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P Giuliani

Publications and source records attributed to P Giuliani.

At least 19 recordsLinked to original sources
PubMed2005

Regional changes in the metabolite profile after long-term hypoxia-ischemia in brains of young and aged rats: a quantitative proton MRS study.

Quantitative proton magnetic resonance spectroscopy (MRS) was used to determine region-specific metabolic changes in young and aged animals subjected to a long-term hypoxic-ischemic injury. Focal ischemia, which was studied as an experimental stroke model, was induced in 3- and 24-month-old rats by unilateral common carotid artery occlusion associated with 24 h of hypoxia. Eight metabolites were quantified from extracts in three different brain regions (hippocampus, frontoparietal and occipital cortices) from both the ipsilateral and contralateral sides. Our findings showed significant differences in lactate and myo-inositol concentration values in the hippocampus of the aged rats as compared to the same area of the young adult group under normoxic conditions. After hypoxia-ischemia (HI), the most relevant changes in metabolite concentrations were found in the hippocampal region of both young and aged groups as compared to their age-matched controls. Of the three brain areas under investigation, the hippocampus proved to be particularly susceptible to the prolonged hypoxia-ischemia perturbation. The effects were more evident in the aged animals.

Age Factors↗
PubMed2001

Cascades in helical turbulence.

We suggest the existence of a characteristic inner scale xi for helicity dissipation in a regime of hydrodynamic fully developed turbulence and estimate it on dimensional grounds. This scale is always larger than the Kolmogorov scale eta and their ratio eta/xi vanishes in the high Reynolds number limit, so the flow will always be helicity free in the small scales. These ideas are illustrated in a shell model of turbulence.

Journal Article↗
PubMed2001

AIT-082 is neuroprotective against kainate-induced neuronal injury in rats.

4-[[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzoic acid (AIT-082) is an hypoxanthine derivative that stimulates in vitro neurite outgrowth and the production of adenosine and neurotrophins from astrocytes. These effects may predict an in vivo neuroprotective activity of the drug. Thus, we evaluated whether AIT-082 protected against a long-term excitotoxicity of hippocampal neurons following status epilepticus induced in rats by i.p. injection of kainate (12 mg/kg). The epileptogenic effect of kainate was evaluated by monitoring behavioral signs and by electroencephalographic (EEG) recording (80% of the animals showed status epilepticus with a latency of 96.8 +/- 7.4 min starting from the injection). In surviving rats (40% of the injected animals) the neurotoxic effect was evaluated by measuring glutamic acid decarboxylase (GAD) activity, as an index of loss of hippocampal GABAergic neurons, by evaluating the body weight after 7 days and by histological examination of hippocampi. The GAD activity was reduced by 44 +/- 8%, and neuronal loss (about 70%) was found in the CA3c, the CA1 area, and in the dentate gyrus. A single dose of diazepam (20 mg/kg; i.p., 20 min before the kainate injection) almost completely inhibited both seizures and neurotoxicity, ensuring survival of animals. AIT-082 (60 mg/kg/day; i.p., for 7 days, starting from 20 min before the kainate injection) did not modify the seizures caused by kainate but, like diazepam, it decreased kainate-induced mortality, the reduction of GAD activity, and the loss of hippocampal neurons. These data confirm that AIT-082 is of potential interest for the experimental therapy of neurodegenerative disorders.

Aminobenzoates↗
PubMed2000

Cultured astrocyte proliferation induced by extracellular guanosine involves endogenous adenosine and is raised by the co-presence of microglia.

Extracellular adenosine (Ado) and ATP stimulate astrocyte proliferation through activation of P(1) and P(2) purinoceptors. Extracellular GTP and guanosine (Guo), however, that do not bind strongly to these receptors, are more effective mitogens than ATP and Ado. Exogenous Guo, like GTP and 5'-guanosine-betagamma-imidotriphosphate (GMP-PNP), dose-dependently stimulated proliferation of rat cultured astrocytes; potency order GMP-PNP > GTP > or = Guo. The mitogenic effect of Guo was independent of the extracellular breakdown of GTP to Guo, because GMP-PNP, a GTP analogue resistant to hydrolysis, was the most mitogenic. In addition to a direct effect on astrocytes, Guo exerts its proliferative activity involving Ado. Exogenous Guo, indeed, enhanced the extracellular levels of endogenous Ado assayed by HPLC in the medium of cultured astrocytes. Culture pretreatment with Ado deaminase (ADA), that converts Ado into inosine, reduced but did not abolish Guo-induced astrocyte proliferation whereas erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), that inhibits ADA activity, amplified Guo effect. Moreover, the mitogenic activity of Guo was partly inhibited by 8-cyclopentyl-1,3-dipropylxanthine and alloxazine, antagonists of Ado A(1) and A(2B) receptors, respectively. Also microglia seem to be a target for the action of Guo. Indeed, the mitogenic effect of Guo on astrocytes was: i) increased proportionally to the number of microglial cells present in the astrocyte cultures; ii) amplified when purified cultures of astrocytes were supplemented with conditioned medium deriving from Guo-pretreated microglial cultures. These data indicate that the mitogenic effects exerted by exogenous Guo on rat astrocytes are mediated via complex mechanisms involving extracellular Ado and microglia-derived soluble factors.

Adenosine↗
PubMed1999

Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia.

Brain ischemia stimulates release from astrocytes of adenine-based purines, particularly adenosine, which is neuroprotective. Guanosine, which has trophic properties that may aid recovery following neurological damage, is present in high local concentrations for several days after focal cerebral ischemia. We investigated whether guanine-based purines, like their adenine-based counterparts, were released from astrocytes and whether their release increased following hypoxia/hypoglycemia. HPLC analysis of culture medium of rat astrocytes showed spontaneous release of endogenous guanine-based purines at a higher rate than their adenine-based counterparts. The concentration of guanosine (approximately 120 nM) and adenosine (approximately 43 nM) in the culture medium remained constant, whereas concentrations of adenine and guanine nucleotides, particularly GMP, and their metabolites increased with time. Exposure of the cultures to hypoxia/hypoglycemia for 30 min increased the extracellular concentration of adenine-based purines by 2.5-fold and of guanine-based purines by 3.5-fold. Following hypoxia/hypoglycemia extracellular adenine nucleotide levels increased further. Adenosine concentration increased, but not proportionally to nucleotide levels. Accumulation of adenosine metabolites indicated it was rapidly metabolized. Conversely, the concentrations of extracellular guanine-based nucleotides remained elevated and the concentration of guanosine continued to increase. These data indicate that astrocytes are a major source of guanine-based purines, the release of which is markedly increased following hypoxia/hypoglycemia, permitting them to exert neurotrophic effects.

Adenosine↗
PubMed1998

[Effect of A1 adenosine receptor blockade on postischemic damage to the coronary microcirculation].

Recent studies suggest that A1 adenosine receptor antagonists may prevent reperfusion injury in the lung and heart. The pathophysiology of this protective effect is unclear; a possible inhibition of superoxide anion release from neutrophils, or leukocyte activation and platelet aggregation are reported. We tested the hypothesis of a blood-independent cardioprotection following A1 adenosine receptor antagonism with 1,3 dipropyl,8-cyclopentylxanthine (DPCPX). Isolated working rat hearts were submitted to 10 and 20 min global ischemia in order to assess functional alterations, necrosis enzyme and purine release in coronary effluent, arrhythmias, heart weight, ultrastructural morphometry and microvascular permeability by FITC-albumin diffusion technique. DPCPX (100 nM) was administered to the perfusion buffer before ischemia. In untreated hearts we detected a significant impairment of function, associated with a significant enzyme and purine release, myocardial edema and ultrastructural damage. In DPCPX-treated hearts functional and histological damage was significantly reduced compared to controls. Moreover, a significant reduction in postischemic endothelial permeability (FITC-albumin diffusion, p < 0.02) and ultrastructural damage was observed. Our data suggest that A1 adenosine receptor antagonism with DPCPX significantly reduces ischemia-reperfusion damage in isolated, crystalloid perfused rat heart by a direct reduction of endothelium damage, fluid diffusion within the interstitium and improvement of coronary microcirculation.

Adenosine↗
PubMed1997

Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments. Their synthesis and purification by the isoelectric focusing technique carried out in solution.

The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoelectricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required, (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters, (iii) Very little manipulation is needed, and each focusing requires 3-5h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

Amino Acid Sequence↗
PubMed1996

Rat astroglial P2Z (P2X7) receptors regulate intracellular calcium and purine release.

Treatment of rat astrocyte cultures with 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a P2X7 agonist, but not with adenosine 5-[alpha, beta methylene] triphosphate (alpha, beta meATP), a P2X agonist, increased influx of extracellular Ca2+ and [Ca2+]i. Lucifer yellow, a small molecule which permeates P2X7 receptor-induced pores, entered BzATP-treated but not control astrocytes. BzATP also stimulated efflux of [3H]purine from cultured astrocytes. The P2X7 receptor antagonist oxidized ATP abolished the effects of BzATP on [Ca2+]i, lucifer yellow permeation and [3H]purine release, indicating that these effects were due to P2X7 receptor activation. In neurological diseases or injuries extracellular ATP may activate P2X7 receptors further enhancing [3H]purine release, with important pathophysiological consequences.

Animals↗
PubMed1996

Interaction between A1 adenosine and class II metabotropic glutamate receptors in the regulation of purine and glutamate release from rat hippocampal slices.

Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time-dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [14C]glutamate and [3H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8-cyclopentyl-1,3-dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)-alpha-methyl-4-carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2-chloro-N6-cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG-IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG-IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.

Adenosine↗
PubMed1995

Mycobacterium tuberculosis chaperonin 10 forms stable tetrameric and heptameric structures. Implications for its diverse biological activities.

The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures. We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M. tuberculosis lysate. At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric. Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected. These differences in structural assembly between species may explain differences in biological activity such as antigenicity. Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation. CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E. coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel beta-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins.

Amino Acid Sequence↗
PubMed1995

Sequence and structural homologies between M. tuberculosis chaperonin 10 and the MHC class I/II peptide binding cleft.

The peptide corresponding to the C-terminal half of M.tuberculosis hsp10 was synthesised based on the prediction that it might represent an independent structural region of the protein. This hypothesis was confirmed by aggregation and CD studies using this peptide and longer sequences of the protein. The peptide shares about 40-50% sequence homology with alpha 2 and beta 1 chains of MHC class I and II antigens. This and the CD results which indicated that the peptide at acidic pHs folds into an anti-parallel beta-sheet were used to generate a 3D model which has the same "W" fold contained in the MHC peptide binding groove. These data suggest that the hypothesis of molecular mimicry proposed to be one of the mechanisms which triggers autoimmune diseases may be extended to hsp10 proteins. Furthermore the suggested evolutionary relationship between hsp's and MHC antigens may find support from these data.

Amino Acid Sequence↗
PubMed1995

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells.

[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca(2+)-free medium + 0.5 mM ethylene glycol-bis(beta-aminoethylether)N,N,N',N'-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 microM modified [3H]purine outflow whereas 0.1 microM omega-conotoxin and 0.03-0.1 microM nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 microM omega-conotoxin + 0.1 microM nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor omega-conotoxin influenced the uptake of 45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the 45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 microM Fura-2/AM. 10-30 microM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 microM thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC, seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Protective effects of ITF 296 in the isolated rabbit heart subjected to global ischemia.

The anti-ischemic action of ITF 296 was evaluated in isovolumic, electrically driven rabbit heart preparations subjected to temporary ischemia and reperfusion. Reduction of perfusion rate from 20 to 0.2 ml/min produced a sharp decrease of peak systolic pressure, left ventricular (LV)-developed pressure, and LV dP/dt, which culminated in complete ventricular arrest within 3-4 min. Thereafter, LV end-diastolic pressure (LVEDP) progressively increased, suggesting a severe ischemic episode. Reperfusion with 20 ml Krebs solution/min after 40 min of low-flow perfusion produced only minimal recovery from the rhythm disturbances associated with cardiac mechanical activity. During reperfusion, loss of myocardial elasticity was associated with a significant increase in coronary vascular resistance, as indicated by the augmentation of coronary perfusion pressure. Injection of ITF 296 (0.3-10 microM) into the perfusion system dose-dependently inhibited the increase in LVEDP that took place during ischemia and progressively improved the recovery of a regular rhythm in the isolated heart. Isosorbide dinitrate (ISDN) at a concentration of 10 microM exerted a protective action on the myocardium similar to that obtained with ITF 296 (3 microM). Treatment with ITF 296 (1 and 10 microM) resulted in a dose-dependent increase in the rate of 6-keto-PGF1 alpha biosynthesis during both the ischemia and the reperfusion period (+157% over basal values). Similar results were obtained when hearts were pretreated with ISDN (10 microM). Infusion of a buffer containing NG-monomethyl-L-arginine (L-NMMA; NO synthase inhibitor at 10 microM) just before reduction of flow, markedly exacerbated the effects of ischemia and reperfusion on the myocardium and increased coronary perfusion pressure. The effects of L-NMMA were clearly antagonized by ITF 296 (10 microM) or L-arginine (100 microM). Mechanical activity and sinus rhythm during reperfusion were rapidly and completely restored, and coronary resistance values were close to the preischemic values. As with ISDN, ITF 296 significantly increased the rate of synthesis of 6-keto-PGF1 alpha both under basal conditions and during reperfusion.

6-Ketoprostaglandin F1 alpha↗
PubMed1995

Mechanism of ITF 296-induced vasorelaxation compared to nitroglycerin and isosorbide dinitrate: relationship between relaxation of rabbit aorta and tissue cGMP.

The vasorelaxant properties of ITF 296, a new mononitrate ester, were studied in endothelium-denuded rabbit aortic rings and were compared to nitroglycerin (NTG) and isosorbide dinitrate (ISDN). In norepinephrine-contracted arteries, ITF 296, NTG, and ISDN elicited maximal and concentration-dependent vasodilation with pD2 values of 7.07, 7.95, and 7.2, respectively. The concentration-relaxation curves of ITF 296 were shifted markedly to the right (p < 0.01) in the presence of 10 microM methylene blue (MB) and 3 microM oxyhemoglobin (HbO2), whereas a significant shift to the left (p < 0.01) was observed in the presence of 10 microM M&B-22948 (a specific cGMP phosphodiesterase inhibitor). When KCl (60 mM) was used as contracting agent, a weak relaxation was observed with ITF 296, suggesting the absence of activity on the voltage-dependent Ca2+ channels. A time-dependent increase in cGMP content and a positive correlation between cGMP and vasodilation were observed in norepinephrine-contracted arteries after exposure to a single submaximal concentration of ITF 296 (1 microM). Similar results were obtained with NTG and ISDN, although NTG was found to be more active than ITF 296 or ISDN. The presence of either MB or HbO2 almost completely abolished the increase in cGMP induced by ITF 296, whereas a further increase in cGMP was observed in the presence of isobutylmethylxanthine. No changes in cAMP levels were observed after exposure of the tissues to a concentration of ITF 296 that induced significant elevation in the cGMP content. In the presence of L-cysteine, ITF 296 stimulated semipurified rat lung guanylate cyclase at higher concentrations than those of NTG or ISDN, probably because of its lower rate of nitric oxide (NO) release. These results suggest that, in common with the reference compounds NTG and ISDN, ITF 296-induced vasorelaxation in rabbit aortic rings is mediated by an NO-cGMP mechanism.

Animals↗
PubMed1994

Effects of exogenous ATP and related analogues on the proliferation rate of dissociated primary cultures of rat astrocytes.

The effects of ATP (5-500 microM) were evaluated on the proliferation rate of cultured astrocytes by measuring 3H-thymidine incorporation and by flow cytometric analysis of the cell cycle. Determinations after 16 hours showed that ATP present in the culture medium for the whole period caused a dose-dependent reduction of cell proliferation, while if the exposure to ATP was limited to the first 8 hours, the proliferation was increased (always in a dose-dependent manner). A time course study of 3H-thymidine incorporation showed that, in the presence of ATP, 3H-thymidine was incorporated at a slower rate than in controls; the replacement of the culture medium with an ATP-free fresh medium, at the 8th hour, was followed by a 3H-thymidine incorporation occurring at such a fast rate to overshoot the control values. High performance liquid chromatography (HPLC) analysis, carried out to identify purine compounds present in the culture medium during cell exposure to ATP, indicated that more than 95% of the added ATP was metabolized within 1 hr. Conversely, an increase of purine metabolites was measured, this accumulation being greater at the highest concentrations of added ATP. The presence of high levels of extracellular ATP catabolites suggested that these compounds may act on the regulation of cell replication via the different purine receptors. This hypothesis was tested and confirmed by using agonists and antagonists selective for the P1 and the P2 sites. One hundred microM 2methylthio-ATP (2MeSATP), a P2Y agonist metabolized as fast as ATP, reproduced effects very similar to the ATP-induced ones. On the other hand, the nonhydrolisable ATP analogue, adenosine 5'-(beta, gamma-imido)-triphosphate (AMP-PNP) at 100 microM, induced a mitogenic effect as well as the A2 site stimulation. On the contrary, the activation of A1 receptors by 5 microM R-phenyl-isopropyladenosine (R-PIA) inhibited astrocyte proliferation; moreover, 100 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1 site antagonist, reversed the ATP-induced inhibition of cell proliferation. These results indicate that exogenous ATP, as a consequence of its rapid extracellular breakdown, exerts a dual influence on astrocyte proliferation by the involvement of both P1 and P2Y receptors. These findings might be relevant to such pathological conditions of the central nervous system (CNS), as seizures, hypoxia or ischemia, in which great amounts of purines released in the brain can influence a reactive astrocyte proliferative response to injury.

Adenosine Triphosphate↗
PubMed1993

TMB-8 and thapsigargin modulate purine release from dissociated primary cultures of rat brain astrocytes.

In our previous studies, the evoked purine outflow from rat brain cultured astrocytes was reported to be Na+ independent and K+ and [Ca2+]e partially dependent. Thus, the eventual [Ca2+]i influence on purine astrocyte release was investigated in an attempt to better characterize the ionic requirements of this mechanism in cells which serve many complex and still partly unknown functions within the CNS. TMB-8 and Thapsigargin (drugs described as able to inhibit and increase the ion efflux from its internal stores respectively) and BAPTA/AM (able to chelate the cytoplasmic free Ca2+), were used. TMB-8 and BAPTA/AM decreased, whereas Thapsigargin enhanced glial purine outflow. These findings suggest a significant [Ca2+]i dependence of the electrically evoked purine efflux from cultured astrocytes even though further investigations using fluorescent probes are needed.

Adenosine↗