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Abstract: Several paramagnetic chemical exchange saturation transfer magnetic resonance imaging (PARACEST MRI) agents for the potential detection of nitric oxide synthase (NOS) have been synthesized and evaluated. These agents are [...]

The reaction of 5-aminosalicylic acid (5-ASA) with nitrous acid has been studied at low pH under conditions that simulate a gastric environment. The course of the reaction was followed by UV-visible and fluorescence spectroscopy and the products were analyzed by high performance liquid chromatography (HPLC) with UV-visible and mass spectroscopic detectors. In addition, the formation of nitric oxide (NO) was estimated electrochemically. 5-ASA was readily consumed in a process catalyzed by chloride and thiocyanate, whose rate is first order in 5-ASA and second order in nitrous acid. 2-Hydroxy-5-diazonium benzoic acid (diazonium derivative) and NO were detected as products of the reaction. From the NO formation profiles, it is concluded that NO is produced as a minor product in a process parallel to the path that leads to generation of the diazonium derivative. While the formation of NO could be beneficial for the protection of the stomach, the generation of the diazonium derivative could be considered a potentially toxic process. Key words: 5-aminosalicylic acid, mesalazine, nitrous acid, nitrite, diazonium derivative, nitric oxide. On a etudie la reaction de l'acide 5-aminosalicylique (5-AAS) avec l'acide nitreux, a un pH faible, dans des conditions qui simulent un environnement gastrique. On a suivi l'evolution de la reaction par spectroscopies UV-visible et de fluorescence et on a analyse les produits par chromatographie liquide a haute performance (CLHP) avec des detecteurs par spectroscopie UV-visible et de masse. De plus, on a fait appel a une methode electrochimique pour evaluer la formation d'oxyde nitrique (NO). L'acide 5-aminosalicylique est rapidement consomme dans un processus catalyse par les ions chlorure et thiocyanate et pour lequel la vitesse est du premier ordre en 5-AAS et du second ordre en acide nitreux. On a detecte de l'acide 2-hydroxy-5-diazoniumbenzoique (derive diazonium) et du NO comme produis de la reaction. D'apres les profils de formation du NO, on en conclut que le NO se forme comme produit mineur dans un processus parallele a la voie qui conduit a la formation du derive diazonium. Alors que la formation du NO pourrait etre benefique pour la protection de l'estomac, la formation du derive diazonium peut etre consideree comme un processus potentiellement toxique. Mots-cles : acide 5-aminosalicylique, mesalazine, acide nitreux, nitrite, derive diazonium, oxyde nitrique., Introduction Chronic inflammatory bowel pathologies such as ulcerative colitis and Crohn's disease cause inflammatory changes and ulcers of different types in the gastrointestinal mucosa. (1) 5-Aminosalicylic acid (5-ASA), the active [...]

Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide ([N.sub.2]O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron ([Fe.sub.B]) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side. 10.1126/science.1195591

This paper reports a real-time study of the codynamical changes in the release of endogenous nitric oxide (NO) and oxygen ([O.sub.2]) consumption in a rot neocortex in vivo upon electrical stimulation using an amperometric NO/[O.sub.2] dual microsensor. Electrical stimulation induced transient cerebral hypoxia due to the increased metabolic demands that were not met by the blood volume inside the stimulated cortical region. A NO/ [O.sub.2] dual microsensor was successfully used to monitor the pair of real-time dynamic changes in the tissue NO and [O.sub.2] contents. At the onset of electrical stimulation, there was an immediate decrease in the cortical tissue [O.sub.2] followed by a subsequent increase in the cortical tissue NO content. The averages of the maximum normalized concentration changes induced by the stimulation were a 0.41 ([+ or -] 0.04)-fold decrease in the [O.sub.2] and a 3.6 ([+ or -] 0.9)-fold increase in the NO concentrations when compared with the corresponding normalized basal levels. The peak increase in NO was always preceded by the peak decrease in [O.sub.2] in all animals (n = 11). The delay between the maximum decrease in [O.sub.2] and the maximum increase in NO varied from 3.1 to 54.8 s. This rather wide variation in the temporal associations was presumably attributed to the sparse distribution of NOS-containing neurons and the individual animal's differences in brain vasculatures, which suggests that a sensor with fine spatial resolution is needed to measure the location-specific real-time NO and [O.sub.2] contents. In summary, the developed NO/[O.sub.2] dual mierosensor is effective for measuring the NO and [O.sub.2] contents in vivo. This study provides direct support for the dynamic role of NO in regulating the cerebral hemodynamics, particularly related to the tissue oxygenation. 10.1021/ac1013496

The ability to perform a fluorescence-based quantitative determination of a biologically important analyte directly released from mammalian cells using a standard microtiter plate reader to measure wells integrated into a microfluidic device is reported. Specifically, the amount of nitric oxide (NO) released from flowing erythrocytes (ERYs) exposed to a hypoxic buffer is measured using a fluorescein-based probe. The ERYs are pumped through channels in one layer of the poly(dimethylsiloxane) (PDMS) device; as these cells release NO, it flows through a porous polycarbonate membrane to the probe. The device is then placed into a standard microliter plate reader for measurement, with the entire calibration and analyte determination occurring simultaneously. Using this method, NO release from hypoxic ERYs was determined to be 6.9 [+ or -] 1.8 [micro]M, a significantly increased value in comparison to that from normoxic ERYs of 0.60 [+ or -] 0.04 [micro]M (p < 0.001, n = 4 rabbits). Furthermore, the reproducibility (reported as a %RSD) of measuring fluorescence standards was 3.5%. Detection limits, dynamic range, and optimal membrane pore diameters are also reported. This device enables the use of a standard high-throughput tool (the plate reader) to measure analytes in a microfluidic device, the ability to improve the quantitative determination of a relatively unstable molecule (NO), and the incorporation of a flow component and blood constituent into a system that can be combined with microtiter plate technology. 10.1021/ac101130s

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated cellular proteins or changes in proteasome expression, suggesting that Rpn13 must have a specialized role in proteasome function. Thus, Rpn13 participation in protein degradation, by recruiting UCH37, is rather selective to specific proteins whose degradation critically depends on UCH37 deubiquitination activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn13 is involved in iNOS degradation and is required for iNOS interaction with the deubiquitination protein UCH37. Furthermore, we discovered that I[kappa]B-[alpha], a protein whose proteasomal degradation activates the transcription factor NF-[kappa]B, is also a substrate for the Rpn13/UCH37 complex. Thus, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway. proteasome | ubiquitin | UCH37 | inflammation | lung doi/ 10.1073/pnas.0913495107

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming ( time-resolved Raman spectroscopy | allostery | structural dynamics doi/ 10.1073/pnas.0912938107

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain--containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections. doi/ 10.1083/jcb.200912087

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative [Fe.sub.B] site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which Iowered the heme reduction potential by ~110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ~100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the [Fe.sub.B] site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues. biomimetic models | heme-copper oxidase | metalloprotein | protein design | protein engineering doi/10.1073/pnas.1000526107

This article describes a thin amperometric nitric oxide (NO) sensor that can be microchannel embedded to enable direct real-time detection of NO produced by cells cultured within the microdevice. A key for achieving the thin (~1 mm) planar sensor configuration required for sensor-channel integration is the use of gold/indium--tin oxide patterned electrode directly on a porous polymer membrane (pAu/ITO) as the base working electrode. The electrochemically deposited Au--hexacyanoferrate layer on pAu/ITO is used to catalyze NO oxidation to nitrite at lower applied potentials (0.65-0.75 V vs Ag/AgCl) and stabilize current output. Furthermore, use of a gas-permeable membrane to separate internal sensor compartments from the sample phase imparts excellent NO selectivity over common interfering agents (e.g., nitrite, ascorbate, ammonia, etc.) present in culture media and biological fluids. The optimized sensor design reversibly detects NO down to the ~1 nM level in stirred buffer and 10.1021/ac100085w

Nonadiabatic coupling of nuclear motion to electronic excitations at metal surfaces is believed to influence a host of important chemical processes and has generated a great deal of experimental and theoretical interest. We applied a recently developed theoretical framework to examine the nature and importance of nonadiabatic behavior in a system that has been extensively studied experimentally: the scattering of vibrationally excited nitric oxide molecules from a Au(111) surface. We conclude that the nonadiabatic transition rate depends strongly on both the N-O internuclear separation and the molecular orientation and, furthermore, that molecule-surface forces can steer the molecule into strong-coupling configurations. This mechanism elucidates key features of the experiments and provides several testable predictions regarding the dependence of vibrational energy transfer on the initial vibrational energy, molecular orientation, and incident angle. 10.1126/science.1179240

A fungal elicitor prepared from the cell debris of the plant-pathogenic ascomycete Alternaria alternata induces multiple responses by Inonotus obliquus cells, including an increase in generation of nitric oxide (NO), activity of phenylalanine ammonia lyase (PAL) and accumulation of total mycelial phenolic compounds (TMP), but does not trigger production of oxylipins or jasmonic acid (JA). The role of NO in TMP production was investigated via the effects of the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO) and the nitric oxide synthase (NOS) inhibitor aminoguanidine (AG). TMP profiles were assayed using [sup.1]H NMR spectroscopy combining multivariate pattern recognition strategies. Pretreatment of L obliquus mycelia with cPITO or AG suppressed not only elicitor-enhanced NO generation and PAL activity, but also the elicitor-induced increase in TMP production. This TMP reduction by either a NO scavenger or a NOS inhibitor was reversed by exogenous addition of either a NO donor, sodium nitroprusside, or JA separately. NMR-based metabonomic analysis of TMP profiles showed that the induced TMP were hispidin analogues including inoscavins, phelligridins, davallialactone and methyldavallialactone, which possess high antioxidant activities. Thus, NO mediates an elicitor-induced increase in production of antioxidant polyphenols in I. obliquus via a signalling pathway independent of oxylipins or JA, a mechanism which differs from those in some higher plants. DOI 10.1099/mic.0.030650-0

News, Views and Reviews provides focused updates, in-depth topic reviews and editorials concerning the latest developments in dermatologic therapies. Since first identified in 1986 as the endothelium derived relaxing factor, [...]

The effects of exercise training on the nitric oxide synthase {NOS) isoform profile in aging fast-twitch (white gastrocnemius [WG]) and slow-twitch (soleus [SOL]) muscle have not been investigated. Six-month and 27-month male Fischer-344 rats were divided into the following groups: young sedentary (YS), young treadmill exercise trained for 12 weeks. old sedentary (OSI, and old exercise trained (OE). Inducible NOS (iNOS) protein expression and activity were significantly higher in OS compared with YS. whereas exercise training significantly reduced iNOS protein and activity levels in the WG. Neuronal NOS protein expression decreased with aging in WG but was upregulated significantly with exercise training in OE for both WG and SOL. Endothelial NOS (eNOS) protein leveis were depressed in WG of old rats but were higher in OE than in OS. eNOS was unaffected by aging or exercise in the SOL. Our results indicate that endurance exercise training attenuates age-induced alterations of NOS isoforms with a greater response in fast-twitch compared with slow-twitch muscle. Key Words: Aging--Skeletal muscle--Exercise Nitric oxide synthase--nNOS--eNOS--iNOS.

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the [PDZ.sub.2] domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA--mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS--PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

In intestinal cells, arginine (Arg) is 1 of the 2 most potent amino acid activators of [p70.sup.s6k], a key regulator of 5'-terminal oligopyrimidine mRNA translation, a necessary condition for increased cell migration. To investigate the mechanism of response to Arg, we used the rat crypt cell line cdx2-transformed IEC-6 cells (cdx2-IEC) and measured cell migration, immunocytochemical analysis of [p70.sup.s6k] activation in response to Arg, and production of nitric oxide (NO). When treated with Arg, cdx2-IEC increased in phosphorylation on Thr-389 of [p70.sup.s6k] ([pp70.sup.s6k]) compared with control (P < 0.01). Phospho-Thr-421/Ser-424-[p70.sup.s6k] was located in the nucleus shortly after Arg treatment. Arg enhanced [pp70.sup.s6k], cell migration (55% wound coverage), and NO production. In comparison, the branched-chain amino acid leucine (Leu) activated [pp70.sup.s6k], was a weaker stimulator of migration (23% coverage), and did not increase NO. A total of 25 [micro]mol/L DETA-NONOate (DETA/NO) did not significantly enhance phosphorylation of [p70.sup.s6k] but enhanced the rate of cell migration by ~25%. Wound coverage with Leu plus DETA/NO (25 [mu]mol/L) was greater than coverage with DETA/NO alone (P < 0.01). These and our previous studies lead to a model in which Arg must stimulate both [pp70.sup.s6k] (in the nucleus) and NO release to enhance intestinal epithelial cell migration, which may be relevant to diseases that involve intestinal villous injury.

The mechanisms through which moderate wine consumption reduces ischemic cardiovascular events are not yet fully unraveled. Grape extracts or a mixture of the polyphenols contained in wine were previously shown to increase nitric oxide (NO); however, little information is available on the effect of resveratrol, one of the main polyphenols of wine, on platelet NO production. We assessed the effects of resveratrol, at the concentrations attainable after moderate wine intake, on platelet NO production and the mechanism of this activity. Twenty healthy volunteers were studied before and after 15 d of controlled white or red wine intake (300 mL/d). After wine intake, plasma resveratrol and the release of NO by stimulated platelets increased significantly. Resveratrol, at the concentrations detected in plasma after wine intake, was incubated in vitro with washed platelets and several variables related to NO production and to signal transduction were measured. Resveratrol in vitro enhanced significantly the production of NO by stimulated platelets, the activity of platelet NO synthase (NOS), phosphorylation of protein kinase B, an activator of the endothelial NOS (eNOS), and phosphorylation of vasodilator-activated protein (VASP), an expression of the biologic activity of NO in platelets. Simultaneously, we observed decreased phosphorylation of P38 mitogen-activated protein kinase (p38MAPK), a proinflammatory pathway in human platelets, a reduction of the activity of NADPH oxidase, a major source of reactive oxygen species (ROS) and of the generation of [O.sub.2.sup.-] radicals, as detected by cytochrome C reduction. In conclusion, resveratrol, at concentrations attainable after moderate wine intake, activates platelet eNOS and in this way blunts the proinflammatory pathway linked to p38MAPK, thus inhibiting ROS production and ultimately platelet function. This activity may contribute to the beneficial effects of moderate wine intake on ischemic cardiovascular disease.

The effect of water addition on NO formation in counterflow C[H.sub.4]/air premixed fames was investigated by numerical simulation. Detailed chemistry and complex thermal and transport properties were employed. The results show that the addition of water to aflame suppresses the formation of NO primarily due to flame temperature drop. Among a lean, a stoichiometric, and a rich premixed flame, the effectiveness of water addition is most significant for the stoichiometric flame and least for the rich flame. The addition of water also reduces the formation of NO in a flame because of the chemical effect. Compared to the stoichiometric flame, the chemical effect is intensified in the lean and rich flames. [DOI: 10.1115/1.2432890] Keywords: premixed flame, water addition, numerical simulation, N[O.sub.x]

Nitric oxide (NO) modulates vasodilation in cerebral cortex during sensory activation. NO is known to inhibit the synthesis of 20-HETE, which has been implicated in arteriolar constriction during astrocyte activation in brain slices. We tested the hypothesis that the attenuated cerebral blood flow (CBF) response to whisker stimulation seen after NO synthase (NOS) inhibition requires 20-HETE synthesis and that the ability of an epoxyeicosatrienoic acids (EETs) antagonist to reduce the CBF response is blunted after NOS inhibition but restored with simultaneous blockade of 20-HETE synthesis. In anesthetized rats, the increase in CBF during whisker stimulation was attenuated after the blockade of neuronal NOS with 7-nitroindazole. Subsequent administration of the 20-HETE synthesis inhibitor N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) restored the CBF response to control levels. After the administration of 7-nitroindazole, the inhibitory effect of an EETs antagonist 14, 15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on the CBF response was lost, whereas the simultaneous administration of 7-nitroindazole and HET0016 restored the inhibitory effect of 14,15-EEZE. The administration of HET0016 alone had only a small effect on the evoked CBF response in rats. Furthermore, in neuronal NO[S.sup.+/+] and NO[S.sup.-/-] mice, HET0016 administration did not increase the CBF response to whisker stimulation. In neuronal NO[S.sup.+/+] mice, HET0016 also blocked the reduction in the response seen with acute NOS inhibition. These results indicate that 20-HETE synthesis normally does not substantially restrict functional hyperemia. Increased NO production during functional activation may act dynamically to suppress 20-HETE synthesis or downstream signaling and permit EETs-dependent vasodilation. With the chronic loss of neuronal NOS in mice, other mechanisms apparently suppress 20-HETE synthesis or signaling. eicosanoids; epoxyeicosatrienoic acid; functional activation; mouse; rat; vibrissae; 20-hydroxyeicosatetraenoic acid

A new fragment-based approach, fragment hopping, is described for de novo inhibitor design by focusing on ligand diversity and isozyme selectivity. This new approach has incorporated the concept of early 'ADME/Tox' considerations and has provided a basic platform for medical chemistry-driven efforts.

Computer simulation methods are used for studying the nitric oxide (NO) release mechanism of nitrophorin 4 (NP4) at a molecular level. The results have shown that NP4 has evolved a cage mechanism that has trapped the NO at low pH and has released it upon cage opening when the pH has increased.

Contrary to many reports, sodium methoxide is shown to react with nitric oxide (NO). Sodium trimethylsilanoate is considered as a substitute for sodium methoxide as the base used to achieve the replacement of active hydrogens by the diazeniumdiolate functional group via the Traube reaction.

Analysis of the molecular factors determining hepatocyte survival or death in response to inflammatory stimuli is essential for understanding the pathogenesis of inflammatory liver disease and for identifying novel therapeutic approaches, c-Jun N-terminal kinase (JNK) is a major mediator of cytokine-induced cell death during hepatitis, but the signaling pathways downstream of JNK remain less well defined. Here we show that the transcription factor c-Jun/AP-1, a prototypic target of JNK, is strongly expressed in the liver of patients with acute liver injury. The molecular function of c-Jun in inflammatory liver disease was analyzed in mice by using the Con A model of T cell-mediated hepatitis. Mice lacking c-Jun in hepatocytes display increased liver cell death and mortality upon Con A injection. This phenotype is caused by impaired expression of inducible nitric oxide synthase (nos2), a direct transcriptional target of c-Jun, and reduced production of hepatoprotective nitric oxide (NO). Moreover, increased hepatotoxicity in mutant mice is likely caused by hypoxia and oxidative stress and can be rescued pharmacologically by liver-specific NO delivery. These findings demonstrate that c-Jun/AP-1 is hepatoprotective during acute hepatitis by regulating nos2/NO expression and thus functionally antagonizes the cell death-promoting functions of JNK. activator protein 1 | concanavalin A | hypoxia

A planar-type amperometric dual microsensor for simultaneous detection of nitric oxide and carbon monoxide is presented. The sensor consists of a dual platinum microdisk-based working electrode (WE) and a Ag/AgCl counter/ reference electrode covered with an expanded poly(tetrafluoroethyiene) (Tetra-tex) gas-permeable membrane. The dual WE possesses two different platinized platinum disks (WE1 and WE2, 250 and 25 /ira in diameter, respectively). The larger WE1 is further modified with electrochemical deposition of tin. Use of two sensing disks different in their size as well as in their surface modification produces apparently different sensitivity ratios of NO to CO at WE1 and at WE2 (~2 and ~10, respectively) that are induced by favorable CO oxidation on the surface of tin versus platinum. Anodic currents independently measured at WE1 and at WE2 are successfully converted to the concentrations of NO and CO in the co-presence of these gases using the differentiated sensitivities at each electrode. The sensor is evaluated in terms of its analytical performance: respectable linear dynamic range (sub nM to [micro]M); low detection limit (~1 nM for NO and

The results from the molecular dynamics simulations of nitrophorin 4 (NP4) at pH 5 and pH 7, modeled by selective deprotonation of acidic groups reveal that release rate of diatomic molecules from heme proteins could be varied by several orders of magnitude through modest adjustments in geminate rebinding and gating behavior.

The study is done to determine the heme coordination states of inducible nitric oxide synthase with a Re (I) sensitizer coupled to nitro-L-arginine by an imidazole-alkyl molecular wire. It is demonstrated that Re (I) when applied can initiate quick photoreduction of the ferriheme in the active site of the enzyme.

A new kinetic mechanism of the NO + N[H.sub.3] reaction is used on Pt{100} in order to simulate the spatiotemporal traveling waves. The Irurzun, Mola and Imbihl (IMI model) has predicted the presence of macroscopic oscillations in the partial pressures of the reactants coexisting with front wave patterns on the surface.

The kinetics of interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains in a rat neuronal nitric oxide synthesis (nNOS) holoenzyme in the presence and absence of added calmodulin using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct is reported. The results attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex.

Highly correlated ab initio molecular orbital calculations were used to study the gas-phase N-nitrosation of a series of acyclic and heterocyclic amines. The results have suggested the possibility of controlling the production of nitrosamines in processes such as food preparation and rubber manufacture by limiting the concentration of N[O.sub.2] to significantly low levels.

The formation of the elusive active species Compound l (Cpd I) of nitric oxide synthase (NOS) from the oxyferrous intermediate is analyzed by quantum mechanical / molecular mechanical (QM/MM) methods. The outcomes reveal that two protons are required to initiate a reaction that is sufficiently exothermic and results to the appearance of a radical on the tetrahydrobiopterin cofactor.

Nitric oxide (NO) is quantitatively determined in platelets prior to, and after, stimulation with adenosine triphosphate (ATP) or activation with adenosine diphosphate (ADP). Platelets obtained from the whole blood of rabbits were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent NO production was measured as a fluorescent benzotriazole. Experiments were performed to determine the effect of probe concentration and probe incubation time in the platelets prior to measurement of the fluorescence. This information, combined with the method of multiple standard additions, was then employed to determine the moles of intracellular NO in the platelets (2.7 [+ or -] 0.3) x [10.sup.-16] mol of NO/platelet and the basal level of extracellular NO in the platelet sample (9.9 [+ or -] 2.2) x [10.sup.18] mol of NO/platelet. Moreover, this method was used to quantitatively determine the amount of NO released from platelets whose NO production was stimulated with ATP (a nitric oxide synthase stimulus) or ADP, a substance known to result in NO production through platelet aggregation. When stimulated with ATP, the NO released from the platelets was determined to be (2.0 [+ or -] 0.1) x [10.sup.-17] mol of NO/platelet. When activated with ADP, the platelets released (2.8 [+ or -] 0.3) x [10.sup.-17] mol of NO/platelet. The difference between the extracellular basal levels of NO and that after stimulation with either ATP or ADP is in agreement with current estimates of NO release from platelets. Therefore, we conclude that a fluorescence determination of NO using the DAF family of probes, in combination with the method of multiple standard additions, can be employed to quantitatively determine the basal levels of NO in platelets, as well as the amount of NO released from stimulated and/or activated platelets.

The Raman studies of the Fe[(NO).sub.2] core of dinitrosyl-iron complexes (DNICs) are described. The feasibility of using a v(Fe-NO)/v(NO) correlation diagram to extract the electronic configurations and charges on metals is shown by using the vibrational data of nitrosyl-iron complexes.

Previous studies have demonstrated that pregnancy prevents protective hypertension-induced remodeling of cerebral arteries using nitric oxide synthase (NOS) inhibition to raise mean arterial pressure (MAP). In the present study, we investigated whether this effect of pregnancy was specific to NOS inhibition by using the Dahl salt-sensitive (SS) rat as a model of hypertension. Nonpregnant (n = 16) and late-pregnant (n = 17) Dahl SS rats were fed either a high-salt diet (8% NaCl) to raise blood pressure or a low-salt diet ( gestation; hypertension; brain

Interest in elucidating the mechanisms of action of various classes of anticancer agents and exploring the pathways of the induced-nitric oxide (NO) release provides an impetus to conceive a better designed approach to locally detect NO in tumors, in vivo. We report here on the first use of an electrochemical sensor that allows the in vivo detection of NO in tumor-bearing mice. In a first step, we performed the electrochemical characterization of a stable electroactive probe, [K.sup.4]Fe[(CN).sup.6], directly injected into the liquid microenvironment especially created around the electrode in the tumor. Second, the ability of the inserted electrode system to detect the presence of NO itself in the tumoral tissue was achieved by using the chemically modified Pt/Ir electrode as NO sensor and two NO donor molecules: diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium 1,2-diolate (DEA-NONOate) and (Z)-1-[N-(2-aminopropyl)-N-(2-ammonio propyl)amino]diazen-1-ium 1,2-diolate (PAPA-NONOate). These two NO donor molecules allowed proving the electrochemical detection of (i) directly injected exogenous NO phosphate buffer solution into the tumor (decomposed DEA-NONOate) and (ii) biomimetically induced endogeneous release of NO in the tumoral tissue, upon injection of PAPA-NONOate into the tumor. This approach could be applied to the in vivo study of candidate anticancer drugs acting on the NO pathways.

A study prepared a series of eight pi-conjugated polymers (CPs) composed of phenylenevinylene, phenyleneethynylene, fluorene, and thiophene derivatives with bipyridyl or terpyridyl substituents with the pi-conjugated backbone or at side-chain positions. The positive emission response is largest when Cu(II) is bound to the CP through bipyridyl units within the backbone, making these materials the best candidates for NO sensing by a turn-on emission mechanism.

The ability of the endothelium to produce nitric oxide is essential to maintenance of vascular homeostasis; disturbance of this ability is a major contributor to the pathogenesis of vascular disease. In vivo studies have demonstrated that expression of endothelial nitric oxide synthase (eNOS) is vital to endothelial function and have led to the understanding that eNOS expression is subject to modest but significant degrees of regulation. Subsequently, numerous physiological and pathophysiological stimuli have been identified that modulate eNOS expression via mechanisms that alter steady-state eNOS mRNA levels. These mechanisms involve changes in the rate of eNOS gene transcription (transcriptional regulation) and alteration of eNOS mRNA processing and stability (posttranscriptional regulation). In cultured endothelial cells, shear stress, transforming growth factor-[beta]1, ysophosphatidylcholine, cell growth, oxidized linoleic acid, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, and hydrogen peroxide have been shown to increase eNOS expression. In contrast, tumor necrosis factor-[alpha], hypoxia, lipopolysaccaride, thrombin, and oxidized LDL can decrease eNOS mRNA levels. For many of these stimuli, both transcriptional and posttranscriptional mechanisms contribute to regulation of eNOS expression. Recent studies have begun to further define signaling pathways responsible for changes in eNOS expression and have characterized cis- and trans-acting regulatory elements. In addition, a role has been identified for epigenetic control of eNOS mRNA levels. This review will discuss transcriptional and posttranscriptional regulation of eNOS with emphasis on the molecular mechanisms that have been identified for these processes. gene regulation; mRNA stability; transcription; endothelium; 3'-untranslated region

A new model for the P450 enzyme carrying a [SO.sub.3][.sup.-] ligand coordinated to iron(III) reversibly binds NO to yield the nitrosyl adducts. The thermodynamic and kinetic data for NO binding to the new model complexes of P450 are discussed in reference to earlier results obtained for closely related nitrosylation reactions of cytochrome [P450.sub.cam] and a thiolate-ligated iron(III) model complex.

The neuronal nitric oxide synthases (nNOSr) and nNOSfnr proteins are used to investigate in detail the effects of [Asp.sup.1393] mutations on electron-transfer reactions, NADP(H) binding, FAD thermodynamics, and the regulatory system of nNOSr. In D1393V nNOSr, hydride transfer from NADPH to FAD is so slow that it compromises all downstream electron-transfer events while in D1393N nNOSr, the increased oxidation of reduced flavins by [O.sub.2] and thermodynamics destabilization of the FAD semiquinone uncouples or limits electron transfer to an extent that it inhibits downstream catalysis.

The binding of thyroid hormone to the thyroid hormone receptor (TR) mediates important physiological effects. However, the transcriptional effects of TR mediated by the thyroid response element (TRE) cannot explain many actions of thyroid hormone. We postulate that TR can initiate rapid, non-TRE-mediated effects in the cardiovascular system through cross-coupling to the phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase Akt pathway. In vascular endothelial cells, the predominant TR isoform is TR[[alpha].sub.1]. Treatment of endothelial cells with L-3,5,3'-triiodothyronine ([T.sub.3]) increased the association of TR[[alpha].sub.1] with the p85[alpha] subunit of PI3-kinase, leading to the phosphorylation and activation of Akt and endothelial nitric oxide synthase (eNOS). The activation of Akt and eNOS by [T.sub.3] was abolished by the PI3-kinase inhibitors, LY294002 and wortmannin, but not by the transcriptional inhibitor, actinomycin D. To determine the physiological relevance of this PI3-kinase/Akt pathway, we administered [T.sub.3] to mice undergoing transient focal cerebral ischemia. Compared with vehicle, a single bolus infusion of [T.sub.3] rapidly increased Akt activity in the brain, decreased mean blood pressure, reduced cerebral infarct volume, and improved neurological deficit score. These neuroprotective effects of [T.sub.3] were greatly attenuated or absent in [eNOS.sup.-/-] and TR[[alpha].sub.1.sup.-/-][[beta].sup.-/-] mice and were completely abolished in WT mice pretreated with LY294002 or a [T.sub.3] antagonist, NH-3. These findings indicate that the activation of PI3-kinase/Akt pathways can mediate some of the rapid, non-TRE effects of TR and suggest that the activation of Akt and eNOS contributes to some of the acute vasodilatory and neuroprotective effects of thyroid hormone. nitric oxide | phosphatidylinositol 3-kinase | protein kinase Akt | stroke

A study of the reaction of aquacobalamin, Cbl(H2O) with nitric oxide (NO) as a function of pH and NO and HNO2 concentrations is performed. Unequivocal evidence is presented which states that NO does not interact with Cbl(H2O) at low pH at all but that the observed reactions are induced by traces of nitrite impurities present in aqueous solutions of NO under selected conditions.

The changes in the abundance of inducible nitric oxide synthase (iNOS) and calmodulin (CaM) in response to exposing macrophages to the bacterial endotoxin is examined to understand cellular regulation of iNOS and its relationship to antibacterial activity of macrophages. The results revealed an important role for CaM as a central regulator in mediating macrophage activation.

The discovery of tissue protective effects of erythropoietin has stimulated significant interest in erythropoietin (Epo) as a novel therapeutic approach to vascular protection. The present study was designed to determine the cerebral vascular effects of recombinant Epo in vivo. Recombinant adenoviral vectors ([10.sup.9] plaque-forming units/animal) encoding genes for human erythropoietin (AdEpo) and [beta]-galactosidase (AdLacZ) were injected into the cisterna magna of rabbits. After 48 h, basilar arteries were harvested for analysis of vasomotor function, Western blotting, and measurement of cGMP levels. Gene transfer of AdEpo increased the expressions of recombinant Epo and its receptor in the basilar arteries. Arteries exposed to recombinant Epo demonstrated attenuation of contractile responses to histamine ([10.sup.-9] to [10.sup.-5] mol/l) (P < 0.05, n = 5). Endothelium-dependent relaxations to acetylcholine ([10.sup.-9] to [10.sup.-5] tool/l) were significantly augmented (P < 0.05, n = 5), whereas endothelium-independent relaxations to a nitric oxide (NO) donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt remained unchanged in AdEpo-transduced basilar arteries. Transduction with AdEpo increased the protein expression of endothelial NO synthase (eNOS) and phosphorylated the S1177 form of the enzyme. Basal levels of cGMP were significantly elevated in arteries transduced with AdEpo consistent with increased NO production. Our studies suggest that in cerebral circulation, Epo enhances endothelium-dependent vasodilatation mediated by NO. This effect could play an important role in the vascular protective effect of Epo. recombinant adenoviral vectors; vasodilatation; gene transfer; rabbit doi:10.1152/ajpheart.00045.2006