allowed the localization of interleukin-1beta (IL-1beta) immunoreactivity in the mitochondria and cytosol of neocortical tissue preparations from the brain of naive, untreated, rats and rats receiving a single daily injection into one lateral cerebral ventricle (i.c.v.) of bovine serum albumin (BSA; 100 ng/day) for seven consecutive days. Interestingly, seven days i.c.v. treatment with the HIV-1 coat protein gp120 (100 ng/day) enhances IL-1beta immunoreactivity in the cellular fractions studied. Elevation of mitochondrial immunoreactive IL-1beta levels seems to originate from the conversion operated by the interleukin converting enzyme (ICE) of mitochondrial pro-IL-1beta; in fact, IL-1beta increases reported in the ELISA experiments were paralleled by a decrease of the mitochondrial pro-IL-1beta 31-kDa band in conjunction with enhanced expression of the p20 component of activated ICE. In conclusion, the present results demonstrate that gp120-enhanced neocortical expression of IL-1beta originates, at least in part, from in situ cleavage of mitochondrial pro-IL-1beta and suggest that this, together with the central role of the mitochondrion in the expression of programmed cell death, may be important for apoptosis induced by the viral coat protein in the brain of rats.

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes. At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th2 cytokines. In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the Th1 cytokines IL-12 and IFN-γ reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and 2-arachidonoylglycerol inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.

A number of pathological conditions caused by oxidative stress have been reported in uremic patientsundergoing maintenance hemodialysis (HD). Enhanced lipid peroxidation was previously observed in peripheral blood mononuclear cell's (PBMCs) of HD patients. Upregulation of 5-lipoxygenase (5-Lox) activity and protein content with enhanced production of leukotriene B 4 (LTB 4 ) and membrane lipoperoxides was also shown in PBMCs of HD patients. Administration of free vitamin E specifically inhibited 5-Lox activity without affecting gene expression at the protein level. To assess whether oral or Intramuscular (IM) administration of vitamin E may suppress 5-Lox in HD patients, PBMCs from 16 subjects on maintenance HD therapy for at least 6 months were investigated before and after a short course of IM or oral administration of vitamin E (8 patients per group). PBMCs from 13 healthy controls were also evaluated and assumed as the reference standard. Vitamin E significantly reduced lipid peroxidation, LTB 4 content, and 5-Lox activity in PBMCs, whereas 5-Lox gene expression at the protein level was not affected. There were no significant differences in these parameters between patients treated with IM or oral vitamin E. PBMCs of HD patients showed enhanced membrane lipid peroxidation and release of LTB 4 , both linked to upregulation of 5-Lox. 5-Lox activity and related oxidative stress were significantly (although not completely) suppressed by vitamin E regardless of the administration route.

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.

Endocannabinoids are lipid mediators thought to modulate central and peripheral neural functions. We report here gas chromatography-electron impact mass spectrometry analysis of human brain, showing that lipid extracts contain anandamide and 2-arachidonoylglycerol (2-AG), the most active endocannabinoids known to date. Human brain also contained the endocannabinoid-like compounds N-oleoylethanolamine, N-palmitoylethanolamine and N-stearoylethanolamine. Anandamide and 2-AG (0.16 +/- 0.05 and 0.10 +/- 0.05 nmol/mg protein, respectively) represented 7.7% and 4.8% of total endocannabinoid-like compounds, respectively. N-Palmitoyethanolamine was the most abundant (50%), followed by N-oleoyl (23.6%) and N-stearoyl (13.9%) ethanolamines. A similar composition in endocannabinoid-like compounds was found in human neuroblastoma CHP100 and lymphoma U937 cells, and also in rat brain. Remarkably, human meningioma specimens showed an approximately six-fold smaller content of all N-acylethanolamines, but not of 2-AG, and a similar decrease was observed in a human glioblastoma. These ex vivo results fully support the purported roles of endocannabinoids in the nervous system.

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.

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 effects of a single dose of the HIV-1 coat protein gp120 given into one lateral cerebral ventricle (i.c.v.) on the expression of cyclooxygenase type 2 (COX-2) and PGE(2) levels have been studied using Western blotting and ELISA techniques applied to brain tissue extracts obtained from the neocortex of individual rats, one of the regions of the central nervous system where the viral protein causes apoptosis. The results demonstrate that COX-2 expression is almost doubled 6 h after a single dose (100 ng) of gp120 and this is paralleled by a statistically significant elevation of PGE(2). Enhanced COX-2 expression is implicated in the mechanisms of apoptosis evoked by gp120 because the latter is prevented by NS398 (10 mg/kg i.p.), a selective inhibitor of COX-2 activity. Protection is also afforded by NMDA receptor antagonists, such as MK801 (0.3 mg/kg i.p.) and CGP040116 (10 mg/kg i.p.), and by the free radical scavenger, U-74389G (10 mg/kg i.p.), supporting a glutamate-mediated, excitotoxic, mechanism of apoptotic death induced by gp120. These data together with the observation that MK801 failed to prevent gp120-enhanced COX-2 expression indicate that products of the arachidonic cascade may be responsible for elevation of synaptic glutamate leading neocortical cells to oxidative stress and excitotoxic apoptosis.

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.

A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 +/- 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca2+, Mg2+, and Mn2+, whereas Cu2+, Zn2+, and Ni2+ ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (Km=200±20 nM, Vmax=25±3 pmol min−1 mg protein−1), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (Km=5.0±0.5 μM, Vmax=160±15 pmol min−1 mg protein−1) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.

The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

Anandamide (arachidonoylethanolamide, AnNH) is shown to activate human platelets, a process which was not inhibited by acetylsalicylic acid (aspirin). Unlike AnNH, hydroperoxides generated thereof by lipoxygenase activity, and the congener (13-hydroxy)linoleoylethanolamide, were unable to activate platelets, though they counteracted AnNH-mediated stimulation. On the other hand, palmitoylethanolamide neither activated human platelets nor blocked the AnNH effects. AnNH inactivation by human platelets was afforded by a high-affinity transporter, which was activated by nitric oxide-donors up to 225% of the control. The internalized AnNH could thus be hydrolyzed by a fatty acid amide hydrolase (FAAH), characterized here for the first time.

Escherichia coli JM101 cells were subjected to pore-forming electric fields, irradiation with ultraviolet light or oxidative stress by either the lipoxygenase products 9- and 13-hydroperoxyoctadecadienoic acids (9- and 13-HPOD) or hydrogen peroxide. It was found that all chemico-physical stresses enhanced ultraweak light emission from the bacterial cells, the most effective treatment being electroporation (up to 20-fold increase in luminescence compared to the control value), followed by oxidative stress with 9- or 13-HPOD (up to 4-fold increase) and irradiation with UV light (up to 2.8-fold increase). Bacterial luminescence was always in the red edge of the spectrum and was paralleled by changes in membrane oxidative index and specific activity of catalase and superoxide dismutase. © 1998 John Wiley & Sons, Ltd.

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105m−1cm−1 and 6000 m−1cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

The effect of subchronic intracerebroventricular (i.c.v.) injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100 ng, given daily for up to 7 consecutive days) on cyclooxygenase type 2 (COX-2) expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (100 ng, given i.c.v. for up to 7 days) treated animals (n = 6), a single daily injection of the viral protein for 7 consecutive days enhanced the number of COX-2 immunoreactive cells in the brain cortex of rats (n = 6 per group) and this was accompanied by a 50% increase over control PGE2 content in whole brain tissue homogenates (n = 6). In another series of experiments, pretreatment of rats (n = 6) with indomethacin (6.0 mg/kg given i.p. 1 h before gp120 injection), an inhibitor COX activity, prevented apoptotic death typically produced by gp120 in the neocortex of rat suggesting that enhancement of COX-2 expression may be involved in the mechanisms of apoptosis yielded by the HIV-1 coat protein.

Several lipoxygenase products were able to enhance ultraweak light emission and membrane permeability of human erythroleukemia K562 cells. In particular, 13-hydroperoxyoctadecadienoic acid (13-HPOD) was more effective than hydrophilic hydroperoxides, like H2O2 and tert-butyl hydroperoxide. The enhancement of luminescence induced by 13-HPOD was inhibited by superoxide and hydroxyl radical scavengers. The effect of 13-HPOD was inhibited by superoxide and hydroxyl radical scavengers. The effect of 13-HPOD was potentiated by the calcium ionophore A23187 and inhibited by the calcium chelator EDTA, and was observed also in liposomes containing unsaturated lipids. Cholesterol enrichment, which decreases the membrane fluidity, did not modify the effect of 13-HPOD on K562 cells.

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.

The transcription factor p63 is critical for many biological processes, including development and maintenance of epidermal tissues and tumorigenesis. Here, we report that the TAp63 isoforms regulate cell metabolism through the induction of the mitochondrial glutaminase 2 (GLS2) gene both in primary cells and tumor cell lines. By ChIP analysis and luciferase assay, we confirmed that TAp63 binds directly to the p53/p63 consensus DNA binding sequence within the GLS2 promoter region. Given the critical role of p63 in epidermal differentiation, we have investigated the regulation of GLS2 expression during this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes.

Transforming growth factor beta 1 (TGF beta 1) and cisplatin induce apoptosis (programmed cell death, PCD) in human erythroleukemia K562 cells in an additive manner. After PCD was induced in K562 cells, analysis of phospholipid composition, fatty acids and cholesterol content in their membranes showed a decrease in phosphatidylethanolamine and an increase in phosphatidylserine, cardiolipin and phosphatidic acid. Moreover, cisplatin but not TGF beta 1 enhanced sphingomyeline levels in apoptotic cells, whereas TGF beta 1 increased the amount of linoleic acid and, more remarkably, of cholesterol. The combination TGF beta 1 + cisplatin produced membrane changes similar to those provoked by each inducer individually. Furthermore, the specific activities of 5-lipoxygenase and cytosolic phospholipase A2, both modulating the physical properties of membranes and membrane-lipid-mediated intracellular signalling, were enhanced by treatment with TGF beta 1 or TGF beta 1 + cisplatin. These findings highlight the profound changes in cell membranes during the biochemical events of the apoptotic pathway.

Lentil seedlings contain at least six lipoxygenase isoenzymes, which are difficult to separate by classical enzyme purification techniques. The aim of this work was to study one particular lentil seedling lipoxygenase, as previous work indicated possible interesting characteristics of this enzyme with respect to oxodiene formation. Since it proved to be difficult to obtain this enzyme in significant quantities in a pure state, we expressed it in Escherichia coli. Using an expression vector based on the T7 RNA polymerase promoter (pET11d) we achieved of a fully functional lentil seedling lipoxygenase in E. coli, which was purified to homogeneity by DEAE ion-exchange chromatography and gel-filtration. The products obtained from linoleic acid were analysed. This recombinant lipoxygenase corresponds to that found in the lower part of the epicotyl and in the hypocotyl of the lentil seedling. It produces predominantly 13-(S)-hydroperoxy-(9Z,11E)-octadecadienoic and minor amounts of 9(S)-hydroperoxy-(10E,12Z)-octadecadienoic acids, as well as significant amounts of C18-oxodienes with a regiospecificity different from hydroperoxide formation. The latter mixture was found to consist of equal amounts of 13-oxo-(9Z,11E)-octadecadienoic and 9-oxo-(10E,12Z)-octadecadienoic acids. It is concluded that (1) oxodienes formed by this lentil enzyme do not originate from a secondary conversion of hydroperoxides, but rather from a different lipoxygenase-catalysed reaction and (2) this lipoxygenase shows similarities to pea lipoxygenase G, with both representing a novel type of legume lipoxygenase.

We have studied the neuropathological characteristics of the brain of rats receiving daily intracerebroventricular administration of freshly dissolved human immunodeficiency virus type 1 recombinant protein gp120 (100 ng per rat per day) given for up to 14 days. Histological examination of serial brain sections revealed no apparent gross damage to the cortex or hippocampus, nor did cell counting yield significant neuronal cell loss. However, the viral protein caused after 7 and 14 days of treatment DNA fragmentation in 10% of brain cortical neurons. Interestingly, reduced neuronal nitric oxide synthase (NOS) expression along with significant increases in nerve growth factor (NGF) were observed in the hippocampus, where gp120 did not cause neuronal damage. No changes in NGF and NOS expression were seen in the cortex, where cell death is likely to be of the apoptotic type. The present data demonstrate that gp120-induced cortical cell death is associated with the lack of increase of NGF in the cerebral cortex and suggest that the latter may be important for the expression of neuropathology in the rat brain. By contrast, enhanced levels of NGF may prevent or delay neuronal death in the hippocampus, where reduced NOS expression may be a reflection of a subcellular insult inflicted by the viral protein.