In the etiology of brain injury associated to ischemia/reperfusion (I/R) and neurodegenerative diseases, a critical involvement of excessive activation of signal transducer and activator of transcription 1 (STAT1) and successive induction of iNOS expression has widely been evidenced. Any compound capable to down-regulate STAT1 activation seems to represent a new, promising anti-inflammatory drug. Among plant compounds, only a few have shown to possess anti-STAT1 activity. Among them, epigallocatechin-3-gallate (EGCG), the main polyphenol present in green tea leaves, efficiently protects heart from I/R injury and this action is strictly correlated to its anti-STAT1 property. Hyperforin, the non-polyphenolic compound present in St. John's wort, attenuates β-cell death induced by interferon-γ (IFN-γ) by strongly down-regulating STAT1 activation. STAT1, therefore, seems to represent a new molecular target of the protective treatment also against brain injury associated to a number of brain pathologies. Either understanding the molecular mechanism of anti-STAT1 action of these compounds or identification of other anti-STAT1 compounds are urged.

Arbutus unedo L. has been for a long time employed in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes. Signal transducer and activator of transcription 1 (STAT1) is a fascinating and complex protein with multiple yet contrasting transcriptional functions. Although activation of this nuclear factor is finely regulated in order to control the entire inflammatory process, its hyper-activation or time-spatially erroneous activation may lead to exacerbation of inflammation. The modulation of this nuclear factor, therefore, has recently been considered as a new strategy in the treatment of inflammatory diseases. In this study, we present data showing that the aqueous extract of Arbutus unedo's leaves exerts inhibitory action on interferon-gamma (IFN-gamma) elicited activation of STAT1, both in human breast cancer cell line MDA-MB-231 and in human fibroblasts. This down-regulation of STAT1 is shown to result from a reduced tyrosine phosphorylation of STAT1 protein. Evidence is also presented indicating that the inhibitory effect of this extract may be mediated through enhancement of tyrosine phosphorylation of SHP2 tyrosine phosphatase. The modulation of this nuclear factor turns out into the regulation of the expression of a number of genes involved in the inflammatory response such as inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1). Taken together, our results suggest that the employment of the Arbutus unedo aqueous extract is promising, at least, as an auxiliary anti-inflammatory treatment of diseases in which STAT1 plays a critical role.

Here, we show that extracorporeal shock waves (ESW), at a low energy density value, quickly increase neuronal nitric oxide synthase (nNOS) activity and basal nitric oxide (NO) production in the rat glioma cell line C6. In addition, the treatment of C6 cells with ESW reverts the decrease of nNOS activity and NO production induced by a mixture of lipopolysaccharides (LPS), interferon‐γ (IFN‐γ) plus tumour necrosis factor‐α (TNF‐α). Finally, ESW treatment efficiently downregulates NF‐κB activation and NF‐κB‐dependent gene expression, including inducible NOS and TNF‐α. The present report suggests a possible molecular mechanism of the anti‐inflammatory action of ESW treatment.

NKT cells derive from the thymus and home to the liver. Liver NKT cells can be divided in two groups: 'classical' and 'non-classical'. The first is CD1d-restricted, the second is CD1d-unrestricted. NKT cells (classical and non-classical) co-express T-cell receptor (TCR) and NK-cell marker (NK1.1), display cytotoxicity and produce IFN-gamma under IL-12 stimulation affecting, thereby, Th1 response and innate immunity. NK1.1(+)TCR alpha/beta(+) cells belong to both groups. NK1.1(+)TCR gamma/delta(+) cells belong to the second group. Anyway, both NKT cell subtypes, via IFN-gamma production, protect against viruses and bacteria from early in life. Immune variations as well as zinc rhythmicity during the circadian cycle confer the immune plasticity, which is essential for successful ageing. Liver NK1.1(+)TCR gamma/delta(+) cells, rather than TCR alpha/beta(+), from young and very old mice display 'in vitro' (under IL-12 stimulation) nocturnal peaks in cytotoxicity and IFN-gamma production. The acrophase of liver NK1.1(+)TCR gamma/delta(+) cells is present in young and very old mice, not in old. The interplay among zinc-bound metallothionein (MT)/IL-6/gp130/poly(ADP-ribose) polymerase-1 (PARP-1) may be involved in conferring plasticity to liver NK1.1(+)TCR gamma/delta(+) cells. IL-6, via sub-unit receptor gp130, induces MTmRNA. At night, gene expressions of MT, IL-6, gp130 are lower in very old mice than old and young MT-I transgenic mice (MT-I*). In very old mice, this phenomenon allows limited sequester of intracellular zinc from MT leading to good free zinc ion bioavailability for immune efficiency and zinc-dependent PARP-1 activity. Indeed (1) in vitro, high IL-6 provokes strong accumulation of MT, impaired cytotoxicity and low zinc ion bioavailability in liver NK1.1(+)TCR gamma/delta(+) cells exclusively from old and MT-I* mice. (2) The ratio total/endogen PARP-1 activity is higher in very old than in old and MT-I* mice, suggesting a higher capacity of PARP-1 in base excision DNA-repair in very old age thanks to low zinc-bound MT. Cytotoxicity and IFN-gamma production from liver NK1.1(+)TCR gamma/delta(+) cells are thus preserved leading to successful ageing. In conclusion, MT/IL-6/gp130/PARP-1 interplay may confer plasticity to liver CD1d-unrestricted NK1.1(+)TCR gamma/delta(+) cells, where MT, IL-6, gp130 are the main upstream protagonists, and PARP-1 is the main downstream protagonist in immunosenescence.

In this study, α-bisabolol, a sesquiterpene alcohol present in natural essential oil, was found to have a strong time- and dose-dependent cytotoxic effect on human and rat glioma cells. After 24h of treatment with 2.5–3.5μM α-bisabolol, the viability of these cells was reduced by 50% with respect to untreated cells. Furthermore, the viability of normal rat glial cells was not affected by treatment with α-bisabolol at the same concentrations as above. Glioma cells treated with high concentration of α-bisabolol (10μM) resulted in a 100% cell death. Judging from hypo-G1 accumulation, poly(ADP-ribose) polymerase cleavage, and DNA ladder formation, the cytotoxicity triggered by α-bisabolol resulted from apoptosis induction. Moreover, the dissipation of mitochondrial-inner transmembrane potential and the release of cytochrome c from mitochondria indicated that, in these glioma cells, apoptosis occurred through an intrinsic pathway. As pointed out by the experimental results, α-bisabolol may be considered a novel compound able to inhibit glioma cell growth and survival.

Metallothioneins (MTs) play pivotal role in zinc-related cell homeostasis because of their high affinity for this trace element which is in turn relevant against oxidative stress and for the efficiency of the entire immune system, including natural killer (NK) cell activity. In order to accomplish this role, MTs sequester and/or dispense zinc during stress and inflammation to protect cells against reactive oxygen species. MTs gene expression is affected by IL-6 for a prompt immune response. Concomitantly, MTs release zinc for the activity of antioxidant zinc-dependent enzymes, including poly(ADP-ribose)polymerase-1(PARP-1), which is involved in base excision DNA-repair. This role of MTs is peculiar in young adult-age during transient stress and inflammation, but not in ageing because stress-like condition and inflammation are persistent. This may lead MTs to turn-off from role of protection in young age to deleterious one in ageing with subsequent appearance of age-related diseases (severe infections). The aim is to study the role played by MTs/IL-6/PARP-1 interplay on NK cell activity in elderly, in old infected patients (acute and remission phases by bronchopneumonia infection) and in health nonagenarian/centenarian subjects. MTmRNA is high in lymphocytes from elderly people coupled with high IL-6, low zinc ion bioavailability, decreased NK cell activity and impaired capacity of PARP-1 in base excision DNA-repair. The same trend in this altered physiological cascade during ageing also occurs in old infected patients (both acute and remission phases) with more marked immune damage, inflammatory condition and very impaired PARP-1 in base excision DNA-repair. By contrast, centenarian subjects display low MTmRNA, good zinc ion bioavailability, satisfactory NK cell activity and higher capacity of PARP-1 in base excision DNA-repair. These findings clearly demonstrate that the sequester of zinc by MTs in ageing is deleterious because leading to low zinc ion bioavailability with subsequent impairment of PARP-1 and NK cell activity and appearance of severe infections. Physiological zinc supply (12 mg Zn(++)/day) for 1 month in elderly and in old infected patients (remission phase) restores NK cells activity with values observed in health centenarians. Therefore, the zinc ion bioavailability by zinc-bound MTs homeostasis is pivotal to reach health longevity and successful ageing.

Metallothioneins (MTs) are involved in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis, which is relevant in normal immune response. Consistent with this role, MTs gene expression (MTmRNA) is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs gene expression is affected by glucocorticoids and IL-6 for a prompt immune response. This protection is peculiar in young-adult age during transient stress and inflammatory condition, but not in ageing because stress-like condition and inflammation are constant for the whole circadian cycle. This may lead MTs to turn-off from role of protection in young age to deleterious one in ageing. The aim is to suggest MTmRNA as potential genetic marker of immunosenescence. Liver MTmRNA, IL-6 and glucocorticoids levels are high, whereas the bioavailability of zinc ions is low and natural killer cells activity is depressed in old and very old mice during the light period as compared to young in the same period. An inversion of nutritional-endocrine–immune profile exclusively occurs in young mice during the night showing the existence of immune plasticity. No inversion occurs in old mice during the night. As a consequence, no immune plasticity in old mice ensues. By contrast, very old mice remodel the altered MTmRNA and immune–endocrine profile during the night up to values of young ones observed during the light period. Therefore, the remodelling of MTmRNA may be involved in the maintenance of immune plasticity with subsequent successful ageing. Thus, MTmRNA, via IL-6 and glucocorticoids, may be potential genetic marker of immunosenescence. This assumption is reinforced by low MTmRNA in lymphocytes of nonagenarians and young-adult people in comparison with elderly and Down's syndrome subjects.

SPECIFIC AIMSEpigallocatechin-3-gallate (EGCG), the main polyphenol component of green tea, exerts inhibitory action on urokinase, metalloproteinase, and inducible nitric oxide synthase (NOS II), accounting for its antitumor and anti-inflammatory action. The aim of the present study was to identify in human cell lines the molecular target of EGCG that modulates the expression of a number of genes involved in inflammatory and neoplastic processes.PRINCIPAL FINDINGS1. EGCG blocks interferon γ (IFN-γ)-elicited activation of signal transducers and activators of transcription 1 (STAT1) by interfering with tyrosine phosphorylation in human MDA MB 231 cell lineTreatment of the estrogen nonresponsive human mammary carcinoma cell line MDA MB 231 with lipopolysaccharide (LPS)/IFN-γ/tumor necrosis factor (TNF)-α/interleukin 1β (IL-1β) (MIX) promptly induced the activation of a nuclear factor, NF-κB, that was not affected by the pretreatment with 20 μM EGCG; 20 μM EGCG did not inhibit the constitutively activated nuc...

Poly(ADP-ribose) polymerase (PARP) is activated in glutamate-induced toxicity of neurons in culture (Cosi et al., 1994). Since injection of the excitatory amino acid, kainic acid (KA) into the rat striatum induces a delayed neuronal death, the effects of this in vivo excitotoxin lesioning procedure on striatal PARP activity was investigated. PARP activity was measured in striatal extracts both in the absence ("endogenous" activity) and presence ("total" activity) of exogenously-added fragmented DNA. KA (5 nmols/1 μl) produced significant and time-dependent changes in striatal PARP activity, compared to saline-injected control animals: no changes at 6 h after intrastriatal KA, a 68% and 48% decrease in endogenous and total PARP activity respectively at 12 h, a doubling in endogenous PARP activity at 24 h, and a 382% and 60% increase in endogenous and total activities at 1 week after KA. PARP cleavage was not detected at any time point. These results suggest a participation of PARP in KA-induced toxicity in the brain in vivo.

European Journal of Heart FailureVolume 2, Issue S1 p. 8-8 AbstractsFree Access Inflammatory molecules induce arginase activity in human endothelial cells Tiziana Bachetti, Tiziana Bachetti Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorAlessandra Carcereri de Prati, Alessandra Carcereri de Prati Dept. of Neurological & Vision Sci., University of Verona, ItalySearch for more papers by this authorElisabetta Cavalieri, Elisabetta Cavalieri Dept. of Neurological & Vision Sci., University of Verona, ItalySearch for more papers by this authorLaura Comini, Laura Comini Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorDaniela Bastianon, Daniela Bastianon Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorMichela Palmieri, Michela Palmieri Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorSalvatore Curello, Salvatore Curello Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorHisanori Suzuki, Hisanori Suzuki Dept. of Neurological & Vision Sci., University of Verona, ItalySearch for more papers by this authorRoberto Ferrari, Roberto Ferrari Chair of Cardiology, University of Ferrara, ItalySearch for more papers by this author Tiziana Bachetti, Tiziana Bachetti Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorAlessandra Carcereri de Prati, Alessandra Carcereri de Prati Dept. of Neurological & Vision Sci., University of Verona, ItalySearch for more papers by this authorElisabetta Cavalieri, Elisabetta Cavalieri Dept. of Neurological & Vision Sci., University of Verona, ItalySearch for more papers by this authorLaura Comini, Laura Comini Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorDaniela Bastianon, Daniela Bastianon Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorMichela Palmieri, Michela Palmieri Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorSalvatore Curello, Salvatore Curello Cardiovascular Research Centre, Fondazione S. Maugeri, IRCCS, Gussago (Brescia), ItalySearch for more papers by this authorHisanori Suzuki, Hisanori Suzuki Dept. of Neurological & Vision Sci., University of Verona, ItalySearch for more papers by this authorRoberto Ferrari, Roberto Ferrari Chair of Cardiology, University of Ferrara, ItalySearch for more papers by this author First published: 19 May 2004 https://doi.org/10.1016/S1388-9842(00)80022-7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume2, IssueS1June 2000Pages 8-8 RelatedInformation

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-gamma (IFN gamma), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFN gamma, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFN gamma increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFN gamma-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.

Evidence for the presence of a type-III nitric oxide synthase-like protein (NOS-III-like protein) in astroglial cells is reported. The mRNA of a NOS-III-like protein is constitutively expressed in human astrocytoma T67 cells, taken as an astroglial model. The nucleotide sequence of the PCR product (422 bp) shares more than 99% identity with the cDNA (from 1588 to 2009) of the human endothelial nitric oxide synthase (NOS-III). The molecular mass of the astroglial NOS-III-like protein is about 140 kDa, as observed for human NOS-III. Moreover, the astroglial NOS-III-like protein is constitutively tyrosine-phosphorylated and associated with caveolin-1. The astroglial NOS-III-like protein is apparently inactive, as reported for phosphorylated human NOS-III associated with caveolin-1.

Previous studies have shown a role for nitric oxide (NO) as a cytotoxic effector. In the present work, two chemically different NO-donors such as glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP) were evaluated for both NO release and cytostatic/cytotoxic properties. Nitrite accumulation in the supernatant of MCF-7 and U251 cell lines indicated a greater and quickly release of NO derived from SNAP. A time-course of hemoglobin absorption spectral changes showed a greater release of NO derived from GTN in presence of cells compared to the values observed in the media, confirming that the release of NO by GTN can be enzymatic and non-enzymatic. On the contrary, SNAP generated NO without contribution of cellular components and saturated oxyhemoglobin quickly, within 2 hours. Both NO-donors inhibited thymidine incorporation in a similar manner and dose-dependently in U251 cells, but not in MCF-7 cells, where SNAP at the highest tested dose of 1000 microM induced only a 33% cytostatic effect. About trypan blue exclusion test, after 24 h GTN and SNAP, releasing similar amounts of NO, showed comparable cytotoxic effects on U251 cells (50% dead cells), but not on MCF-7 cells, where GTN resulted more cytotoxic. From our data, the "in vitro" antitumoral activity of NO-donors seems to be related to the type of tumor cell lines, to the amount and duration of NO release.

Previous results indicate that induction of inducible nitric-oxide synthase (iNOS) expression may be kept suppressed by the endogenous NO level as produced by a constitutive NOS (cNOS) enzyme. In cell types possessing both cNOS and iNOS, this may represent an evident paradox. Here, we report that lipopolysaccharide and interferon-gamma, which are able to strongly induce iNOS in astrocytoma cells, can rapidly inhibit the NO production generated by the constitutive NOS isoform, thus obtaining the best conditions for iNOS induction and resolving the apparent paradox. In fact, a 30-min treatment of T67 cells with the combination of lipopolysaccharide plus interferon-gamma (MM) strongly inhibits the cNOS activity, as determined by measuring [H-3]citrulline production. In addition, the effect of MM is also observed by measuring nitrite, the stable breakdown product of NO: a 30-min pretreatment of T67 cells with MIX is able to reduce significantly the N-methyl-D-aspartate-induced nitrite production. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a 30-min treatment of T67 cells with MM does not affect expression of mRNA coding for the neuronal NOS-I isoform. These results suggest the novel concept of a possible role of a cNOS isoform in astrocytes as a control function on iNOS induction.