Mechanism of inactivation of inducible nitric oxide synthase by amidines. Irreversible enzyme inactivation without inactivator modification

Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrulline and nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding, and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400W was recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected. Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator of iNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W shows both loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation by another amidine-containing compound, N5-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdin produced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation of iNOS with either 1400W or L-NIO to be biliverdin IXalpha out of the four possible regioisomers. Two mechanisms were previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate, leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methyl group followed by attachment to the heme cofactor, which causes the enzyme to catalyze the heme oxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d3-1400W, which produced no d2-1400W. Detection of carbon monoxide as one of the heme-degradation products further excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanism is proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because of the amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing its conversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one that resembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IXalpha. This appears to be the first example of a compound that causes irreversible inactivation of an enzyme without itself becoming modified in any way.     

(CuBr)3P4Se4: A Low Symmetric Variant of the (CuI)3P4Se4 Structure Type

Bright orange (CuBr)₃P₄Se₄ is obtained from the reaction of CuBr, P, and Se in stoichiometric amounts (CuBr : P : Se = 3 : 4 : 4). The composition and the crystal structure of the compound were determined from single crystal X‐ray diffraction data. Lattice constants are a = 33.627(2) Å, b = 6.402(1) Å, c = 19.059(1) Å, β = 90.19(3) °, V = 4103.2(3) ų, and Z = 12. The compound crystallizes in a structure that is related to (CuI)₃P₄Se₄. Cages of β‐P₄Se₄ are stacked along the b‐axis and are separated by columns of copper(I) bromide. However, the coordination of the β‐P₄Se₄ cage molecules to the copper atoms in the CuBr columns in (CuBr)₃P₄Se₄ is quite different from (CuI)₃P₄Se₄. The monoclinic compound (space group: P2₁, no. 4) has an almost orthorhombic metric in combination with a threefold superstructure in [100]. Structural aspects of (CuBr)₃P₄Se₄ are discussed with respect to the heavier homologue (CuI)₃P₄Se₄.     

Liquid-liquid phase equilibria for some polystyrene-methylcyclohexane mixtures

Liquid-liquid cloud point diagrams of solutions of nearly monodisperse samples of polystyrene (PS), and binary mixtures of nearly monodisperse PS’s, both in methylcyclohexane (MCH), were determined for several polymer molecular weights (M_w) at 0.1 MPa. The bimodal mixtures (PS[M_w(1),ρ(1)] + PS[M_w(2),ρ(2)], M_w(1)=90×10³ g/mol, M_w(2)=13×10³ g/mol, 5.78 × 10³ g/mol, and 2.2 × 10³ g/mol, ρ=1.06) were prepared constraining 〈M_w〉=38.6×10³ g/mol, ρ=M_w/M_n is the polydispersity index. In each case the cloud point curves (CPC’s) for the bimodal mixtures are strongly skewed, lying well above CPC for 〈M_w〉 when φ<φ_CRITICAL, and below CPC for 〈M_w〉 when φ>φ_CRITICAL; φ is volume fraction polymer in the polymer/solvent mixture. The experimental results are discussed in the context of empirical and mean-field representations.     

Separation of actinides from Low Level Liquid Wastes (LLLW) by extraction chromatography using novel DMDOHEMA and TODGA impregnated resins

The uptake of several actinides [U(VI), Th(IV), Am(III), Cm(III)] and fission products was investigated from nitric acid solutions by two novel extraction chromatographic sorbents containing 2-(2-hexyloxy-ethyl)-N,N'-dimethyl-N,N'-dioctyl-malonamide (DMDOHEMA) and N,N,N',N'-tetraoctyl-3-oxapentane-1,5-diamide (TODGA), respectively. The kinetics of the uptake of actinides was studied. The sorption of metal ions fromz simulated Low Level Liquid Waste (LLLW) solutions was evaluated. The results of these experiments revealed that the actinides and lanthanides could be separated from the bulk of other fission products in simulated LLLW solutions on both sorbents.This revised version was published online in November 2005 with corrections to the Cover Date.     

Thermal conductivity, thermal effusivity, and specific heat capacity near the lower critical point of the binary liquid mixture n-butoxyethanol-water

Experimental investigations on binary liquid mixtures near the critical mixing point are presently leading to a controversy about the anomaly in the thermal conductivity. A photopyroelectric technique is used to determine the thermal conductivity and the effusivity of the binary liquid mixture n-butoxyethanol-water at its critical concentration near the critical mixing point. It is proven that, contrary to previous reports, there is no critical enhancement in the thermal conductivity. The specific heat capacity is calculated from these results and compared with the results from measurements performed by adiabatic scanning calorimetry.     

Evaluation of automated nano-electrospray mass spectrometry in the determination of non-covalent protein-ligand complexes

The use of electrospray ionization mass spectrometry (ESI-MS) for studying non-covalent interactions between macromolecules and ligands is well established. ESI-MS can be a useful tool for the determination of dissociation constants between molecules in the gas phase. We validate this method by studying the binding of the catalytic domain of cellobiohydrolase I (CBH I) from Trichoderma reesei to the disaccharide inhibitor cellobiose. The method was further applied to study two newly synthesized cellobiose derivatives (m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside and p-benzyloxybenzyl beta-cellobioside). In a titration experiment, peak areas of different charge states of the free enzyme and the complex were summed in order to determine the dissociation constant. For cellobiose and m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside, the calculated values are in good agreement with those reported from either displacement titration or equilibrium binding experiments in solution. Due to non-specific binding, the dissociation constant of p-benzyloxybenzyl beta-cellobioside does not correspond with the solution-based value. Our results indicate the need for careful interpretation of data sets when using nanoESI to study non-covalent interactions.     

Study of the winter and summer changes of the air composition in the church of Szalowa, Poland, related to conservation

The St. Michael Archangel’s Church in Szalowa, Poland, was selected for closer investigation with respect to the indoor/outdoor air exchange and its influence on the air quality and work arts preservation. Chemical composition, size and abundance of particulate matter and concentration of gases NO₂, SO₂, O₃ inside and outside the church were determined. To study seasonal variation of the weather condition (temperature, inversion level, wind direction) and the influence of seasonal sources of the air pollution (like heating of the nearby houses), samples were collected in winter and summer time. It was stated that suspended particulate matter inside the wooden church has in general an outdoor source. Several groups of particles were distinguished such as the organic ones, soil dust, nitrates and sulphates. In case of organic and soil dust particles, the concentration inside was higher than outside. From the results, in comparison to literature data, one can conclude that accumulation of particulate suspended matter in the church is more intense than in other types of buildings. Gaseous pollutants were detected but their concentration was negligible.     

Exploring the optimal site for modifications of pyranmycins with the extended arm approach

[reaction: see text] Continuing from the syntheses and the antibacterial studies of a library of pyranmycins, we further probed the proximity around ring III of pyranmycin by introducing an "extended arm" that has hydroxyethyl or aminoethyl groups at the O-2' ', O-3' ', or O-4' ' positions. The results from the antibacterial studies reveal the optimal structural motif is the attachment of an extended arm with a terminal hydroxyl group at the O-3' ' position.     

Synthesis of homoceramides, novel ceramide analogues, and their lack of effect on the growth of hippocampal neurons

The synthesis of a new series of D-erythro-homoceramide analogues is described. Several synthetic approaches were investigated. Homoceramides can be successfully synthesized from L-homoserine as chiral building block and a protected Weinreb-amide as a key intermediate. The synthesis of short-chain analogues with a heptyl side chain, as well as with a phenyl residue in the sphingoid part (instead of the naturally occurring tridecyl side chain), was effected. The homoceramides 15-17 and 24 were investigated for their potential to reverse the inhibitory effect of fumonisin B(1) on axonal growth. Unfortunately, none of the tested compounds showed any biological activity due to their lack of metabolism to glucosylhomoceramide.     

Comparison of mono- and polyatomic primary ions for the characterization of organic dye overlayers with static secondary ion mass spectrometry

Organic carbocyanine dye coatings have been analyzed by time-of-flight static secondary ion mass spectrometry (TOF-S-SIMS) using three types of primary ions: Ga(+) operating at 25 keV, and Xe(+) and SF(5) (+) both operating at 9 keV. Secondary ion yields obtained with these three primary ions have been compared for coatings with different layer thickness, varying from (sub)-monolayer to multilayers, on different substrates (Si, Ag and AgBr cubic microcrystals). For (sub)-monolayers deposited on Ag, Xe(+) and SF(5) (+) primary ions generate similar precursor ion intensities, but with Ga(+) slightly lower precursor ion intensities were obtained. Thick coatings on Ag as well as mono- and multilayers on Si produce the highest precursor and fragment ion intensities with the polyatomic primary ion. The yield difference between SF(5) (+) and Xe(+) can reach a factor of 6. In comparison with Ga(+), yield enhancements by up to a factor of 180 are observed with SF(5) (+). For the mass spectrometric analysis of dye layers on AgBr microcrystals, SF(5) (+) again proves to be the primary ion of choice.