Biomedical subjects

G Nisticò

Publications and source records attributed to G Nisticò.

At least 19 recordsLinked to original sources

PubMed2000

gp120 induces cell death in human neuroblastoma cells through the CXCR4 and CCR5 chemokine receptors.

To infect target cells, the human immunodeficiency virus (HIV) type I (HIV-1) must engage not only the well-known CD4 molecule, but it also requires one of several recently described coreceptors. In particular, the CXCR4 (LESTR/fusin) receptor allows fusion and entry of T-tropic strains of HIV, whereas CCR5 is the major coreceptor used by primary HIV-1 strains that infect macrophages and CD4(+) T-helper cells (M-tropic viruses). In addition, the alpha chemokine SDF1alpha and the beta chemokines MIP1alpha, MIP1beta, and RANTES, natural ligands of CXCR4 and CCR5, respectively, are potent soluble inhibitors of HIV infection by blocking the binding between the viral envelope glycoprotein gp120 and the coreceptors. Approximately two-thirds of individuals with acquired immunodeficiency syndrome (AIDS) show neurologic complications, which are referred to a syndrome called AIDS dementia complex or HIV-1-associated cognitive/motor complex. The HIV-1 coat glycoprotein gp120 has been proposed as the major etiologic agent for neuronal damage, mediating both direct and indirect effects on the CNS. Furthermore, recent findings showing the presence of chemokine receptors on the surface of different cell types resident in the CNS raise the possibility that the association of gp120 with these receptors may contribute to the pathogenesis of neurological dysfunction. Here, we address the possible role of alpha and beta chemokines in inhibiting gp120-mediated neurotoxicity using the human neuroblastoma CHP100 cell line as an experimental model. We have previously shown that, in CHP100 cells, picomolar concentrations of gp120 produce a significant increase in cell death, which seems to proceed through a Ca(2+) - and NMDA receptor-dependent cascade. In this study, we gained insight into the mechanism(s) of neurotoxicity elicited by the viral glycoprotein. We found that CHP100 cells constitutively express both CXCR4 and CCR5 receptors and that stimulation with phorbol 12-myristate 13-acetate down-regulates their expression, thus preventing gp120-induced cell death. Furthermore, all the natural ligands of these receptors exerted protective effects against gp120-mediated neuronal damage, although with different efficiencies. These findings, together with our previous reports, suggest that the neuronal injury observed in HIV-1 infection could be due to direct (or indirect) interactions between the viral protein gp120 and chemokine and/or NMDA receptors.

Acquired Immunodeficiency Syndrome↗

PubMed1998

The HIV envelope protein gp120 in the nervous system: interactions with nitric oxide, interleukin-1beta and nerve growth factor signalling, with pathological implications in vivo and in vitro.

The neuronal loss often described at post-mortem in the brain neocortex of patients suffering from AIDS has been proposed to be responsible for the development of the AIDS dementia complex. Neuroinvasive strains of the HIV virus infect macrophages, microglial cells, and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products known to initiate a complex network of events that may lead to the death of neurones and to the development of AIDS-associated neurological syndrome. The HIV-1 coat protein gp120, in particular, has been proposed as a likely etiologic agent of the described neuronal loss because it causes the death of neurones in culture. More recently, it has been shown that brain cortical cell death caused in rats by intracerebroventricular injection of gp120 occurs via apoptosis. This observation broadens our knowledge of the pathophysiology of the reported neuronal cell loss and opens a new avenue of experimental research for the development of novel therapeutic strategies for the treatment of patients suffering from AIDS-associated neurological syndrome.

AIDS Dementia Complex↗

PubMed1998

Cytotoxic effect of HIV-1 coat glycoprotein gp120 on human neuroblastoma CHP100 cells involves activation of the arachidonate cascade.

HIV type-1 (HIV-1) coat glycoprotein gp120 causes necrotic death in human neuroblastoma CHP100 cells. Here, we investigated the possible role of the arachidonate cascade and membrane peroxidation in gp120-induced cell necrosis. It is shown that gp120 increases the intracellular concentrations of prostaglandin E2 and leukotriene B4 by up-regulating the activity and expression of the arachidonate-metabolizing enzymes prostaglandin H synthase and 5-lipoxygenase respectively. Consistent with this observation, selective inhibitors of prostaglandin H synthase (i.e. indomethacin) and 5-lipoxygenase (i.e. MK886 and caffeic acid) protected CHP100 cells against gp120-induced necrosis. Treatment with gp120 also enhanced membrane lipid peroxidation and this may be implicated in the execution of cell damage. Interestingly, incubation with exogenous nitric oxide (NO) mimicked the effects of gp120 on necrotic death of CHP100 cells and activation of prostaglandin H synthase and 5-lipoxygenase. This suggests that NO might participate in the mechanism by which gp120 activates the arachidonate cascade.

Arachidonate 5-Lipoxygenase↗

PubMed1998

Melatonin reduces paraquat-induced genotoxicity in mice.

The protection afforded by melatonin against paraquat-induced genotoxicity in both bone marrow and peripheral blood cells of mice was tested using micronuclei as an index of induced chromosomal damage. Melatonin (2 mg/kg) or an equal volume of saline was injected i.p. into mice 30 min prior to the i.p. administration of paraquat (two injections of 15 mg/kg; the paraquat injections were given with a 24 h interval) and thereafter at 6 h intervals to the conclusion of the study (72 h). Using fluorescence microscopy, the number of micronuclei in polychromatic erythrocytes (MN-PCE) per 2000 PCE (1000 PCE/slide) per mouse was counted both in blood and bone marrow, and the ratio of PCE to normochromatic erythrocytes (NCE) (PCE/NCE) was calculated. Paraquat treatment increased the number of MN-PCE at 24, 48, and 72 h, both in peripheral blood and bone marrow cells, while no differences were observed in the PCE/NCE ratio. Melatonin inhibited the paraquat-induced increase in MN-PCE by more than 50% at 48 and 72h. Paraquat toxicity is believed to be due to free radical generation. Since melatonin is known to be an efficient free radical scavenger, it is concluded that melatonin's protection against paraquat-induced genotoxicity is mediated, at least in part, by its free radical scavenging activity.

Animals↗

PubMed1998

Systemic administration of N omega-nitro-L-arginine methyl ester and indomethacin reduces the elevation of brain PGE2 content and prevents seizures and hippocampal damage evoked by LiCl and tacrine in rat.

Administration of tacrine (5 mg/kg i.p.), an anticholinesterase agent, in rats pretreated (24 h beforehand) with lithium chloride (LiCl; 12 mEq/kg i.p.) enhances the expression of neuronal nitric oxide (NO) synthase (NOS), increases NO, and causes seizures and hippocampal damage. Here we report immunohistochemistry evidence showing that in rat LiCl and tacrine enhance the expression of cyclooxygenase type 2 (COX-2) enzyme protein in the dorsal hippocampus and elevate brain PGE2 content during the preconvulsive period. The latter effect, but not enhanced COX-2 expression, is inhibited by previous (30 min before tacrine) administration of N omega-nitro-L-arginine-methyl ester (L-NAME; 10 mg/kg i.p.), an inhibitor of NO synthesis, thus implicating NO in the mechanism of stimulation of COX activity leading to elevation of brain PGE2 content. Indomethacin (10 mg/kg given i.p. 30 min before tacrine), an inhibitor of COX activity, prevented brain PGE2 elevation and abolished the expression of seizures and hippocampal damage thus supporting a role for this metabolite of the arachidonic acid cascade in the mechanisms of LiCl and tacrine-evoked neurotoxicity in rat.

Animals↗

PubMed1998

Intrahippocampal injection of paraquat produces apoptotic cell death which is prevented by the lazaroid U74389G, in rats.

Injection of paraquat, a redox-cycling compound, into the rat hippocampus produces limbic seizures and hippocampal damage. Here we report that a proportion of the neuronal cell death caused by the herbicide occurs via an apoptotic mechanism which appears to be mediated by oxygen free radicals. Adult male Wistar rats (n=12) received a single dose of paraquat (25 nmol/0.5 microl; 0.5 microl/min rate) and were sacrificed 24 h later. Paraquat caused DNA fragmentation, nuclear chromatin marginalization and compaction in all hippocampal subsectors, 24 h after its injection, as revealed by both the TUNEL procedure and hematoxylin eosin staining of coronal brain tissue sections. Pre-treatment with the free radical scavenger lazaroid U74389G (30 mg/kg given i.p. 30 min beforehand) significantly reduced paraquat-induced apoptosis, but did not protect against non apoptotic neuronal cell loss caused by the herbicide.

Animals↗

PubMed1998

Paraquat: a useful tool for the in vivo study of mechanisms of neuronal cell death.

The present article reviews the results of experimental studies on paraquat neurotoxicity, started by our group several years ago--when clinical and experimental reports had increased the interest for the possibility that environmental chemicals, including paraquat, may be related to the development of Parkinson's disease-, and which are still continuing since paraquat appears to be a promising tool to study the mechanisms of neuronal cell death in vivo. Our observations have demonstrated that paraquat causes evident neurotoxic effects after intracerebroventricular or intracerebral injection in experimental animals; however, it seems that the herbicide does not exibit a selective neurotoxicity towards the dopaminergic nigro-striatal system since potent behavioural and electrocortical changes are induced by paraquat after injection in brain areas other than the substantia nigra and caudate nucleus. By studying the mechanisms through which paraquat induces neurotoxic effects in vivo, it was shown that either free radical production and activation of cholinergic and glutamatergic transmission may be regarded as related events which play a crucial role in paraquat-induced neurotoxicity. In addition, it was observed that in rats paraquat penetrates the blood-brain barrier following systemic administration to give rise to a differential brain regional distribution; the latter observation rises some concern over the hazard of paraquat as a potential environmental neurotoxin. Indeed, paraquat, administered systemically in rats produces behavioural excitation and brain damage. The brain damage appears to be selective for the pyriform cortex and this does not seem to be strictly related to the high concentrations reached by the herbicide in this area but to the higher vulnerability of this cortical area to the enhanced cholinergic transmission. The recent observation that paraquat, injected into the rat hippocampus, induces the expression of apoptotic neuronal cell death, appears of valuable interest also with a view to paraquat as an useful experimental model in the development of neuroprotective drugs able to block the molecular events which, once activated, are responsible for the induction of neuronal cell death.

Animals↗

PubMed1997

N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists protect from apoptosis induced in the lateral geniculate nucleus of rabbits exposed to the dark.

Mono or binocular deprivation, during early postnatal development, produces dramatic effects on the anatomy and physiology of the visual system. Here we report that dark exposure induces apoptotic cell death in the lateral geniculate nucleus (LGN) of adult rabbit and this may be related to activation of N-methyl-D-aspartate (NMDA) and non-NMDA subtypes of glutamate receptors. In situ DNA fragmentation (TUNEL) was observed in the LGN of rabbits exposed to dark for 48 h. Morphological changes were confirmed on hematoxylin-eosin stained brain tissue coronal sections. Systemic treatment with CGP 040116 or MK 801, two NMDA receptor antagonists, and with GYKI 52466, a non-NMDA receptor antagonist, prevented in situ DNA fragmentation and nuclear chromatin marginalization and condensation. In no instance was apoptosis seen in rabbits kept under a normal light-dark cycle. Our findings indicate that glutamate, acting on NMDA and non-NMDA receptors, may be involved in the mechanisms of apoptotic cell death induced in the LGN of adult rabbits exposed to darkness.

Animals↗

PubMed1997

Decrease of immunoreactive catalase protein in specific areas of ageing rat brain.

The activity of catalase, the main enzyme responsible for detoxification against hydrogen peroxide, decreases in specific brain areas of aged rats. The reduction of enzyme activity appears to be the consequence of a decreased protein expression rather than an impaired function of the native enzyme. In fact, diminution of the immunoreactive protein parallels enzyme activity decrease. Since the extent of decrease of both activity and protein content was observed to be area dependent, we hypothesise that this phenomenon may underlie, at least in part, the increased susceptibility of specific brain regions to oxidative insults observed in pathological situations related to ageing.

Aging↗

PubMed1997

The human immunodeficiency virus type 1 (HIV-1) glycoprotein gp120 reduces the expression of neuronal nitric oxide synthase in the hippocampus but not in the cerebral cortex and medial septal nucleus of rat.

The expression of neuronal type of nitric oxide synthase (nNOS) has been studied by immunocytochemistry in the brain of rats receiving a single daily (for up to 14 consecutive days) intracerebroventricular (i.c.v.) injection of the HIV-1 recombinant protein gp 120 (100 ng/day). This treatment failed to affect nNOS expression in the cerebral cortex and medial septal nucleus whereas it reduced nNOS immunopositive neurones in the CA1 hippocampal cell layer of rats treated with gp120 for 14 days; the latter effect was accompanied by a parallel decrease in Ca(2+)-dependent NOS enzyme activity in hippocampal brain tissue homogenates. In conclusion, the present data demonstrate that in rats gp120 reduces the expression of nNOS in the hippocampus, an area of the brain involved in memory formation.

Animals↗

PubMed1997

Seizures and hippocampal damage produced by dendrotoxin-K in rats is prevented by the 21-aminosteroid U-74389G.

The epileptogenic and neurodegenerative effects of dendrotoxin K (DTx-K), from Dendroaspis polylepsis, a specific blocker of a noninactivating, voltage-sensitive K+ channel, were studied after focal injection into one dorsal hippocampus in rats pretreated with the 21-aminosteriod U-74389G, a scavenger of free oxygen radicals. Administration of 35 pmol DTx-K elicited in all of the treated animals (n = 6) motor seizures and bilateral electrocortical (ECoG) discharges after a latent period of approximately 5 min. At 24 h, histological examination of brain (n = 6) coronal sections (10 microns; n = 6 per brain) detected bilateral damage to the hippocampal formation. Quantitation of damage revealed significant bilateral neuronal cell loss in the CA1 and CA4 pyramidal cell layer and dentate gyrus granule cell layer relative to the corresponding brain regions of rats (n = 6) injected with bovine serum albumin (300 ng), which per se was ineffective in all respects. DTx-K (35 pmol) also caused a significant loss of CA3 pyramidal neurons ipsilateral to the site of toxin injection. Systemic (i.p.) administration of U-74389G (5 mg/kg given 30 min beforehand) delayed the onset of motor and ECoG seizures and reduced the number of epileptogenic discharges typically observed in rats receiving an injection of DTx-K (35 pmol) alone. Similarly, this treatment prevented the damage inflicted to the hippocampus by the toxin and in no instance was significant neuronal loss observed. At variance with these results, pretreatment with U-74389G (up to 10 mg/kg i.p.) failed to prevent seizures and CA1 hippocampal damage evoked by intra-hippocampal injection of alpha-DTx (35 pmol), a DTx-K homologue which preferentially inhibits a slowly inactivating, voltage-dependent K+ conductance in nerve cells. In conclusion, the present data support a role for free oxygen radicals in mediating hippocampal damage induced by DTx-K, but not alpha-DTx, and confirm the original deduction that these DTx homologues are complementary neurobiological tools to study mechanisms of seizures and neuronal death.

Animals↗

PubMed1997

Suppressive effect of melatonin administration on ethanol-induced gastroduodenal injury in rats in vivo.

1. Melatonin protection against ethanol-induced gastroduodenal injury was investigated in duodenumligated rats. 2. Melatonin, injected i.p. 30 min before administration of 1 ml of absolute ethanol, given by gavage, significantly decreased ethanol-induced macroscopic, histological and biochemical changes in the gastroduodenal mucosa. 3. Ethanol-induced lesions were detectable as haemorrhagic streaks. Ethanol administration damaged 36% and 25% of the total gastric and duodenal surface, respectively. Melatonin treatment reduced ethanol-induced gastric and duodenal damage to 14% and 8%, respectively. When indomethacin was given together with ethanol, the gastric damaged area was 44% of the total surface, while the duodenal damaged area was 35%; melatonin administration reduced the damage to only 13% of the total gastric surface and to 12% of total duodenal surface. 4. Both stomach and duodenum of ethanol-treated animals showed polymorphonuclear leukocyte (PMN) infiltration. The number of PMN increased more than 600 and 200 times in stomach and duodenum, respectively, following ethanol administration. Melatonin treatment reduced ethanol-induced PMN infiltration by 38% in the stomach and 20% in the duodenum. In indomethacin-ethanol-treated rats, the number of PMN increased by 875% compared to control group in the stomach and by 264% in duodenum. Melatonin administration reduced the indomethacin-ethanol-induced PMN rise by 57% in the stomach and 40% in the duodenum. 5. Gastroduodenal total glutathione (tGSH) concentration and glutathione reductase (GSSG-Rd) activity were significantly reduced following ethanol and indomethacin-ethanol administration. Melatonin ameliorated both the decrease in tGSH concentration as well as the reduction of GSSG-Rd activity elicited by ethanol both in the stomach and duodenum; melatonin was effective against indomethacin-ethanol-induced damage only in the stomach. 6. Ethanol-induced gastroduodenal damage is believed to be mediated by the generation of free radicals. Recently, a number of in vivo and in vitro experiments have shown melatonin to be an effective antioxidant and free radical scavenger; thus, we conclude that the protection by melatonin against ethanol-induced gastroduodenal injury is due, at least in part, to its radical scavenging activity.

Animals↗

PubMed1997

Nitric oxide effects on cell growth: GMP-dependent stimulation of the AP-1 transcription complex and cyclic GMP-independent slowing of cell cycling.

1. The role of nitric oxide (NO) in the control of cell growth is controversial since both stimulation and (more often) inhibition have been demonstrated in various cell types. In order to reinvestigate the problem and identify the sites of NO action, we have employed murine NIH-3T3 fibroblasts overexpressing epidermal growth factor (EGF) receptors. 2. The effects of four structurally-unrelated NO donors: S-nitroso-N-acetyl penicillamine, S-nitroso-L-glutathione, 3-morpholinosydnonimine and isosorbide dinitrate (0.01-3 mM) on EGF (10 nM)-stimulated cell growth were estimated by both thymidine incorporation and the colorimetric MTT assay, while those of a messenger generated in response to NO, cyclic GMP, were revealed by the use of 8-Br cyclic GMP (0.01-3 mM) as well as of blockers of guanylyl cyclase and cyclic GMP-dependent kinase I. 3. Studies were focused on: (i) multiple signalling events, including receptor-induced tyrosine phosphorylations, phosphorylation of mitogen-activated protein kinase, activation of the AP-1 transcription complex and deoxyribonucleotide synthesis; (ii) the progression through the cell cycle, dissected out by the use of staurosporine (1 nM), lovastatin (10 microM), mimosine (200 microM), hydroxyurea (1 mM) and nocodazole (1.5 microM). 4. NO was found to have no effects on the phosphorylation events of the growth factor cascade. In contrast, later processes were modified by the messenger but with opposite effects. 5. A cyclic GMP-dependent stimulation of growth was shown to be sustained in part by the activation of the AP-1 transcription complex, while a predominant, cyclic GMP-independent inhibition was found to be mediated by both the negative regulation of ribonucleotide reductase and the marked slowing down of the cell cycle occurring at early and late G1 and during the S phase. 6. Although multiple and apparently conflicting, the effects of NO here described could work coordinately in a general programme of cell growth regulation. In particular, the cyclic GMP-dependent actions might function as rapid modulatory events, while the effects on cell cycle might operate collectively as a multi-switch process whenever growth inhibition is required.

3T3 Cells↗

PubMed1996

NMDA and HIV-1 coat protein, GP120, produce necrotic but not apoptotic cell death in human CHP100 neuroblastoma cultures via a mechanism involving calpain.

Treatment of neuroblastoma cultures with N-methyl-D-aspartate (NMDA) or human immunodeficiency virus type 1 (HIV-1) coat protein, gp120, induces significant cytotoxic effects which are reduced by leupeptin, E-64, N-Ac-Leu-Leu-norleucinal (ALLnL) as well as by N-Ac-Leu-Leu-normethioninal (ALLnM) and this suggests that activation of the Ca(2+)-dependent protease, calpain, is involved. The cell death induced by NMDA and gp120 appears to be of the necrotic type; in fact, analysis of DNA fragmentation by flow cytometry or agarose gel electrophoresis failed to demonstrate signs of apoptosis, such as the presence of apoptotic bodies or internucleosomal cleavage. Similar negative results were also obtained by studying the nuclear morphology of the cells with Hoechst 33258 staining. Altogether the data indicate that neuroblastoma cell death induced by NMDA and gp120 is of the necrotic type and this implicates calpain protease.

Calcium↗

PubMed1996

Nitric oxide enhances amino acid release from immature chick embryo retina.

Nitric oxide (NO) was investigated for its ability to induce amino acid release from immature chick retina. The production of endogenous NO by activation of NO synthase after stimulation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptor caused a significant increase in basal release of gamma-aminobutyric acid (GABA) and glutamine, whereas a more modest increase in the glutamate release was also observed. The exposure of chick retina from 9-day-old embryos to NO-generating compounds, S-nitroso-N-acetylpe-nicillamine (SNAP) and sodium nitroprusside (SNP) produced a dose dependent increase in GABA, glutamine, and glutamate release. This effect was reduced by about 80% by haemoglobin. These results indicate that NO has a stimulatory effect on amino acid release from chick embryo immature retina. However, this effect does not appear to involve a cGMP-related mechanism because 8-bromo-cGMP, a stable analogue of cGMP, failed to affect spontaneous amino acid release and because zaprinast did not enhance NMDA-stimulated release. In conclusion, our present observations may account for a role of NMDA-mediated events in the biochemical maturation under depolarizing conditions.

Amino Acids↗

PubMed1996

The HIV-1 gp120 causes ultrastructural changes typical of apoptosis in the rat cerebral cortex.

We used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) techniques to study the neuropathological effects of intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120 in rats. In brain cortical tissue sections from rats treated with a single daily dose of gp120 (100 ng day-1 for 7 or 14 consecutive days) TEM analysis showed chromatin compaction and marginalization along the inner surface of the nuclear envelope followed by masses of condensed chromatin, ultrastructural signs demonstrating the occurrence of apoptotic cell death. These effects were paralleled by in situ DNA fragmentation, as revealed by application of TUNEL technique to cryostat brain tissue sections from rats treated likewise with the viral coat protein. In no instance was apoptosis seen in the brain cortex of control rats. The present data demonstrate that gp120 given i.c.v. produces apoptosis in the neocortex of rats.

Animals↗

PubMed1996

The type 2 ryanodine receptor of neurosecretory PC12 cells is activated by cyclic ADP-ribose. Role of the nitric oxide/cGMP pathway.

Of two neurosecretory PC12 cell clones that respond to NO donors and 8-bromo-cGMP with similar increases in cADP-ribose and that possess molecularly similar Ca2+ stores, only one (clone 16A) expresses the type 2 ryanodine receptor, whereas the other (clone 27) is devoid of ryanodine receptors. In PC12-16A cells, activation of the NO/cGMP pathway induced slow [Ca2+]i responses, sustained by release from Ca2+ stores. In contrast, PC12-27 cells were insensitive to NO donors. Likewise, in PC12-16A cells preincubated with NO donors, Ca2+ stores were partially depleted, as revealed by a test with thapsigargin, whereas those in clone 27 were unchanged. The NO-induced Ca2+ release was increased synergistically by caffeine, and the corresponding store depletion was magnified by ryanodine. The specificity for the NO/cGMP pathway was confirmed by the effects of two blockers of cGMP-dependent protein kinase I, while the role of cADP-ribose was demonstrated by the effects of its antagonist, 8-amino-cADP-ribose, administered to permeabilized cells. These results demonstrate in neurosecretory cells a ryanodine receptor activation pathway similar to that known in sea urchin oocytes. The signaling events described here could be of great physiological importance, especially in the nervous system.

Adenosine Diphosphate Ribose↗

PubMed1996

Melatonin affords protection against kainate-induced in vitro lipid peroxidation in brain.

Melatonin protection against in vitro kainic acid-induced oxidative damage in rat brain is shown. Brain disrupted cell homogenates were incubated with different concentrations of kainate and with or without different concentrations of melatonin. The concentration of malonaldehyde and 4-hydroxyalkenals was measured as an index of lipid peroxidation. When administered together with kainate, melatonin produced a concentration-dependent decrease in kainate-induced lipid peroxidation ranging from 20% to 100%. Moreover, when added to the reaction mixture alone, melatonin decreased the basal level of lipid peroxidation compared to controls.

Animals↗