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Cysteine (Cys) residues, especially those located on protein surface, are considered to be important for metal coordination, catalysis, protein structure by forming disulfide bonds and protein function regulation. Crucial Cys residues can be involved in the modulation of protein activity and signalling events via reactions of their thiol groups. These reactions can take several forms, such as redox events, chelation of transition metals or S-nitrosylation. In several cases, these disparate reactions can compete with each other for the same thiol group on a single Cys residue, forming a molecular switch composed of possible redox, NO or Zn(2+) modifications to control protein function. This work investigates the basis for these molecular Cys switches in spermine oxidase (SMO), an enzyme involved in the polyamine homeostasis of animal cells, which oxidizes spermine (Spm) to produce spermidine (Spd), 3-aminopropanal and H2O2. The murine SMO enzyme contains 14 Cys (correspond to 2.5% amino acid content), mainly localized on the protein surface. We carried out an in vitro chemical modifications of Cys residues to analyze their effect on the biochemical features of SMOWT (containing 14 Cys) and SMOC263-C429 mutant (containing only 2 Cys) recombinant proteins. Regarding the mutant protein, the rationale was that of mutating all the surface Cys residues into non reactive and isosteric Ser residues, while leaving the Cys263 and Cys429 residues which potentially play an important structural role. We used the NO-generating donor NOR-3 for S-nitrosylation, glutathione (GSH) for S-glutathionylation and S-nitrosoglutathione (GSNO) for both S-glutathionylation and S-nitrosylation. We could observed that treatment with NOR-3, but not with GSH and GSNO, decreased both SMOWT and SMOC263-C429 enzymatic activity.

The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of "functional silencing" of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.

Cystein residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitro sative/oxidative stress. MT from rabbit liver was treated with different concentrations of GSNO, diamide plus GSH or H2O2 plus GSH. Parallel sets of samples were treated with 10 mM DTT for 30 min at 37 degrees C to reduce mixed disulphides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell extracts. Structural aspects of S-glutathionylation of MTs were also examined. Treatment with GSNO, diamide/GSH or H2O2/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulphides. Structural analysis of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance. (C) 2007 Elsevier Inc. All rights reserved.

We present here a brief description of the relationships among metals, nitric oxide metabolism, and ageing. In particular, we will discuss the interactions occurring between redox (copper, iron) and non-redox (zinc) metals and nitric oxide, the metal- and nitric oxide-catalyzed formation of thiol adducts (nitrosothiols, mixed disulphides) and the possible involvement of these species in the ageing process. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the Cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1beta in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1beta, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1beta has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

The action of copper on the nitric oxide (NO) pathway was investigated in rat C6 glioma cells expressing both inducible and constitutive NO synthase (NOS) isoforms, The inducible NOS-II-mediated NO synthesis (i.e., nitrite production induced by LPS plus IFN gamma) was found to be increased upon copper uptake by cells, this effect being attributable to NOS-II mRNA transcriptional over-expression. On the other hand, the constitutive neuronal isoform (NOS-I) was inhibited after copper uptake, as revealed by the decrease of basal intracellular cGMP levels in C6 cells. Consistently, in vitro experiments showed that copper selectively blocked the catalytic activity of NOS-I, but not of NOS-II, The observed modulation of NOS isoforms by copper in C6 cells is in line with the previous hypothesis that selective inhibition of NOS-I leads to enhanced NO production through transcriptional activation of NOS-II. (C) 2000 Academic Press.

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

Nitric oxide (NO) is a polypotent regulatory molecule involved in a variety of activities, such as the modulation of the catalytic activity of cysteine-containing enzymes. The present study reports the modulation of the HIV-1 reverse transcriptase activity by NO, released by the NO donors 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), 4-(phenylsulfonyl)-3-((2-(dimethylamino) ethyl)thio)furoxan oxalate (SNO-102), and sodium nitroprusside (SNP). NO inhibits dose-dependently the HIV-1 reverse transcriptase activity, Likely due to oxidation of Cys residue(s). Present results, representing a new insight into the modulation mechanism of the HIV-1 reverse transcriptase activity, may be relevant to develop new strategies for inhibition of HIV-1 replication. (C) 1999 Academic Press.

Current evidence suggests that integrating diverse warning systems at different biological levels may not only increase the probability of detecting threats but also mitigate their impact. Here, we propose the use of both molecular and morphological descriptors at different biological levels in sessile bivalves (a suitable biological model in monitoring programs) to collect information on the ecosystem health of coastal marine habitats. In this context, studies may be implemented on biomarkers to exploit some population features, with the aim to propose an actual monitoring program that predictively would provide possible scenarios on the species fitness and ecosystem changes. Thus, the use of quality biotic elements may provide an objective environmental monitoring method and facilitate the development of sanitary, economic, and social strategies related to sustainable exploitation.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.

Several biological levels are considered as signals of environmental perturbations so that integrative biological responses have been proposed in environmental monitoring activities. We evaluated the use of markers in Mytilus galloprovincialis at two different levels (protein nitration and S-nitrosylation, and geometric morphometrics) to investigate the sea coastal environmental status. We studied adductor muscle, digestive gland and valves of mussels collected from 3 sampling sites (2 disturbed + 1 control) in eastern Tyrrhenian Sea, and 2 (1 + 1) in western Ionian Sea. Molecular and morphometric markers showed significant variations in relation to environmental conditions, but the most interesting result was evidenced by the analysis of covariation between both protein post-translational modifications and geometric morphometric descriptors. The main interesting finding regards the relationship between protein nitration values (an irreversible molecular marker) of both adductor muscle and digestive gland, and geometric morphometric descriptors (that are Procrustes’ distances) of valve shapes. Molecular and morphometric markers revealed useful warning systems in detecting environmental changes, even if causes and mechanisms altering both cellular metabolism and shape morphology are still unclear. Our findings suggested that pollutants affected the normal cellular metabolic pathways altering the correct functionality at higher biological levels and producing developmental detrimental noise visible on the shell.

Clonidine, clinically used in the treatment of hypertension, is a central alpha(2)-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca(2+)-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca(2+)-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca(2+)-dependent NOS (NOS-III) activity. However, the value of K(i) for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH(4)) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of K(i) for clonidine binding to NOS-I increased (from [7. 9 +/- 0.4] x 10(-5) M to [8.0 +/- 0.4] x 10(-3) M) as BH(4) concentration was increased (between 3.0 x 10(-7) M and 1.0 x 10(-3) M), at pH 7.5 and 37.0 degrees. In addition, clonidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Finally, clonidine (1.0 x 10(-4) M to 1.0 x 10(-3) M) dose dependently increased the levels of LPS/IFN-gamma-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

Members of the family Meloidae are known to produce cantharidin, a highly toxic monoterpene found in their hemolymph and exuded as droplets capable of deterring many predators. As a nuptial gift, males transfer large amounts of cantharidin to females via a spermatophore, which is formed by specific accessory glands containing high concentrations of this terpene. Using light, electron and ion beam microscopy, the ultrastructural features of the three pairs of male accessory glands as well as the glandular part of the vasa deferentia were comparatively investigated in seven species of blister beetles belonging to five different tribes and two subfamilies. All gland pairs examined share common features such as mesodermal derivation, the presence of muscle sheath, a developed rough endoplasmic reticulum, abundant mitochondria, secretory vesicles, and microvillated apical membranes. Within the same species, glands exhibit distinctive features, suggesting that each pair is responsible for the formation of a specific substance. The vasa deferentia, while showing many similarities within the family, often exhibit features unique to each of the individual species investigated, whereas the accessory glands of the first and second pairs display the highest degree of ultrastructural variability. A comparison across the species shows an interesting constancy limited to ultrastructural features in the third pair of accessory glands. The similarities and differences among the species are discussed in the light of the available literature and in relation to the potential role that blister beetles’ male accessory glands could play in the storage and management of cantharidin.

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

Amyloid-β (Aβ) deposition, a hallmark of Alzheimer’s disease, is known to induce free radical production and oxidative stress, leading to neuronal damage. During oxidative stress, several cell types (including astrocytes) can activate the nuclear factor erythroid 2-related factor 2 (Nrf2), a regulator of several phase II detoxifying and antioxidant genes, such as the System Xc− subunit xCT. Here, we studied (i) the effect of the Aβ fragment 25-35 (Aβ25-35) on Nrf2-dependent System Xc− expression in U373 human astroglial cells and (ii) the effect of Aβ25-35-induced astrocytic response on neuronal cell viability using an in vitro co-culture system. We found that Aβ25-35 was able to activate an antioxidant response in astrocytes, by inducing both Nrf2 activation and System Xc− up-regulation. However, this astrocytic response caused an enhanced cell mortality of co-cultured SH-SY5Y cells, taken as a neuronal model. Consistently, the specific System Xc− inhibitor sulfasalazine prevented the increase of both neuronal mortality and extracellular glutamate levels, thus indicating that the neurotoxic effect was due to an augmented release of glutamate through the transporter. The involvement of NMDA receptor activation in this pathway was also demonstrated using the specific inhibitor MK801 that completely restored neuronal viability at the control levels. The present study sheds light on the Nrf2/system Xc− pathway in the toxicity induced by Aβ25-35 and may help to better understand the involvement of astrocytes in neuronal death during Alzheimer’s disease.

Background Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. Results Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. Conclusions The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

Blister beetles owe their name to their ability to release cantharidin, a blistering terpene, the highest concentration of which is retained in male accessory glands. The anatomy and ultrastructure of the three pairs of male reproductive accessory glands and the glandular region of the two vasa deferentia of Meloe proscarabaeus were investigated using light, electron and ion beam microscopy. All of the mesodermal glands here analysed share a common structural organization with an outer muscular layer and an inner glandular epithelium facing a broad lumen in which the secretory products are released. Developed rough endoplasmic reticulum, Golgi systems, abundant mitochondria, numerous secretory vesicles and a microvillated apical membrane are commonly found in the cells of different glandular epithelia, suggesting that all accessory gland pairs as well as the vasa deferentia are involved in an active synthesis. Nevertheless, each pair of glands appears specialized in the production of a specific set of substances, as suggested by the peculiarities in cellular ultrastructure and by the different aspect of the secretions stored in their glandular lumen. The above cited features of male accessory glands of M. proscarabaeus are compared with those of other beetles and some hints on their potential role in producing and/or concentrating cantharidin are provided.

Nitric oxide (NO) may modulate the catalytic activity of cysteine proteases. In the present study, the inhibitory effect of NO, released by the NO-donors (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide and nitroprusside, on papain action is reported. Papain inactivation via NO-mediated nitrosylation of the Cys25 catalytic residue represents a molecular model for cysteine protease inhibition.

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

HIV-associated neurocognitive disorder (HAND) is a common cognitive impairment in AIDS that affects 15 to 50% of adults infected with human immunodeficiency virus (HIV). Excessive amounts of nitric oxide (NO), as produced by inducible NO synthase (iNOS) upon exposure of activated microglia and astrocytes to cytokines and/or viral proteins (e.g., HIV tat and gp120), are assumed to contribute to neuronal abnormalities in HAND. Evidence exists supporting the notion that iNOS induction takes place after an early decline in physiological NO levels (i.e., NO released by constitutive NOS). Here, we demonstrate that HIV-1 gp120 is able to inhibit neuronal NOS through a cytosolic phospholipase A(2) (cPLA(2))-dependent arachidonic acid (AA) production, this response being critical for allowing activation of the transcriptional factor NF-kappa B and subsequent iNOS and interleukin-1 beta transcription in astroglial cells. In this context, AA seems to act as an upstream proinflammatory effector. In view of the pathogenic role of cPLA(2) in HAND, a deeper insight into the molecular and cellular mechanisms of its modulation may be helpful in finding new drugs to manage cognitive impairment in HIV-1 patients.

Production of nitric oxide (NO) in the cell is catalysed by NO synthase (NOS), which using L-arginine and oxygen as substrates, synthesises NO. Normally cells express neuronal and/or endothelial NOS (nNOS and eNOS, respectively) producing nanomolar NO. Under inflammatory conditions cells express inducible NOS (iNOS), which, once expressed, produces micromolar NO. Since micromolar NO could elicit damage to cells, iNOS expression is time-spatially finely regulated. Recent evidence points out the possible interaction between n/eNOS and iNOS. Under inflammatory conditions interferon-γ, tumour necrosis factor-α and interleukin 1 -β rapidly trigger inhibition of e/nNOS activity with successive drop in the amounts of NO. Since NO normally exerts suppressive action on NF- κ B activation, drop in NO may create a favourable conditions for NF- κ B activation and successive iNOS exptression. Evidence in the literature indicates that during ageing nNOS activity gradually drops with concomitant increase in iNOS expression in the brain. However, how nNOS activity may influence iNOS expression remains to be elucidated. This review points out the possible functional link between age-dependent decrease in nNOS activity and increase in iNOS expression in the brain. Evidence for the possible scenario that often underestimated continuous infections to the brain, one of the hallmarks of ageing, may trigger apparently spontaneous iNOS expression is presented. Future treatment of aged people based on this scenario is also described.