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Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. Since we previously demonstrated that LIG3-dependent repair is involved in the genomic instability, drug resistance and survival of MM cells, we here investigated the biological relevance of PARP1, a driver component of Alternative-Non Homologous End Joining (Alt-NHEJ) pathway, in MM. We found a significant correlation between higher PARP1 mRNA expression and poor prognosis of MM patients. PARP1 knockdown or its pharmacological inhibition by Olaparib impaired MM cells viability in vitro and was effective against in vivo xenografts of human MM. Anti-proliferative effects induced by PARP1-inhibition were correlated to increase of DNA double-strand breaks, activation of DNA Damage Response (DDR) and finally apoptosis. Importantly, by comparing a gene expression signature of PARP inhibitors (PARPi) sensitivity to our plasma cell dyscrasia (PC) gene expression profiling (GEP), we identified a subset of MM patients which could benefit from PARP inhibitors. In particular, Gene Set Enrichment Analysis (GSEA) suggested that high MYC expression correlates to PARPi sensitivity in MM. Indeed, we identified MYC as promoter of PARP1-mediated repair in MM and, consistently, we demonstrate that cytotoxic effects induced by PARP inhibition are mostly detectable on MYC-proficient MM cells. Taken together, our findings indicate that MYC-driven MM cells are addicted to PARP1 Alt-NHEJ repair, which represents therefore a druggable target in this still incurable disease.

Liraglutide (LIR) is a novel long-lasting glucagon-like peptide-1 (GLP-1) analogue that facilitates insulin signalling and shows also neuroprotective properties in different brain disease models. In this study, we explored the potential antiepileptogenic effects of LIR in two different animal models; namely, the mouse intrahippocampal kainic acid (KA) model of temporal lobe epilepsy and the WAG/Rij rat model of absence epileptogenesis. Moreover, we evaluated LIR effects on comorbidities in various behavioural tests. Mice with kainate-induced epilepsy were treated with LIR (300 μg/kg/day s.c.) for 4 weeks after status epilepticus and then evaluated for drug effects on seizure development and behavioural alterations, whereas WAG/Rij rats were treated for 17 weeks (starting at 30 days of age, before seizure onset) with LIR (300 μg/kg/day s.c.) in order to investigate whether an early chronic treatment was able to reduce the development of absence seizures and related comorbidities. Our results indicate that LIR was effective in reducing the development of spontaneous seizures in kainate-induced epilepsy; moreover, in this model, it prevented memory impairment and related anxiety-like behaviour in the open field (OF) test while in the forced swimming test (FST), LIR displayed an apparent pro-depressant effect that was instead related to reduced endurance as confirmed by Rotarod test. In contrast, LIR was unable to modify the epileptogenic process underlying the development of absence seizures in WAG/Rij rats while being antidepressant in the FST in this strain. Our results indicate that LIR may represent a promising novel treatment to prevent and treat the epileptogenic process and its associated behavioural and cognitive alterations in some models of convulsive epilepsy characterized by neurodegeneration, since LIR effects are likely secondary to its recognized neuroprotective properties.

Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.

Exposure to environmental endocrine disruptors has been associated with an increased frequency of thyroid pathology. In this study, we evaluated the effects of various concentrations of methylmercury (MeHg) on immortalized, non-tumorigenic thyroid cells (Nthy-ori-3-1). Exposure to MeHg at 2.5 and 5 &micro, M for 24 h caused a reduction in cell viability with a decrease of the cell population in sub-G0 phase, as detected by MTT and flow cytometry. Conversely, MeHg at the lower concentration of 0.1 &micro, M increased the cell viability with a rise of G2/M phase. An immunoblot analysis showed higher expression levels of phospho-ERK and not of phospho-Akt. Further enhancement of the cell growth rate was observed after a prolonged exposure of the cells up to 18 days to MeHg 0.1 &micro, M. The present findings demonstrate the toxicity of high concentrations of MeHg on thyroid cells, while showing that treatment with lower doses of Hg, as may occur after prolonged exposure to this environmental contaminant, exerts a promoting effect on thyroid cell proliferation, by acting on the ERK-mediated pro-oncogenic signal transduction pathway.

Sodiumvalproate (VPA), an antiepileptic drug, may cause dose- and time-dependent hepatotoxicity. However, its iatrogenic molecular mechanism and the rescue therapy are disregarded. Recently, it has been demonstrated that sodium butyrate (NaB) reduces hepatic steatosis, improving respiratory capacity and mitochondrial dysfunction in obese mice. Here, we investigated the protective effect of NaB in counteracting VPA-induced hepatotoxicity using in vitro and in vivo models. Human HepG2 cells and primary rat hepatocytes were exposed to high VPA concentration and treated with NaB. Mitochondrial function, lipid metabolism, and oxidative stress were evaluated, using Seahorse analyzer, spectrophotometric, and biochemical determinations. Liver protection by NaB was also evaluated in VPA-treated epileptic WAG/Rij rats, receiving NaB for 6months. NaB prevented VPA toxicity, limiting cell oxidative and mitochondrial damage (ROS, malondialdehyde, SOD activity, mitochondrial bioenergetics), and restoring fatty acid oxidation (peroxisome proliferator-activated receptor α expression and carnitine palmitoyl-transferase activity) in HepG2 cells, primary hepatocytes, and isolated mitochondria. In vivo, NaB confirmed its activity normalizing hepatic biomarkers, fatty acid metabolism, and reducing inflammation and fibrosis induced by VPA. These data support the protective potential of NaB on VPA-induced liver injury, indicating it as valid therapeutic approach in counteracting this common side effect due to VPA chronic treatment.

The measurement of Trans-Epidermal Water Loss (TEWL) allows to evaluate the integrity of Stratum Corneum Epidermis (SCE) barrier after topical application of colloidal nanocarriers by using a non-invasive method. The temporarily modifications of SCE lipids are important for the passage of colloidal nanocarriers across the skin; this passage causes a modification of TEWL values. Niosomes, ethosomes®, and transfersomes® are used as topical drug delivery systems due to their biopharmaceutical properties, and capability to permeate intact through the SCE. In vitro and in vivo evaluation of TEWL values was studied for niosomes, ethosomes® and transfersomes® in occlusive and non-occlusive conditions. TEWL values in vivo, using healthy human volunteers, are ∼12 g/m2∙× h for all nanoformulations after 72 h, due to the rearrangement of lipids forming the SCE membranes. Conversely, TEWL values of healthy human volunteers, that are topically treated with niosomes, ethosomes® and transfersomes®, in non-occlusive conditions, are ∼20 g/m2∙× h. This data was lower than those obtained in occlusive conditions (∼35 g/m2∙× h). In vitro studies agreed results which are obtained in occlusive conditions using healthy human volunteers. SCE lipids of the skin restore their native structure after 72 h of nanocarrier application. In vitro and in vivo results showed that niosomes, ethosomes®, and transfersomes® interact with the skin in a temporary and reversible mode, and they can be used as suitable colloidal nanocarriers to increase the percutaneous permeation of drugs after topical application without damaging the native structure of the skin.

Agnese Gagliardi,1 Donatella Paolino,1 Michelangelo Iannone,2 Ernesto Palma,3 Massimo Fresta,3 Donato Cosco3 1Department of Experimental and Clinical Medicine, University “Magna Græcia” of Catanzaro, Catanzaro, Italy; 2CNR, Section of Pharmacology, Institute of Neurological Sciences, Borgia, Italy; 3Department of Health Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy Background: The use of biopolymers is increasing in drug delivery, thanks to the peculiar properties of these compounds such as their biodegradability, availability, and the possibility of modulating their physico-chemical characteristics. In particular, protein-based systems such as albumin are able to interact with many active compounds, modulating their biopharmaceutical properties. Zein is a protein of 20–40 kDa made up of many hydrophobic amino acids, generally regarded as safe (GRAS) and used as a coating material. Methods: In this investigation, zein was combined with various surfactants in order to obtain stable nanosystems by means of the nanoprecipitation technique. Specific parameters, eg, temperature, pH value, Turbiscan Stability Index, serum stability, in vitro cytotoxicity and entrapment efficiency of various model compounds were investigated, in order to identify the nanoformulation most useful for a systemic drug delivery application. Results: The use of non-ionic and ionic surfactants such as Tween 80, poloxamer 188, and sodium deoxycholate allowed us to obtain nanoparticles characterized by a mean diameter of 100–200 nm when a protein concentration of 2 mg/mL was used. The surface charge was modulated by means of the protein concentration and the nature of the stabilizer. The most suitable nanoparticle formulation to be proposed as a colloidal drug delivery system was obtained using sodium deoxycholate (1.25% w/v) because it was characterized by a narrow size distribution, a good storage stability after freeze-drying and significant feature of retaining lipophilic and hydrophilic compounds. Conclusion: The sodium deoxycholate-coated zein nanoparticles are stable biocompatible colloidal carriers to be used as useful drug delivery systems. Keywords: nanoparticles, sodium deoxycholate, stabilizers, zein

The topical administration of active compounds represents an advantageous strategy to reach the various skin components as well as its appendages. Pilosebaceous follicles are skin appendages originating in the deeper skin layers. They are very difficult to target, and hence higher active dosages are generally required to achieve effective biological responses, thus favoring the rise of side effects. The aim of this work was to design a supramolecular colloidal carrier, i.e., a liquid crystal nanocarrier, for the selective delivery of active compounds into the pilosebaceous follicle. This nanocarrier showed mean sizes of ~80 nm, a good stability, a negative surface charge, and great safety properties. In vitro studies highlighted its ability to contain and release different substances and to successfully permeate the skin. Minoxidil was encapsulated in the nanocarriers and the in vivo biological effect was compared with a conventional dosage form. Minoxidil-loaded liquid crystal nanocarrier was able to selectively reach the pilosebaceous follicle, thus allowing an increased biological effectiveness of the delivered active in terms of biological response, duration of the biological effects, and reduction of collaterals. Our investigation showed that liquid crystal nanocarriers represent a promising device for the treatment of different pilosebaceous follicular impairments/diseases.

Studies have fully demonstrated that a diet rich in fruit and vegetables may reduce the incidence of tumors. In particular, Grape Seed Extract (GSE) has been shown to carry on chemopreventive and antitumor activity thanks to the numerous beneficial substances it contains. The purpose of this work was to create a biocompatible matrix containing GSE in order to obtain microparticles able to modulate its biopharmaceutical parameters. The spray-drying technique was chosen in order to realize chitosan microparticles characterized by a mean diameter of 4–10 μm and a positive surface charge that decreased after GSE encapsulation. The evaluation of the pharmacological activity of the GSE and these GSE-loaded chitosan microparticles on different cancer cells together with CLSM investigation evidenced an increase in the antitumor effect promoted by the polysaccharide as a consequence of the enhanced cell interaction. Therefore GSE-loaded chitosan microparticles may be an innovative drug delivery system useful for the treatment of certain cancer-related diseases.

Carlotta Marianecci,1 Federica Rinaldi,1 Luisa Di Marzio,2 Marica Mastriota,3 Stefano Pieretti,3 Christian Celia,2,4 Donatella Paolino,5 Michelangelo Iannone,6,7 Massimo Fresta,5 Maria Carafa11Department of Drug Chemistry and Technologies, University Sapienza of Rome, Rome, 2Department of Pharmacy, University G d'Annunzio of Chieti of Pescara, Chieti, 3Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Rome, Italy; 4Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX, USA; 5Department of Health Sciences, University Magna Graecia of Catanzaro, University Campus S Venuta, Building of BioSciences, Germaneto, 6ARPA Calabria, Environmental Epidemiology Center, 7CNR, Neuroscience Institute, Pharmacology Section, Complesso "Nini Barbieri", Roccelletta di Borgia, ItalyBackground: Liquorice extracts demonstrate therapeutic efficacy in treating dermatitis, eczema, and psoriasis when compared with corticosteroids. In this work, nonionic surfactant vesicles (niosomes, NSVs) containing polysorbate 20 (Tween 20), cholesterol, and cholesteryl hemisuccinate at different molar concentrations were used to prepare monoammonium glycyrrhizinate (AG)-loaded NSVs. The anti-inflammatory properties of AG-loaded NSVs were investigated in murine models.Methods: The physicochemical properties of the NSVs were characterized using dynamic light scattering. The fluidity of the lipid bilayer was evaluated by measuring the fluorescence intensity of diphenylhexatriene. The drug entrapment efficiency of AG was assessed using high-performance liquid chromatography. The physicochemical stability of the NSVs was evaluated as a function of time using dynamic light scattering combined with Turbiscan Lab® Expert analysis. Serum stability was determined by incubating the NSVs with 10% v/v fetal bovine serum. The cytotoxic effects of the NSVs were investigated in human dermal fibroblasts using the Trypan blue dye exclusion assay (for cell mortality) and an MTT assay (for cell viability). Release profiles for the AG-loaded NSVs were studied in vitro using cellulose membranes. NSVs showing the most desirable physicochemical properties were selected to test for in vivo anti-inflammatory activity in murine models. The anti-inflammatory activity of the NSVs was investigated by measuring edema and nociception in mice stimulated with chemical agents.Results: NSVs showed favorable physicochemical properties for in vitro and in vivo administration. In addition, they demonstrated long-term stability based on Turbiscan Lab Expert analysis. The membrane fluidity of the NSVs was not affected by self-assembling of the surfactants into colloidal structures. Fluorescence anisotropy was found to be independent of the molar ratios of cholesteryl hemisuccinate and/or cholesterol during preparation of the NSVs. The anti-inflammatory AG drug showed no effect on the stability of the NSVs. In vivo experiments demonstrated that AG-loaded NSVs decreased edema and nociceptive responses when compared with AG alone and empty NSVs. In vitro and in vivo results demonstrated that pH sensitive and neutral NSVs show no statistical significant difference.Conclusion: NSVs were nontoxic and showed features favorable for potential administration in vivo. In addition, neutral NSVs showed signs of increased anti-inflammatory and anti-nociceptive responses when compared with AG.Keywords: niosomes, ammonium glycyrrhizinate, pH sensitivity, cytotoxicity, inflammation

BACKGROUND AND PURPOSE Tianeptine is an antidepressant affecting the glutamatergic system. In spite of its proven clinical efficacy, molecular effects of tianeptine are not entirely clear. Tianeptine modulates cytokine expression in the CNS and protects the hippocampus from chronic stress effects. HIV infection is associated with inflammation and neuronal loss, causing HIV-associated dementia (HAD). The human immunodeficiency virus type-1 glycoprotein gp120 has been proposed as a likely aetiological agent of HAD. In this study, we determined whether tianeptine protects astroglial cells from the neurodegenerative effects of gp120. EXPERIMENTAL APPROACH Human astroglial cells were treated with gp120 and tianeptine, and viability and apoptosis was monitored by TUNEL, annexin V, and activated caspase-3 staining and flow cytometry. Protein levels of glutamine synthase (GS), inducible and constitutive nitric oxide synthases (iNOS, cNOS) and nuclear factor κB (NF-κB) pathway were determined by Western blot analysis. The respective activities were assessed indirectly by measuring glutamine and nitrite concentrations or by luciferase reporter assays. KEY RESULTS Tianeptine showed an anti-apoptotic effect and prevented caspase-3 activation by gp120. The mechanism of tianeptine's action involved GS and cNOS stabilization and iNOS suppression. Moreover, tianeptine increased IκB-α levels in the absence of gp120 and blocked its degradation in response to gp120. This correlated with the suppression of basal and gp120-induced NF-κB transcriptional activity. CONCLUSIONS AND IMPLICATIONS Tianeptine clearly exerts neuroprotective effects in vitro by suppressing the molecular pro-inflammatory effects of gp120. Studies in animal models should be performed to evaluate the potential of tianeptine as a treatment for HAD.

The systemic efficacy of the chemotherapeutic agents presently used to treat solid tumors is limited by their low therapeutic index. Previously, our research group improved the in vitro antitumoral activity of gemcitabine, an anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine, entrapping it into unilamellar pegylated liposomes made up of 1,2-dipalmitoyl-snglycero-3-phosphocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio). In this work, we investigated the in vivo efficiency of the gemcitabine liposomal formulation (5mg/kg) with respect to the antitumoral commercial product GEMZAR (50mg/kg) on an anaplastic thyroid carcinoma xenograft model obtaining similar effects in terms of inhibition of tumor mass proliferation after 4weeks of treatment. The investigation of the carrier biodistribution and the drug pharmacokinetic profile furnished the rationalization of the efficacy of the vesicular system containing the active compound 10-fold less concentrated; in fact, liposomes promoted the concentration of the drug inside the tumor and they increased its plasmatic half-life. In addition, no signs of blood toxicity were observed when vesicular devices of effective doses of the drug were used.

In this manuscript we report an integrated study to develop simple choline esters as cholinergic agents potentially useful against the Alzheimer disease. In previously reported experiments we demonstrated the capability of the pivaloylcholine to exert cholinergic effects into the Central Nervous System, so we decided to explore small variants of choline esters. The knowledge of crystallographic models of the enzymes involved in the hydrolysis of the acetylcholine allowed to consider the steric compatibility of some choline derivatives within their catalytic sites. The purpose of the work was to find out analogues with increased selectivity toward the acetylcholinesterase versus the butyrrylcholinesterase. Theoretical models were compared to enzymatic tests carried out with both enzymes and two different methods. In this screening we have selected two candidates for the in vivo experiments with pre-treated rats. Their electroencephalographic profiles were recorded and averaged before and after the intraperitoneal treatment with two compounds in comparison to the pivaloyl lead ester. The results demonstrated that one of the esters can exert biological effects similar to the parent compound.

The potent herbicide paraquat and three other analogues MPP+, MPDP+ and MPTP have a known toxicological profile linked to the ability to damage dopaminergic neurons. Other biological effects were recently addressed to this class of compounds, including the ability to interact with enzymatic targets involved in the Central Nervous System, such as the acetylcholinesterase (AChE) and the butyrylcholinesterase (BuChE). A combined molecular modelling and enzymatic study focusing onto their interaction against the AChE and BuChE is reported. The former study was performed by docking techniques using target known co-crystallographic models. The latter study was carried out by the widely adopted Ellman's method. In both studies the anti-Alzheimer FDA approved drug tacrine was used as reference inhibitor. Our results indicate that paraquat, MPTP, MPDP+ and MPP+ recognize both enzymatic cleft in a similar fashion compared to the reference inhibitor. A structure-activity correlation was found with the net charge of the ligands, indicating a major role of the electrostatic term in the recognition and inhibition of these compounds. Our data completed their enzymatic profile, added new information on the molecular mechanisms underlying their neurotoxicity useful for the rational design of new cholinesterase inhibitors.

The potential therapeutic advantages of the encapsulation of idebenone within pegylated liposomes were investigated in vitro on primary cortical astrocytes of rats. In particular, both the concentration-dependent effects and the therapeutic effectiveness toward excitotoxic injury, elicited by chronic treatment with ethanol (100 microM) for 12 days, were evaluated. The following parameters were taken into consideration to assay free or liposomally entrapped idebenone: lactic dehydrogenase release, respiratory capacity measured by tetrazolium salt conversion, glutamine synthetase, and the levels of constitutive and inducible 70-kDa heat shock proteins. To evaluate the effects on astrocytes, three different drug concentrations were used (0.5 microM, 5 microM, and 50 microM). At the highest concentration used (50 microM), a toxic effect of the free and liposomally entrapped drug was observed. Toxic effects seem to be due to a cellular membrane perturbation, as demonstrated by (45)Ca(2+) permeation. The therapeutic effect of free or liposomally entrapped idebenone on ethanol-induced injury of primary cortical astrocytes was evaluated as a function of the drug concentration. The drug liposome formulation was much more effective than the free drug in counteracting the ethanol-induced damage in astrocytes, i.e., 10-times-lower doses of liposomally entrapped idebenone are able to provide a greater protective action than the free drug. The improved action of idebenone-loaded liposomes is probably due to the greater drug bioavailability at the cellular level.

Bartonella quintana, an emerging gram-negative pathogen, may cause trench fever, endocarditis, cerebral abscess and bacillary angiomatosis usually with the absence of septic shock in humans. B. quintana lipopolysaccharide (LPS), a deep rough endotoxin with strong reactivity in the limulus amebocyte lysate (LAL)-assay, was studied in human whole blood and in a rat model. A significant (P

Herbicides, including paraquat, may produce neurodegenerative effect when given both peripherally and into the brain though the pathophysiological mechanism is still unknown. Microinfusion of paraquat into the Substantia Nigra (50 mug) produced increased motor activity, jumping and circling opposite to the injection site, associated with ECoG desynchronization, high voltage epileptogenic spikes, and with neuropathological effects. These effects were accompanied by increase of malondialdehyde (MDA) levels in the Substantia Nigra, suggesting that paraquat was able to induce oxidative stress when injected directly into the rat brain. Pre-treatment of rats with M40401, a non peptidyl superoxide dismutase (SOD) mimetic given directly into the Substantia Nigra or i.p. prevented both behavioural, electrocorticogram and neuropathological effects and MDA elevation. Taken together, these results demonstrate that paraquat produces brain damage via abnormal formation of oxygen free radicals and that this effect may be counteracted by novel SOD mimetics. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Apoptosis of neurons and astrocytes has been found in patientsundergoing AIDS dementia complex. We demonstrated that supernatantsfrom human primary macrophages (M/M) infected by HIV-1 lead humanastroglial cells to oxidative stress, as shown by elevated levels of malondialdehyde, and then to apoptosis. Electron microscopy ofastrocytes shortly incubated with HIV-1-infected M/M supernatantsshowed apoptotic blebbing, cytoplasmic loss, and chromatincondensation. Apoptosis was antagonized by pretreating astrocytes withthe nonpeptidic superoxide dismutase (SOD) mimetic M40401 but notwith anti-HIV-1 compounds, thus showing that apoptosis of astrocytesdriven by HIV-1-infected M/M supernatants is mainly mediated byabnormal production of superoxide anions without relationship to HIV-1replication in such cells. Overall results support the role ofoxidative stress mediated by HIV-1-infected M/M as one of the leadingcauses of neurodegeneration in patients with HIV-1 and suggest the useof nonpeptidic SOD mimetics to counteract HIV-1-related neurologicaldisorders.

1. The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) was investigated. 2. Incubation of T 67 astroglial cell line with IL-beta (10 ng ml(-1)) and TNF-alpha (500 u ml(-1)) produced a significant (P

In previous experiments we have shown that systemic or intracerebroventricular administration of N ω -nitro- l -arginine methyl ester ( l -NAME), an inhibitor of nitric oxide (NO) synthase, is able to significantly reduce sound-evoked electrocortical (ECoG) desynchronization in rats. The present experiments were aimed at identifying the site(s) of the brain through which these effects are mediated. l -NAME (200 and 300 nmol), oxyhaemoglobin (200 and 300 nmol), a NO-trapping agent, and methylene blue (100 and 150 nmol), an inhibitor of guanylate cyclase and NO synthase, given bilaterally into the inferior colliculi, but not in other relay stations of the acoustic pathway, prevented the reduction in ECoG amplitude induced by sound stimulation in rats. Significant reduction of sound-evoked ECoG desynchronization has also been observed in rats receiving injection of CGP37849 (125 and 500 pmol) and LY274614 (125 pmol), two competitive N -methyl- d -aspartate receptor antagonists into the inferior colliculi. The present results show that the inferior colliculus represents the main site where sound-evoked ECoG desynchronization is prevented by l -NAME and provide further support for the hypothesis that NO may play a role at this level in the control of the measured response.

The behavioural, electrocortical (ECoG) and neurodegenerative effects of intrahippocampal injection of paraquat, a well-known free radical producing agent, were studied in rats. Injection of paraquat (100 nmol) into one dorsal hippocampus produced limbic motor and ECoG seizures. These effects were accompanied at 24 h by severe damage to CA1, CA3 and CA4 hippocampal pyramidal neurones and dentate gyrus granule cells. In comparison to the cell number counted in control, untreated, side of the hippocampus, significant (P0.05) neuronal loss was observed in the CA1 and CA3 pyramidal cell layers of the treated hippocampus. A lower dose of the herbicide (10 nmol) did not produce consistent motor and ECoG effects and in no instance was significant neuronal loss observed. A pretreatment with U74389F [21-4-(2,6-di-l-pyrrodinyl-4-pyridinyl)-1-piperazinyl-pregna-1,4,9 (11)triene-3,20-dione monomethansulfonate] (30 mg/kg i.p., 15 min before paraquat) completely protected rats from motor and ECoG epileptogenic effects induced by intrahippocampal paraquat (100 nmol). This dose of U74389F also reduced the hippocampal damage typically produced by paraquat and no significant neuronal loss was reported in the CA1 and CA3 pyramidal cell layers. A lower dose of U74389F (10 mg/kg i.p.) did not afford any protection against the epileptogenic effects produced by paraquat (100 nmol); in these animals hippocampal damage was still evident though neuronal loss did not reach statistical significance. In conclusion, the present data show that systemic administration of U74389F possesses neuroprotective effects against seizures and neurodegeneration typically elicited by intrahippocampal injection of paraquat.

Olive leaves extracts are a natural source of polyphenols, mainly oleuropein, widely considered to be potentially beneficial for health. This study focused on evaluation of the anti-tumoural activities of some oleuropein peracetylated derivatives, obtained with "green chemical" methodologies, against two human breast cancer cell lines. MCF-7 and T-47D cells were treated with oleuropein, peracetylated oleuropein, peracetylated aglycone and peracetylated hydroxytyrosol and the effects on growth and viability were investigated. Antioxidant effects were analysed after treatment with hydrogen peroxide. The peracetylated compounds exerted higher antiproliferative effects than oleuropein, by an arrest of cell cycle progression, associated with a strong antioxidant activity. Our results demonstrate that olive leaves, a by-product of olive manufacture, may provide a precious source of chemical derivatives, obtainable by peracetylation of oleuropein derivatives, which provide beneficial properties for human health. (C) 2011 Elsevier Ltd. All rights reserved.

The behavioural and neuropathological effects of both systemic and intrahippocampal injections of paraquat dichloride (1,1'-dimethyl 4,4'-bipyridinium dichloride) were studied in rats. Paraquat (0.1-1.0 mumol) injected into the dorsal hippocampus, produced limbic motor seizures within a few minutes of injection followed by neuronal damage in the CA1 and CA3 pyramidal cell layers, pyriform cortex, dentate granule cell layer and in the hilus fascia dentata at 24 hr (n = 9 rats). A smaller dose of paraquat (10 nmol) was ineffective. The effects of intrahippocampal injections of paraquat (1 mumol) were prevented by administering it together with atropine (50 nmol; n = 6 rats) or by giving it 60 min. after MK 801 (0.3 mg.kg-1 intraperitoneally). Systemic injections of paraquat (20-100 mg.kg-1) also produced forelimb clonus and rearing in 10 out of 15 animals. Neuronal cell death was found 24 hr later in 9 of these rats and was restricted to the pyriform cortex, the brain region with the highest concentrations of paraquat. Atropine (150 mg.kg-1 intraperitoneally given 60 min. previously) completely prevented the motor seizures but cell death still occurred in 2 of the 6 animals tested. In conclusion, both systemic and intrahippocampal injections of paraquat produced behavioural excitation accompanied 24 hr later by brain damage and antagonist studies suggested involvement of muscarinic and NMDA receptors in the neurotoxic mechanism.

The effects of tacrine (5 mg/kg i.p.), a potent acetylcholinesterase inhibitor, were studied in rats pretreated (24 h beforehand) with a single dose (12 mEq/kg i.p.) of LiCl. Tacrine and LiCl were ineffective when given individually. Tacrine elicited seizures and brain damage in 90% of the rats treated. The intracerebroventricular microinfusion of N omega-nitro-L-arginine methyl ester (300 micrograms given 24 h after LiCl administration) significantly reduced the seizures and brain damage produced by tacrine (given 15 min later). These experiments suggest that the seizures and brain damage elicited by tacrine may be due, in part, to increased nitric oxide production in the brain.

The neuropathological effects of various doses of paraquat, a widely used herbicide, given directly into different areas of the rat brain, were studied. Paraquat, microinfused into the pars compacta of the substantia nigra (3.2, 16, 32 and 160 nmol), i.e. concentrations of 3.2 to 160 mmol l-1 applied at 1 microliter min-1 for 1 min, produced dose-dependent neuropathological lesions culminating in neuronal necrosis. A particular feature of paraquat neurotoxicity after its microinfusion into the substantia nigra (3.2 mmol per 1 at 1 microliter per min for 1 min) or into the ventral tegmental area (1.6 mmol per 1 at 1 microliter per min for 1 min), but not into other areas of the brain, was a selective vulnerability of hippocampal CA3 neurones consisting initially of a significant decrease of dendritic spines and later in neuronal degeneration and cell loss. No damage occurred after microinfusion of paraquat into other areas of the brain near or distant from the infusion sites. In addition, similar neuropathological alterations occurred in other non-dopaminergic areas, such as the locus coeruleus and some raphe nuclei after direct microinfusion of paraquat into these sites. In conclusion, the above neuropathological findings show that paraquat possesses marked neurotoxicity which, despite its chemical similarity to MPTP, is not selective for dopaminergic neurones.

The effects of lipid composition and preparation conditions on the physicochemical and technological properties of gemcitabine-loaded liposomes, as well as the in vitro anti-tumoral activity of various liposome formulations were investigated. Three liposome formulations were investigated: DPPC/Chol/Oleic acid (8:3:1 molar ratio, liposomes A), DPPC/Chol/DPPS (6:3:1 molar ratio, liposomes B) and DPPC/Chol/DSPE-MPEG (6:3:1 molar ratio, liposomes C). Multilamellar liposomes were prepared by using the TLE, FAT and DRV methods, while small unilamellar liposomes were obtained by extrusion through polycarbonate filters. Light scattering techniques were used to characterize liposome formulations. Loading capacity and release profiles of gemcitabine from various liposome formulations were also investigated. Caco-2 cells were used to evaluate in vitro the antitumoral activity of gemcitabine-loaded liposomes with respect to the free drug and also the intracellular drug uptake. Preparation methods and liposome lipid composition influenced both physicochemical parameters and drug delivery features. Liposomes with a size ranging from 200 nm to 7 microm were obtained. The gemcitabine entrapment was higher than that expected probably due to an interaction with the liposome lipid components. The following decreasing loading capacity order was observed: liposome B>liposome C>liposome A. Gemcitabine release from various liposome formulations is modulated by two different processes, i.e. desorption from and permeation through liposomal bilayers. MTT assay showed a greater cytotoxic effect of gemcitabine-loaded liposomes with respect to the free drug. The following decreasing anticancer activity order was observed between the various liposome formulations: liposome C>liposome A>liposome B. The increased anticancer activity is correlated to the ability of the colloidal carrier to increase the intracellular drug uptake. Due to the encouraging results and to the high liposome modularity various applications of potential therapeutic relevance can be envisaged for liposomes.

The aim of this study was to assess the effects of dexamethasone (DEX) on the inducible bioconversion of glyceryl trinitrate (GTN) into nitric oxide in cultured smooth muscle cells, endothelial cells, and the J774 macrophage cell line as well as in vivo and ex vivo in rats either untreated or pretreated with Escherichia coli lipopolysaccharide. In vitro, an increased bioconversion of GTN to nitrite and an elevation of cyclosine guanosine 3,5;-monophosphate (cGMP) levels occurred after treatment with lipopolysaccharide (LPS) (0.5 microg/ml, 18 h). This effect was ablated by co-incubation with DEX (10 microM, 18 h). Rats treated with an intraperitoneal (IP) injection of LPS (4 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (1 mg/kg, intravenously [IV]) and this was prevented when DEX (4 mg/kg, IP) was given together with LPS. Progesterone (50 mg/kg, IP) had no effect on GTN-induced hypotensive response. Conversely, exposure of rat aortic strips obtained from animals pretreated with LPS produced an enhanced vasorelaxant response in LPS- treated rats. Also, this effect was inhibited by pretreatment with DEX. Thus, the induction of the pathway leading to the formation of nitric oxide from GTN is blocked by DEX both in vitro and in vivo, and this may represent a useful tool in the assessment of the enhanced bioconversion of organic nitrates into nitric oxide occurring via inflammatory mechanisms.

In rats chronically implanted with cannulae into one lateral cerebral ventricle and recording electrodes onto the fronto-parietal cortex, the effects of systemic or intraventricular administration of the nitric oxide (NO) synthesis inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), on electrocortical (ECoG) arousal response evoked by sound stimulation were studied. In control animals, a single acoustic stimulation (80 dB for 15 s) produced a significant decrease in ECoG total voltage power lasting approximately 25 s. No tolerance developed after repeating the same sound stimulation at 15, 30, 60 min and 24 h intervals. Under these experimental conditions, pretreatment with L-NAME, given systemically (10 mg kg-1, i.p.) or intracerebroventricularly (300 micrograms), significantly reduced the sound-evoked arousal response 1 h and 15 min later, respectively. In conclusion, the present data are in favour of a physiological role of NO in the control of arousal mechanisms.

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.

Nitric oxide may influence pathophysiology of brain ischemia in a complex way depending on the sources of its production either from neurons or endothelial cells. We investigated whether inhibition of nitric oxide synthesis affects postischemic neuronal death in hippocampus. Moreover, we evaluated whether the presence of nitric oxide synthase activity in specific neurons protects these against ischemia in the hippocampus, striatum, and sensorimotor cortex. To inhibit nitric oxide synthase, several dosing regimens of NG-nitro-L-arginine methyl ester (L-NAME) were used (5 or 50 mg/kg IP, twice a day for 4 days, or 30 mg/kg IV) in gerbils. Control animals received either the isomer NG-nitro-D-arginine methyl ester or the vehicle. The gerbils underwent 10-minute occlusion of carotid arteries under ether anesthesia and controlled body temperature while physiological parameters were monitored. Neuronal damage was assessed 5 days after ischemia using Nissl-stained sections of hippocampus. Nitric oxide synthase neurons were histochemically stained for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity. L-NAME treatments, but not the chronic one at 5 mg/kg, induced elevation of blood pressure (30% to 80% greater than the control level, P < .01), as observed shortly before and after bilateral carotid occlusion. Postischemic neuronal loss in the CA1 through CA4 sectors was worsened by chronic pretreatment with L-NAME at 50 mg/kg (eg, CA1 neuronal counts per 100-microns length: 3.2 +/- 2.74, mean +/- SD; n = 19; P < .01). After the acute (30 mg/kg) or chronic pretreatment at lower dosage (5 mg/kg) with L-NAME, neuronal loss was comparable to that of animals treated with the D-isomer or the vehicle (CA1 counts in vehicle-treated animals: 7.65 +/- 6.51, mean +/- SD; n = 14). None of the L-NAME treatments affected postischemic survival of NADPH diaphorase-positive neurons in hippocampus, striatum, and sensorimotor cortex. These observations demonstrate that inhibition of endothelial and neuronal nitric oxide synthase activity does not modify resistance of nitric oxide-producing neurons to transient ischemia. The severe inhibition of nitric oxide production aggravates postischemic neuronal death in the hippocampus, whereas the mild inhibition is ineffective.

The effects of ageing on the activity of copper-zinc superoxide dismutase (SOD), selenium-dependent and independent glutathione peroxidase (GSH-Px) and catalase in several areas of the brain in 3-, 12-, and 24-month-old rats were studied. In addition, the effects of a subacute intracerebroventricular treatment of NGF (1 microgram daily for 28 consecutive days) on SOD, GSH-Px, and catalase activity in the same areas of the brain were assessed. The effects of ageing on the activities of antioxidant enzymes varied considerably in the different brain areas studied. Copper-zinc SOD was alone in being unaffected by ageing. Intraventricular infusion of NGF significantly increased SOD activity in the prefrontal cortex, hypothalamus, caudate nucleus, and mesencephalon of 24-month-old rats. Selenium-dependent GSH-Px activity did not significantly change in 12-month-old rats but it increased in the lower brain stem of 24-month-old animals. In comparison to vehicle-treated rats, NGF significantly increased selenium-dependent GSH-Px activity in all brain areas studied in 12- and 24-month-old rats. Catalase activity decreased significantly in the majority of the brain areas studied in 12- and 24-month-old rats. NGF completely restored the fall in catalase activity in 12- and 24-month-old animals to levels similar to those occurring in young rats. In conclusion, the present experiments show, for the first time, that long-term intraventricular administration of NGF significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of superoxide radicals and hydrogen peroxide.

The effects of subchronic administration of nerve growth factor (NGF) into the lateral ventricle on catalase and selenium-dependent glutathione-peroxidase (GSH-Px) activity in several areas of the brain in 3-, 12- and 24-month-old rats were studied. NGF given daily (1 microgram for 28 consecutive days) produced in all brain areas studied a significant increase in catalase activity in 12- and 24-month-old rats. The most important finding was a complete restoration in 12- and 24-month-old rats of catalase activity to levels similar to those occurring in young (3-month-old) rats. In addition, NGF produced in comparison to 3-month-old rats and to same age vehicle-treated rats a significant increase in selenium-dependent GSH-Px in all the brain areas studied in 12- and 24-month-old animals, whereas selenium-independent GSH-Px was unaffected. In conclusion, the present results show that long-term administration of NGF into the lateral ventricle significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of hydrogen peroxide.

In adult rats, with cannulae chronically implanted by a stereotactie instrument into the substantia nigra (pars compacta), the electrocortical (ECoG) and behavioural effects elicited by intranigral infusion of paraquat and the prevention of these effects by prior administration into the same site of different types of superoxide dismutase, were studied. Paraquat (50 μg) produced an intense pattern of behavioural stimulation, contralateral circling and repetitive discharges of high voltage ECoG spikes. The effects of paraquat were abolished in all of the animals pretreated into the same site with copper-free superoxide dismutase. Pretreatment with native Cu, Zn-superoxide dismutase prolonged significantly the latency of onset but did not prevent the behavioural stimulation and ECoG spikes evoked by paraquat. On the contrary, pretreatment with albumin or saline did not confer any protection against the neurotoxicological changes induced by paraquat. In conclusion, the present experiments showed that motor, ECoG and lethal effects of paraquat were completely prevented by Cu-free Superoxide dismutase, suggesting that the central effects of this herbicide are in some way related to the release in the brain of copper and/or other transition metal ions.

The normal serum and tissue levels of many micronutrients are remarkably affected by the therapeutical administration of several substances. In the present study, we have evaluated the modifications of serum selenium levels in different groups of patients under corticosteroid treatment. Such therapy was significantly associated with increased serum selenium levels, with a dose-dependent relationship in subjects treated with methylprednisolone. The reasons for this association are unknown. However, a reduced renal excretion of selenium, due to the mineralcorticoid activity of the corticosteroids, can be inferred.

Overproduction of free radical species has been shown to occur in brain tissues after ischemia-reperfusion injury. However, most of free radical scavengers known to antagonize oxidative damage (e.g. superoxide dismutase, catalase), are unable to protect against ischemia-reperfusion brain injury when given in vivo, an effect mainly due to their difficulty to gain access to brain tissues. Here we studied the effect of a low molecular weight superoxide dismutase mimetic (M40401) in brain damage subsequent to ischemia-reperfusion injury in Mongolian gerbils. In animals undergoing ischemia-reperfusion injury, neuropathological and ultrastructural changes were monitored for 1–7 days either in the presence or in the absence of M40401 after bilateral common carotid artery occlusion (BCCO). Administration of M40401 (1–40 mg/kg, given i.p. 1 h after BCCO) protected against post-ischemic, ultrastructural and neuropathological changes occurring within the hippocampal CA1 area. The protective effect of M40401 was associated with a significant reduction of the levels of malondialdehyde (MDA; a marker of lipid peroxidation) in ischemic brain tissues after ischemia-reperfusion. Taken together, these results demonstrate that M40401 provides protective effects when given early after the induction of ischemia-reperfusion of brain tissues and suggest the possible use of such compounds in the treatment of neurological dysfunction subsequent to cerebral flow disturbances.

A Ca2�-dependent, transglutaminase-like activity has been detected both free in the human seminal plasma and bound on the spermatozoon surface. A marked variability of the two enzymatic activities in the semen of different normal subjects was observed; but limited changes occurred in various ejaculates of the same in dividual. Moreover, we report evidence of the ability of several seminal plasma proteins to act as acyl donor substrates for endogenous transglutaminase, whereas human ejaculated spermatozoa have been shown to possess polyamine-binding sites specifically involved in transglutaminase-catalyzed reactions. It is postulated that semen transglutaminase may play a role in suppressing sperm antigenicity and in the male gamete’s acquiring biological features of a fully differentiated and fertile cell.

Behavioural, ECoG and neuropathological effects after intracerebral injection of MPP+ and paraquat in rats