Giorgio Venturini

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid beta (Abeta) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt-Jakob disease, while Abeta is involved in the pathogenesis of Alzheimers disease. Synthetic fragments of both proteins, PrP106-126 and beta25-35 (beta25-35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106-126 and beta25-35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca2+]i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca2+]i concentration as observed after treatment with PrP106-126 and beta25-35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106-126 and beta25-35.

A pharmacological study of cocaine activity in planaria.

Planaria has been proposed as a suitable research model in neurobiology because of its relatively simple organization. Dopaminergic agonists induce in this flatworm typical hyperkinesias that can be antagonized by dopaminergic blocking agents. The neurochemical basis of the effects of cocaine in vertebrates has not been fully elucidated, but the inhibition of catecholamine reuptake at a presynaptic level seems to play an important role. In this study we analyzed the involvement of the dopaminergic system in the mechanism of action of cocaine in planaria. The dose-related effects of cocaine on planaria motility and the response to cocaine treatment associated with the administration of specific D1 or D2 dopamine agonists and antagonists were investigated. The effects of reuptake inhibitors on cocaine activity were also studied. Planaria specimens treated with low doses of cocaine become motionless, whereas high doses induce a typical behavioural response, identical to the response induced by specific D2 agonists. This response is inhibited by a D2 selective blocking agent. Nomifensine, a specific dopamine reuptake inhibitor, induces a mixed D1/D2 response. The results of these experiments are discussed, also in relation with the conservation of dopaminergic receptors during evolution.

Mitochondrial type I nitric oxide synthase physically interacts with cytochrome c oxidase

Nitric oxide (NO) regulates key aspects of cell metabolism through reversible inhibition of cytochrome c oxidase (CcOX), the terminal electron acceptor (complex IV) of the mitochondrial respiratory chain, in competition with oxygen. Recently, a constitutive mitochondrial NOS corresponding to a neuronal NOS-I isoform (mtNOS-I) has been identified in several tissues. The role of this enzyme might be to generate NO close enough to its target without a significant overall increase in cellular NO concentrations. An effective, selective, and specific NO action might be guaranteed further by a physical interaction between mtNOS-I and CcOX. This possibility has never been investigated. Here we demonstrate that mtNOS-I is associated with CcOX, as proven by electron microscopic immunolocalization and co-immunoprecipitation studies. By affinity chromatography, we found that association is due to physical interaction of mtNOS-I with the C-terminal peptide of the Va subunit of CcOX, which displays a consensus sequence for binding to the PDZ domain of mtNOS-I previously unreported for CcOX. The molecular details of the interaction have been analyzed by means of molecular modeling and molecular dynamics simulations. This is the first evidence of a protein-protein interaction mediated by PDZ domains involving CcOX.

Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity

Abstract Nitric oxide (NO), produced in different cell types through the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NOreactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acidbase catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2 position has been observed in some NOreactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691 696]. Here, we analyse the threedimensional structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.

Nitric oxide in invertebrates.

Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases. The presence of NO is well demonstrated in all vertebrates. The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells. In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms. Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems. All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.

Inhibition of Cysteine Protease Activity by NO-donors

Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed.

S-nitrosylation of viral proteins: Molecular bases for antiviral effect of nitric oxide

Nitric oxide (NO) is considered an important signaling molecule implied in various different physiological processes, including nervous transmission, vascular regulation, and immune defence, as well as the pathogenesis of several diseases. NO reportedly also has an antiviral effect on several DNA and RNA virus families. The NO‐mediated S‐nitrosylation of viral and host (macro)molecules appears to be an intriguing general mechanism for the control of the virus life cycle. In this respect, NO is able to nitrosylate cysteine‐containing enzymes (e.g., proteases, reverse transcriptase, and ribonucleotide reductase). Moreover, zinc‐fingers and related domains present in enzymes (e.g., HIV‐1 encoded integrase or herpes simplex virus type‐1 heterotrimeric helicase primase complex) or nucleocapsid proteins may beconsidered as NO targets. Also, NO may regulate both host (e.g., nuclear factor‐kappaB) and viral‐encoded (e.g., HIV‐1 tat protein or Epstein‐Barr virus Zta) transcriptional factors that are involved in virus replication. Finally, NO‐mediated S‐nitrosylation of cysteine‐containing glycoproteins and hemagglutinin may also occur. Here, NO targets are summarised, and the molecular bases for the antiviral effect of NO are discussed.

NO donors inhibit Leishmania infantum cysteine proteinase activity.

Nitric oxide (NO) releasing drugs (e.g., glyceryl trinitrate) were successfully used in the treatment of cutaneous leishmaniasis in man. In the present study, the effect of NO donors on the catalytic activity of the cysteine proteinase from promastigotes of Leishmania infantum, an agent of Old World visceral and cutaneous leishmaniases, is reported. In particular, one equivalent of NO, released by the NO donors S-nitrosoglutathione, glyceryl trinitrate, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, 3-morpholinosydnonimine, S-nitrosoacetylpenicillamine and sodium nitroprusside, inhibited one equivalent of the parasite cysteine proteinase. As expected, NO-deprived compounds did not affect the catalytic activity of the parasite cysteine proteinase. Furthermore, the absorption spectrum of the (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide-treated inactive L. infantum enzyme displayed a maximum in the 330-350 nm wavelength range. The reducing agents dithiothreitol and L-ascorbic acid completely prevented parasite cysteine proteinase inhibition by NO, fully restored the catalytic activity, and reversed the NO-induced absorption spectrum of the inactive enzyme. Moreover, S-nitrosoacetylpenicillamine displayed a leishmanicidal effect, inhibiting the cysteine proteinase activity in vivo. As expected, the NO-deprived compound N-acetylpenicillamine did not affect significantly the parasite viability and the enzyme activity in vivo. These data suggest that the L. infantum cysteine proteinase undergoes NO-mediated S-nitrosylation, thereby representing a possible mechanism of antiparasitic host defence.

Effects of dopaminergic agents on monoamine levels and motor behaviour in planaria

1. Dopamine, serotonin and, in lesser amounts, norepinephrine were detected in Dugesia gonocephala using electrochemical detection coupled with liquid chromatography (LCED). 2. Treatment with L-dopa induced hyperkinesias, and a rise in dopamine, serotonin and norepinephrine content, whereas reserpine reduced motor activity and the concentrations of all three monoamines. 3. Haloperidol reduced motor activity and dopamine and norepinephrine levels. 4. Apomorphine induced hyperkinesias without altering monoamine levels.

Radioimmunological and immunocytochemical demonstration of Met-enkephalin in planaria.

1. An anti-Met-enkephalin serum has been used for radioimmunological and immunocyto-chemical demonstration of Met-enkephalin in planaria. 2. The serum showed a high degree of sensitivity and specificity. 3. Radioimmunoassay demonstrated in planaria extracts a considerable concentration of Met-enkephalin. 4. Met-enkephalin was localized by immunocytochemical methods in neurons and neuropil.