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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

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

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.

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.

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.

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.

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 molecular mechanisms underlying the interactions between nitric oxide (NO) and human recombinant Indoleamine 2,3-dioxygenase (hIDO) were systemically studied with optical absorption and resonance Raman spectroscopies. The data presented provides glimpse of possible regulatory mechanism of hIDO by NO in the cell at molecular level and suggest that NO-dependent regulation is modulated by cellular factors.

The preparation and characterization of nitric oxide (NO) releasing dendrimer conjugates from primary and secondary amine functionalized dendrimers are reported. It is observed that the total NO released from secondary amine conjugates is significantly greater than from the primary amine parent dendrimers due to the enhanced stability of secondary amine diazeniumdiolates.

The radical reaction mechanism of FCO + NO on the ground electronic state energy surface is studied at the G2M level of theory based on the geometric parameters optimized at the B3LYP/6-311+G(d) level of theory. The theoretical result shows that the predicted total rate constants exhibit a negative-temperature dependence and positive-pressure effect at lower temperatures.

The best currently available values for the enthalpy of formation of H[O.sub.2], and new Active Thermochemical Table (ATcT)values for several other species, such as NO, N[O.sub.2], and OH were used on the basis of ATcT approach. The experiments were performed in [[C.sub.2][H.sub.5]I/N[O.sub.2]] mixtures, using the reflected shock tube technique and OH-radical electronic absorption detection and using a multipass optical system.

The formation yields of 2- and 3-pentyl nitrate from the reactions of 2- and 3-pentyl peroxy radicals with NO are measured at room temperature using OH radical-initiated reaction of n-pentane to generate the pentyl peroxy radicals. The influence of 2- and 3-pentyl nitrate formation from the reaction of 2- and 3-pentoxy radicals with N[O.sub.2] was investigated by conducting experiments with the initial C[H.sub.3]ONO and NO concentrations being varied by a factor of 5-10.

An experimental study of the mutual sensitization of the oxidation of NO and a natural gas blend was conducted in a fused silica jet-stirred reactor (JSR) from fuel-lean to fuel-rich conditions. A detailed chemical kinetic reaction mechanism was validated for the interaction between NO and pure hydrocarbons and clarify the relative importance of the reactions of NO with H[O.sub.2] and R[O.sub.2] in the No-sensitized oxidation of natural gas under high pressure.

Density functional theory is used to characterize the bimetallic-bridging intermediate in the reduction of NO to N2O by heme-copper oxidoreductases. The information of the electronic and vibrational structure of the putative intermediate, which forms the basis for a quantitative accurate atomic-level description of the key steps in the N-N bond formation and the N-O bond cleavage mechanism is provided.

The in situ polarization-modulation infrared absorption spectroscopy is used to study the adsorption/ dissociation of NO on Rh(111). While these studies are not conclusive regarding the detailed surface structures formed during adsorption, they have provided important new information on the dissociation of NO on Rh(111).

A novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS) are designed and expressed. Laser flash photolysis is used to investigate the kinetics of the intramolecular electron transfer (IET) between the FMN and the heme domains in a novel two-domain murine inducible iNOS oxyFMN construct in which only the FMN and oxygenase domains are expressed.

The formation, reaction dynamics and kinetics and mechanism of the reaction between nitrous acid and N-acetylpenicillamine (NAP) to produce S-nitroso-N-acetylpenicillamine (SNAP) is analyzed in acidic medium. The nitrosation of NAP is accomplished through the direct reaction of nitrous acid with NAP as well as through the formation of the nitrosyl ion, which is abundant only in highly acidic environments.

The structure and environment of Bacillus subtilis (bsNOS) heme were examined with resonance Raman (RR) and ATR-FTIR spectroscopies, to better understand the actual function and role of bacterial NO synthase (NOS)-like proteins, which are similar in structural homology to the oxygenase domain of mammalian NOSs (mNOSs). The bsNOS and mNOS heme pocket structures are found similar, but differ in distinct heme environments, greater electron-donation by bsNOS ligand and the absence of a marked influence of tetrahydrobiopterin on bsNOS heme properties.

The synthesis, photochemistry and spectroscopic properties of a new fluorocein-derivatized iron sulfur nitrosyl compound, the Roussin's red salt ester bis((mu-S mu-S')-fluorescein-2-thioethyl-ester)-tetranitrosyldiiron (Fluor-RSE) is described. Irradiation of the trichromophoric complex with UV-vis light results in cluster decomposition with modest quantum yields and the subsequent release of NO, ferrous ions and a very bright thiolated ethyl fluorescein analogue.

Two color resonance-enhanced multiphoton ionization (REMPI) scheme of NO2 through the E (super 2)SIGMA(sub u)(super +) Rydberg state was used to prepare NO2(super +) in its ground and vibrational states. A model is proposed to account for both the collision energy and the vibrational state dependence.

Four peptides were synthesized corresponding to the first 28 amino acids of N-terminal region of endothelial nitric oxide synthase. Since three acylated peptides exerted some degree of perturbation on thermal transition of vesicles, no conclusion could be drawn about the role of acylation in the translocation to sphigomyelin- and cholesterol-enriched caveolar subdomains.

The initial step in the second half-reaction of the mechanism of nitric oxide synthases (NOSs) is investigated by employing density functional theory method. A detailed and comprehensive density functional theory (DFT) based-investigation on the key initial step of the second half-reaction of the NOS is performed.

The formation of NO(sub x) and N(sub 2)O during plasma destruction of a range of compounds including chlorinated, brominated, fluorinated, and iodinated methanes, with air as carrier gas are studied. It is found that the amount of NO(sub x) formed is unaffected by the addition of a few hundred parts per million of a simple hydrocarbon (e.g. methane).

The room-temperature reactions of nitric oxide with 46 atomic cations are surveyed using inductively-coupled plasma/selected-ion flow tube tandem mass spectrometer. The substantial kinetic results obtained demonstrate that atomic cations initiate a very rich chemistry in their reactions with nitric oxide in the gas phase at room temperature.

The selectivity of electrochemical nitric oxide (NO) reduction by heme-didodecyldimethylammonium bromide (DDAB) and myoglobin-DDAB films as a function of potential and pH are examined. The selectivity of the pathway depends on whether a proton or NO molecule is transferred to a Fe(super II)-NO(super -) reaction intermediate and is influenced by pH, NO concentration and potential.

The study demonstrates the detection of His-heme a3(super 2+)-NO/CU(sub B)(super 1+) species in the reduction of NO to N2O by ba3-oxidase. It is suggested that the reduction of NO to N2O by ba3-oxidase proceeds by the fast binding of the first NO molecule to heme a3 with high-affinity, and the second NO molecule binds to Cu(sub B) with low affinity, producing the temporal co-presence of two NO molecules in the hemecopper center.

The kinetics of the binding and release of NO is studied as a function of pH, temperature, and pressure by stopped-flow and laser flash photolysis techniques. The mechanistic interpretation is further substantiated by the results of water-exchange studies performed on the polyanionic porphyrin complex as a function of pH, temperature and pressure.

3-5'Ether-linked pseudooligopentose derivatives are synthesized for the first time from readily available carbohydrate precursors. The 1,2-isopropylidene-protected ether-linked oligopentoses are potentially important as precursors of novel RNA analogues.

The ferrous [Fe(super 2+)] complexes of nitrophorin 4 (NP4) with nitric oxide (NO), CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy are reported. The ferrous complexes exhibit absorption spectra that are similar to the analogous derivatives of myoglobin and exhibit heme coordination geometry that is consistent with a reduced form of the protein.

The enediamine tautomer of a variety of substituted amidine free bases reacts with nitric oxide (NO) to produce compounds containing a carbon-bound diazeniumdiolate [R(super 1)R(super 2)R(super 3)C-N(O)=NO(super -)] functional group. The reaction should be applicable to a variety of pharmaceutical agents, including NO synthesis inhibitors, antitumor agents, and antibacterials.

The sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria is examined. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.

The mechanism of electrocatalytic reduction of nitric oxide on Pt(100)-(1 x 1) in acidic media is studied using voltammetry, in-situ infrared spectroscopy, and on-line mass spectroscopy, considering the effects of surface defects, NO coverage, and the nature of the supporting electrolyte. It is suggested that HNO(sub ads) is the intermediate appearing both in hydroxylamine (HAM) reduction/oxidation and in NO(sub ads) reduction.

Several 4-substituted-2,6-dimethylnitrobenzenes are characterized as a new type of nitric oxide donor. The computational study and ultraviolet-visible-light spectrum highlights the importance of the conformation of the nitro group, the intensity of light energy absorption, and the length of the conjugated pi-electron system.

A study of the denitrosation mechanism of N(super 1)-nitrosomelatonin (NOMe1) as a model for N(super 1)-nitrosoindoles is presented. The results revealed that NOMe1 slowly decomposes spontaneously in aqueous solution, generating melatonin as the main organic product.

An investigation of the kinetics of the reaction of several alkene-derived chloroalkylperoxy radicals with NO is described. The measurements are conducted at 100 Torr and 298 K under pseudo-first-order kinetics conditions via a turbulent flow tube coupled to a high-pressure chemical ionization mass spectrometer that allowed for direct detection of the chloroalkylperoxy radicals.

The chemical fixation of nitric oxide reacting with alkynyllithium to produce 5-methyl-3-oxide-1,2,3-oxadiazole is investigated. The findings suggest that the title reaction is stepwise and exothermic.