Human iNOS Research Articles

Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS), based on a difluorobenzene ring linked to a 2-aminopyridine scaffold with different functionalities at the 4-position, is reported. In our efforts to develop novel nNOS inhibitors for the treatment of neurodegenerative diseases, we discovered 17, which showed excellent potency toward both rat (Ki 15 nM) and human nNOS (Ki 19 nM), with 1075-fold selectivity over human eNOS and 115-fold selectivity over human iNOS. 17 also showed excellent permeability (Pe = 13.7 × 10-6 cm s-1), a low efflux ratio (ER 0.48), along with good metabolic stability in mouse and human liver microsomes, with half-lives of 29 and >60 min, respectively. X-ray cocrystal structures of inhibitors bound with three NOS enzymes, namely, rat nNOS, human nNOS, and human eNOS, revealed detailed structure-activity relationships for the observed potency, selectivity, and permeability properties of the inhibitors.

INOS expressing macrophages co-localize with nitrotyrosine staining after myocardial infarction in humans.

Inducible nitric oxide synthase (iNOS) produces micromolar amounts of nitric oxide (NO) upon the right stimuli, whose further reactions can lead to oxidative stress. In murine models of myocardial infarction (MI), iNOS is known to be expressed in infiltrating macrophages, which at early onset enter the infarcted zone and are associated with inflammation. In contrast cardiac tissue resident macrophages are thought to enhance regeneration of tissue injury and re-establish homeostasis. Both detrimental and beneficial effects of iNOS have been described, still the role of iNOS in MI is not fully understood. Our aim was to examine cell expression patterns of iNOS and nitrotyrosine (NT) production in human MI. We examined in postmortem human MI hearts the iNOS mRNA expression by means of qPCR. Further we performed immunohistochemical stainings for cell type identification. Afterwards a distance analysis between iNOS and NT was carried out to determine causality between iNOS and NT production. iNOS mRNA expression was significantly increased in infarcted regions of human MI hearts and iNOS protein expression was detected in resident macrophages in infarcted human hearts as well as in controls hearts, being higher in resident macrophages in MI hearts compared to control. Furthermore in MI and in healthy human hearts cells showing signs of NT production peaked within 10-15 µm proximity of iNOS+ cells. These results indicate that, unexpectedly, resident macrophages are the main source of iNOS expression in postmortem human MI hearts. The peak of NT positive cells within 10-15 µm of iNOS+ cells suggest an iNOS dependent level of NT and therefore iNOS dependent oxidative stress. Our results contribute to understanding the role of iNOS in human MI.

Insights into human eNOS, nNOS and iNOS structures and medicinal indications from statistical analyses of their interactions with bound compounds.

83 Structures of human nNOS, 55 structures of human eNOS, 13 structures of iNOS, and about 126 reported NOS-bound compounds are summarized and analyzed. Structural and statistical analysis show that, at least one copy of each analyzed compound binds to the active site (the substrate arginine binding site) of human NOS. And binding features of the three isoforms show differences, but the binding preference of compounds is not in the way helpful for inhibitor design targeting nNOS and iNOS, or for activator design targeting eNOS. This research shows that there is a strong structural and functional similarity between oxygenase domains of human NOS isoforms, especially the architecture, residue composition, size, shape, and distribution profile of hydrophobicity, polarity and charge of the active site. The selectivity and efficacy of inhibitors over the rest of isoforms rely a lot on chance and randomness. Further increase of selectivity via rational improvement is uncertain, unpredictable and unreliable, therefore, to achieve high selectivity through targeting this site is complicated and requires combinative investigation. After analysis on the current two targeting sites in NOS, the highly conserved arginine binding pocket and H4B binding pocket, new potential drug-targeting sites are proposed based on structure and sequence profiling. This comprehensive analysis on the structure and interaction profiles of human NOS and bound compounds provides fresh insights for drug discovery and pharmacological research, and the new discovery here is practically applied to guide protein-structure based drug discovery.

Molecular interplay promotes amelioration by quercetin during experimental hepatic inflammation in rodents

Protein modulation is a process of changing protein structure which is either derived or deliberately induced. This leads to variation in protein activity during protein-protein interactions and can be utilized to understand disease mechanisms or to develop novel therapeutics. In this context, COX-2, iNOS and GST along with basic liver biochemistry were examined in experimentally injured rats and quercetin (QC) supplementation. Diethylnitrosamine (10 ml/kgbwt of 1%DEN) was used to generate liver injury in animals. Quercetin was administered at 60 mg/kgbwt daily in DEN-treated animals to investigate the effect. Hepatic AST/ALT/ALP/γGT, bilirubin, glycogen, LPO, CAT, SOD, GST, collagen levels, protein oxidation and nitrites were examined in all animals. Besides, histopathology and immunohistochemistry of COX-2 and iNOS was performed in tissue sections. Molecular docking of quercetin with human COX-2, iNOS and GST was performed by Autodock 4.2.6 software while drug likeliness and the properties of the ligand by SWISS-ADME and admetSAR and Molinspiration respectively. Our results demonstrate disturbed primary liver function concomitant with disconcert liver biochemistry and anatomy in DEN-treated animals (p < 0.05). Immunohistochemistry exhibited up-regulation of COX-2 and iNOS during liver injury. Molecular docking showed quercetin as acceptable drug as it passes Lipinski rule and shows binding with GST, iNOS, COX-2. In conclusion, COX-2, iNOS and GST appears to be the dynamic molecular targets of Quercetin action to restore tissue biochemistry and anatomy during experimental liver injury.

Inhibitory effects of Indonesian temu kunci (Boesenbergia rotunda) rhizome extract on nitric oxide synthase production and on the kidneys of Wistar rats

Nitric oxide synthases (NOSs) are enzymes that function by generating nitric oxide (NO) from its substrate, L‑arginine, in human tissues. There are three known isoforms of NOS, eNOS (endothelial NOS), nNOS (neuronal NOS), and iNOS (inducible NOS). iNOS is released in response to inflammation. The human iNOS inhibitor, S‑ethylisothiourea (SEITU), has been found to attach to a Glu377 residue within the active‑site cavity of the enzyme. The discovery of iNOS inhibitors is always challenging. <em>Boesenbergia rotunda</em> (<em>B. rotunda</em>) has been reported to reduce inflammation in an animal model. The present study examined the inhibitory effects of <em>B. rotunda</em> rhizome ethanolic extract (BRE) on iNOS using the fluorometric method and investigated the histopathological effects of higher concentrations of BRE (1,000, 2,000 and 4,000 mg/kg body weight) on the kidneys of male Wistar rats. Molecular docking simulation of quercetin and kaempferol towards iNOS was also performed. The phytochemical screening of BRE indicated the presence of polyphenolics, flavonoids, triterpenoids, and saponins. The <em>in vitro</em> experiment confirmed that BRE reduced NO production with a half‑maximal inhibitory concentration value of 79.06 µg/ml. Both quercetin and kaempferol interacted with Glu377 by building hydrogen bonds. Furthermore, the animal experiment resulted in no mortality and no marked morphological changes in the rat kidneys at all concentrations, indicating the safety of BRE as regards this organ. Taken together, BRE may be further developed as a safe anti‑inflammatory drug candidate.

Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase.

Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN-heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.

2-Aminopyridines with a shortened amino sidechain as potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood–brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (Ki = 46 nM) and human nNOS (Ki = 48 nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (Pe = 17.3 × 10-6 cm s-1) through a parallel artificial membrane permeability assay, a model for blood–brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood–brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure–activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases.

Olea europea L. Leaves and Hibiscus sabdariffa L. Petals Extracts: Herbal Mix from Cardiovascular Network Target to Gut Motility Dysfunction Application.

It is well known that diet and nutrition play a critical role in the etiopathogenesis of many disorders. On the other hand, nutrients or bioactive compounds can specifically target and control various aspects of the mechanism underlying the pathology itself, and, in this context, diseases related to intestinal motility disorders stand out. The Herbal Mix (HM) consisting of Olea europea L. leaf (OEE) and Hibiscus sabdariffa L. (HSE) extracts (13:2) has been proven to be a promising nutraceutical option for many diseases, but its potential in inflammatory-driven gastrointestinal disorders is still unexplored. In this study, HM effects on guinea-pig ileum and colon contractility (induced or spontaneous) and on human iNOS activity, as well as on human colorectal adenocarcinoma Caco-2 cells, were studied. Results showed that the HM can control the ileum and colon contractility without blocking the progression of the food bolus, can selectively inhibit iNOS and possesses a strong pro-apoptotic activity towards Caco-2 cells. In conclusion, the present results suggest that, in some diseases, such as those related to motility disorders, an appropriate nutritional approach can be accompanied by a correct use of nutraceuticals that could help not only in ameliorating the symptoms but also in preventing more severe, cancer-related conditions.

The Role of Nitric Oxide in Cancer: Master Regulator or NOt?

Nitric oxide (NO) is a key player in both the development and suppression of tumourigenesis depending on the source and concentration of NO. In this review, we discuss the mechanisms by which NO induces DNA damage, influences the DNA damage repair response, and subsequently modulates cell cycle arrest. In some circumstances, NO induces cell cycle arrest and apoptosis protecting against tumourigenesis. NO in other scenarios can cause a delay in cell cycle progression, allowing for aberrant DNA repair that promotes the accumulation of mutations and tumour heterogeneity. Within the tumour microenvironment, low to moderate levels of NO derived from tumour and endothelial cells can activate angiogenesis and epithelial-to-mesenchymal transition, promoting an aggressive phenotype. In contrast, high levels of NO derived from inducible nitric oxide synthase (iNOS) expressing M1 and Th1 polarised macrophages and lymphocytes may exert an anti-tumour effect protecting against cancer. It is important to note that the existing evidence on immunomodulation is mainly based on murine iNOS studies which produce higher fluxes of NO than human iNOS. Finally, we discuss different strategies to target NO related pathways therapeutically. Collectively, we present a picture of NO as a master regulator of cancer development and progression.

An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase.

Intraprotein interdomainelectron transfer (IET) between the flavin mononucleotide (FMN) and heme centers is an obligatory step in nitric oxide synthase (NOS) enzymes. An isoform-specific pivotal region near Leu406 in the heme domain of human inducible NOS (iNOS) was proposed to mediate the FMN-heme domain-domain alignment (J Inorg Biochem 153:186-196, 2015). The FMN-heme IET rate is a measure of the interdomain FMN/heme complex formation. In this work, the FMN-heme IET kinetics in the wild type (wt) human iNOS oxygenase/FMN (oxyFMN) construct were directly measured by laser flashphotolysis with added synthetic peptide related to the pivotal region, in comparison with the wt construct alone. The IET rates were decreased by the iNOS HKL peptide in a dose-saturable fashion, and the inhibitoryeffect was abolished by a single L406 → E mutation in the peptide. A similar trend in change of the NO synthesis activity of wt iNOS holoenzyme by the peptides was observed. These data, along with the kinetics and modeling results for the L406T and L406F mutant oxyFMN proteins, indicated that the Leu406 residue modulates the FMN-heme IET through hydrophobic interactions. Moreover, the IET rates were analyzed for the wt iNOS oxyFMN protein in the presence of nNOS or eNOS-derived peptide related to the equivalent pivotal heme domain site. These results together indicate that the isoform-specific pivotal region at the heme domain specifically interacts with the conserved FMN domain surface, to facilitate proper interdomain docking for the FMN-heme IET in NOS.

INOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

BackgroundIschemia and reperfusion (I/R) induces cytokines, and up-regulates inducible nitric oxide synthase (iNOS), interferon regulatory factor-1(IRF1) and p53 up-regulated modulator of apoptosis (PUMA), which contribute to cell death and tissue injury. However, the mechanisms that I/R induces IRF1-PUMA through iNOS/NO is still unknown.MethodsIschemia was induced by occluding structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes for 60 min, and reperfusion was initiated by removal of the clamp. Induction of iNOS, IRF1 and PUMA in response to I/R were analyzed. I/R induced IRF1 and PUMA expression were compared between iNOS wild-type and iNOS knockout (KO) mice. Human iNOS gene transfected-cells were used to determine iNOS/NO signals targeting IRF1. To test whether HDAC2 was involved in the mediation of iNOS/NO-induced IRF1 transcriptional activities and its target gene (PUMA and p21) expression, NO donors were used in vitro and in vivo.ResultsIRF1 nuclear translocation and PUMA transcription elevation were markedly induced following I/R in the liver of iNOS wild-type mice compared with that in knock-out mice. Furthermore, I/R induced hepatic HDAC2 expression and activation, and decreased H3AcK9 expression in iNOS wild-type mice, but not in the knock-out mice. Mechanistically, over-expression of human iNOS gene increased IRF1 transcriptional activity and PUMA expression, while iNOS inhibitor L-NIL reversed these effects. Cytokine-induced PUMA through IRF1 was p53 dependent. IRF1 and p53 synergistically up-regulated PUMA expression. iNOS/NO-induced HDAC2 mediated histone H3 deacetylation and promoted IRF1 transcriptional activity. Moreover, treating the cells with romidepsin, an HDAC1/2 inhibitor decreased NO-induced IRF1 and PUMA expression.ConclusionsThis study demonstrates a novel mechanism that iNOS/NO is required for IRF1/PUMA signaling through a positive-feedback loop between iNOS and IRF1, in which HDAC2-mediated histone modification is involved to up-regulate IRF1 in response to I/R in mice.

First Contact: 7-Phenyl-2-Aminoquinolines, Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors That Target an Isoform-Specific Aspartate.

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against human nNOS, and only modest selectivity for nNOS over related enzymes. In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and assayed them against rat and human nNOS, human eNOS, and murine and (in some cases) human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this residue.

Variability within the human iNOS gene and Achilles tendon injuries: Evidence for a heterozygous advantage effect

ObjectivesThe aim of this case control genetic association study was to explore whether two variants within the inducible nitric oxide synthase (iNOS) gene, rs2779249 (C/A) and rs2248814 (A/G), influenced the risk of Achilles tendinopathy in a British population. DesignCandidate gene, case control association study. MethodWe recruited 145 individuals diagnosed with Achilles tendon pathology and 132 asymptomatic controls. All participants were genotyped for the iNOS variants using qPCR and significant associations were discovered using a combination of Chi squared and ANOVA type analysis. ResultsThe CA genotype of the iNOS rs2779249 variant was protective and conformed to a heterozygous advantage model of inheritance as it was overrepresented in the control participants (p=0.009). In sex specific analysis the protective association persisted in male participants (p=0.016) but not in females. Unlike the rs2779249 variant, the rs2248814 variant was not associated with Achilles tendinopathy or Achilles tendon rupture. ConclusionThe rs2779249 CA genotype within the human iNOS gene appears to protect individuals from Achilles tendinopathy. This study further supports a genetic contribution to modifying the risk of Achilles tendon problems. The study also infers an important role for nitric oxide in tendon healing and/or degradation.

Molecular docking analysis of triptoquinones from genus Tripterygium with iNOS and in silico ADMET prediction

This paper presents an investigation on the binding interaction of triptoquinones identified from genus Tripterygium to iNOS. In silico methods are adopted to predict ADME parameters, pharmacokinetic properties, drug-likeliness and acute toxicity of these identified compounds. A total of 20 triptoquinones are currently identified from genus Tripterygium. Most of these triptoquinones are found to bind to the key human iNOS residues involved in inhibitor binding. All the compounds are considered having drug-likeliness properties with no violation against Lipinski's rule of 5 and are under safe category when administered orally. Twelve out of the 20 triptoquinones are predicted as passively crossing the blood-brain barrier. Eight of the given compounds are predicted to be pumped out by the p-glycoprotein. CYP2C19 and CYP2C9 are the significant isoforms influenced by the investigated triptoquinones from genus Tripterygium. As a result, triptoquinone ingredients from genus Tripterygium may be promising candidates for the development of drugs preventing inflammatory diseases.

Regulation of human inducible nitric oxide synthase expression by an upstream open reading frame

The human inducible nitric oxide synthase (iNOS) gene contains an upstream open reading frame (uORF) in its 5′-untranslated region (5′-UTR) implying a translational regulation of iNOS expression. Transfection experiments in human DLD-1 cells revealed that the uORF although translatable seems not to inhibit the translation start at the bona fide ATG. Our data clearly show that human iNOS translation is cap-dependent and that the 5′-UTR of the iNOS mRNA contains no internal ribosome entry site. Translation of the bona fide coding sequence is most likely mediated by a leaky scanning mechanism.The 5′-UTR is encoded by exon 1 and exon 2 of the iNOS gene with the uORF stop codon located in front of the first intron indicating an involvement of the nonsense mediated RNA decay (NMD) in iNOS regulation. SiRNA-mediated down-regulation of Upf1 resulted in enhanced endogenous cytokine iNOS expression in human DLD-1 cells. Transfection of constructs containing iNOS exon 1, intron 1 and exon 2 in front of a luciferase gene showed a clear effect of the mutation of the uORF-ATG on luciferase reportergene expression.Our data indicate that the uORF in the 5′-UTR sequence of human iNOS gene reduces its expression via the NMD mechanism.

Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at −4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.

Inducible Nitric Oxide Synthase Is a Key Host Factor for Toxoplasma GRA15-Dependent Disruption of the Gamma Interferon-Induced Antiparasitic Human Response.

Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host's NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor.IMPORTANCEToxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.

Abstract TP254: Effects of Inducible Nitric Oxide Synthase on Behavior and Functional Outcomes in a Novel “Humanized” Transgenic Mouse Model of Ischemic Stroke

Introduction: Inducible nitric oxide synthase (iNOS) is a critical regulator of brain and systemic inflammatory responses in many neurological disorders. Intriguingly, iNOS null mice have produced equivocal outcomes in mouse models of ischemic stroke, a result that may be due, in part, to species-specific differences between the mouse and human iNOS genes. Due to numerous differences at the gene regulatory level, mouse iNOS naturally produces much higher levels of nitric oxide (NO) than the human gene. To resolve the functional differences between mouse and human iNOS in ischemic stroke, we employed a novel mouse that expresses human iNOS on a mouse iNOS null genetic background, termed the HN mouse. Our study objective was to compare functional and behavioral outcomes following ischemic stroke between wildtype (WT) and HN mice. Hypothesis: We hypothesized that post-stroke functional outcomes would be more severe in HN male mice than in WT mice. Methods: Male C57BL/6J (WT) and HN mice (4-6 months) were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion and behavioral assessments at seven-day intervals for 30 days. Laser-Doppler flowmetry pre- and post-tMCAO was used to assess the effect of iNOS on blood flow. A comprehensive neurological battery was used to assess sensorimotor ability, reflexes, and balance, followed by open field, wire hang, and rotarod testing to evaluate locomotor activity, limb strength, and coordination. Results and Conclusion: Blood flow was significantly decreased in the ipsilateral hemisphere of both WT and HN mice following acute ischemic stroke; however, HN mice exhibited a sustained global decrease in blood flow following reperfusion. HN mice also demonstrated decreased locomotor activity and coordination, as well as greater neurological deficits compared to WT mice. Overall, these results suggest that the HN mouse model recapitulates more neurological deficits associated with human ischemic stroke than WT mice. Thus, the HN mouse may represent a more “humanized” model of ischemic stroke than more commonly used mouse models that express the mouse iNOS gene.

Characterization and gene expression analysis of pacu (Piaractus mesopotamicus) inducible nitric oxide synthase (iNOS) following Aeromonas dhakensis infection

Nitric oxide (NO) is an important effector molecule which is involved in a myriad of biological processes, including immune responses against pathogens such as parasites, virus and bacteria. During the inflammatory processes in vertebrates, NO is produced by the inducible nitric oxide synthase (iNOS) enzyme in practically all nucleated cells to suppress or kill intracellular pathogens. The aim of the present study was to characterize the full coding region of the iNOS gene of pacu (Piaractus mesopotamicus), an economically and ecologically important South American fish species, and to analyze mRNA expression levels following intraperitoneal infection with the pathogenic bacterium Aeromonas dhakensis by means of quantitative real time PCR (qPCR). The results showed that the pacu iNOS transcript is 3237 bp in length, encoding a putative protein composed of 1078 amino acid residues. The amino acid sequence showed similarities ranging from 69.03% to 94.34% with other teleost fish and 57.70% with the human iNOS, with all characteristic domains and cofactor binding sites of the enzyme detected. Phylogenetic analysis showed that the iNOS from the red-bellied piranha, another South American characiform, was the closest related sequence to the pacu iNOS. iNOS transcripts were constitutively detected in the liver, spleen and head kidney, and there was a significant upregulation in the liver and spleen at 12, 24 and 48 h after infection with A. dhakensis. No significant variations were observed in the head kidney during the periods analyzed. These results show that iNOS expression was induced by A. dhakensis infection and suggest that this enzyme may be involved in the response to this bacterium in pacu.

Erratum to: Evaluation of INOS, ICAM-1, and VCAM-1 gene expression: A study of adult T cell leukemia malignancy associated with HTLV-1.

The main aim of this study was to evaluate the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and inducible nitric oxide synthase (iNOS) as host factors, and proviral load as the viral parameter, in adult T-cell leukemia/lymphoma (ATLL) individuals and healthy carrier (HC(s)) groups. Peripheral blood mononuclear cells (PBMC) from ATLL patients (n = 17) and HC subjects (as the control group, n = 17) were evaluated using real-time PCR to determine the levels of HTLV-1 proviral load and mRNA expression of ICAM, VCAM-1, and iNOS. ICAM-1 was significantly lower in ATLL patients than in control subjects. Although the expression of VCAM-1 was higher in ATLL individuals, there was no significant difference between the studied groups. In addition, no iNOS expression was found in ATLL patients, when compared to the HCs subjects, while ATLL patients demonstrated a higher level of proviral load when compared to the control group. Considering the importance of ICAM-1 in facilitating immune recognition of infected cells, it is posited that reduction of ICAM-1 expression is a unique strategy for circumventing appropriate immune responses that are mediated by different accessory proteins. Additionally, as the viral regulatory protein Tax and the NF-κB pathway play pivotal roles in expression of iNOS, lack of the latter in ATLL patients may be related to the level of Tax expression, disruption of the NF-κB pathway, or the occurrence of epigenetical mechanisms in the human iNOS promoter. Further studies are recommended to gain a better understanding of the interaction between host and viral factors in HTLV-1 pathogenesis and to identify a possible therapeutic target for ATLL.