C60-Fullerene monomalonate adducts selectively inactivate neuronal nitric oxide synthase by uncoupling the formation of reactive oxygen intermediates from nitric oxide production.

C(60)-Fullerene monomalonate adducts inactivate selectively the neuronal nitric oxide synthase isoform in a manner completely preventable by the concurrent presence of superoxide dismutase and catalase. This inactivation is time-, fullerene concentration-, and turnover-dependent and is not reversible by dilution. The di(carboxypropan-3-ol)methano-[60]-fullerene (diol adduct) has no effect on NADPH consumption by nNOS as measured in the absence of arginine substrate, but dramatically increases NADPH consumption in the presence of arginine. This fullerene-enhanced NADPH consumption is linked to oxygen as electron acceptor and is accompanied by the increased production of hydrogen peroxide. These effects of fullerene monomalonate adducts are unique to the nNOS isoform and are not observed using either the iNOS or the eNOS isoform. The inhibitory effects of fullerene monomalonate adducts are unaltered and insurmountable by increased concentrations of arginine, tetrahydrobiopterin, or calmodulin. These observations indicate that fullerene monomalonate adducts uncouple in the presence of arginine the formation of reactive oxygen intermediates from NO production by nNOS. These reactive oxygen intermediates dissociate from the enzyme and, acting from solution, inactivate NOS NO forming activity.

Alkaloid-fullerene systems through photocycloaddition reactions.

The photocycloaddition of tertiary amines to ¿60fullerene (C(60)) is an interesting and useful reaction. We wished to extend the applications of this type of reaction through an investigation of the photoaddition of alkaloids to C(60) for the purpose of synthesizing novel and complex photoadducts that are difficult to obtain by usual methods. Irradiation of tazettine (2) or gramine (3) with C(60) in toluene leads to formation of one monoadduct (6 or 7), whereas scandine (1a) or 10-hydroxyscandine (1b) reacts with C(60) photochemically to give two products, the expected ¿6,6 monoadduct (5a, 5b) and a new type of monoadduct with a bis-¿6, 6 closed structure (4a, 4b). These new structures were characterized by UV-vis, FT-IR, (1)H NMR, (13)C NMR, (1)H-(1)H COSY, ROESY, HMQC (heteronuclear multiple-quantum coherence), and HMBC (heteronuclear multiple-bond connectivity) spectroscopy. The techniques of time-of-flight secondary ion MS (TOF-SIMS) and field desorption MS (FD-MS) were used for the mass determination. (3)He NMR analysis of the product mixture from photoaddition of 1a to C(60) containing a (3)He atom ((3)He@C(60)) led to two peaks at -9.091 and -11.090 ppm relative to gaseous (3)He, consistent with formation of a ¿6, 6-closed monoadduct and a bis-¿6,6 closed adduct. Presumably, the bis-¿6, 6 closed adducts are formed by an intramolecular ¿2 + 2 cycloaddition of the vinyl group to the adjacent 6,6-ring junction of C(60) after the initial photocycloaddition.

Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts.

C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.

Modeling Mercury Removal by Sorbent Injection.

Sorbents for removing mercury from flue gases of coal-fired power plants are presently being evaluated due to potential regulation of mercury emissions under Title III of the 1990 Clean Air Act Amendments. Laboratory tests have been conducted to evaluate the adsorption characteristics of potential sorbents and the effects of flue gas constituents on these characteristics. This paper presents a theoretical model that combines the adsorption characteristics measured in the lab with mass transfer considerations to predict mercury removal by the duct injection process in actual flue gas streams. The model was used to determine the effect of various sorbent properties on mercury removal when injecting a powdered sorbent upstream of either an electrostatic precipitator (ESP) or fabric filter. Mercury removal is expected to differ between these configurations since the mass transfer conditions are different in an ESP and fabric filter. The model was used to determine when mercury removal is limited by mass transfer and when it is limited by sorbent capacity. This information defines conditions when removal can be improved by reducing particle size or increasing sorbent capacity. In both cases, removal can be increased by injecting more sorbent.

Neuroanatomical distribution of vasotocin in a urodele amphibian (Taricha granulosa) revealed by immunohistochemical and in situ hybridization techniques.

Immunohistochemical and in situ hybridization techniques were used to investigate the neuroanatomical distribution of arginine vasotocin-like systems in the roughskin newt (Taricha granulosa). Vasotocin-like-immunoreactive neuronal cell bodies were identified that, based on topographical position, most likely, are homologous to groups of vasopressin-immunoreactive neuronal cell bodies described in mammals, including those in the bed nucleus of the stria terminalis, medial amygdala, basal septal region, magnocellular basal forebrain-including the horizontal limb of the diagonal band of Broca, paraventricular and supraoptic nuclei, suprachiasmatic nucleus, and dorsomedial hypothalamic nucleus. Several additional vasotocin-like-immunoreactive cell groups were observed in the forebrain and brainstem regions; these observations are compared with previous studies of vasotocin- and vasopressin-like systems in vertebrates. Arginine vasotocin-like-immunoreactive fibers and presumed terminals also were widely distributed with high densities in the basal limbic forebrain, the ventral preoptic and hypothalamic regions, and the brainstem ventromedial tegmentum. Based on in situ hybridization studies with synthetic oligonucleotide probes for vasotocin and the related neuropeptide mesotocin, as well as double-labeling studies with combined immunohistochemistry and in situ hybridization, we conclude that the vasotocin immunohistochemical procedures used identify vasotocin-like, but not mesotocin-like, elements in the brain of T. granulosa. The distribution of arginine vasotocin-like systems in T. granulosa is greater than the distribution previously reported for any other single vertebrate species; however, it is consistent with an emerging pattern of distribution of vasotocin- and vasopressin-like peptides in vertebrates. Complexity in the vasotocinergic system adds further support to the conclusion that this peptide regulates multiple neurophysiological and neuroendocrinological functions.

The influence of histatin-5 fragments on the mineralization of hydroxyapatite.

The adsorption of histatin 5 on hydroxyapatite (HAP) was determined and compared to that of several fragments of histatin 5, such as residues 1-16 (N16), 7-16 (M10), 9-24 (C16), 11-24 (C14), 13-24 (C12), 15-24 (C10). The influence of the adsorbed peptides on the seeded crystal growth of HAP was investigated with the constant composition method. The adsorption affinity of the peptides as well as their ability to inhibit mineralization was influenced by the length of the peptide chain. Histatin 5 showed the highest affinity, as determined by a Langmuir model, whereas the smaller C10 and C12 displayed the lowest equilibrium uptake. The smaller C10 and C12 peptides were, on the other hand, more effective as crystal growth inhibitors, indicating a more efficient coverage of surface active sites. Electrophoretic mobility data indicated an increase in the positive charge at the HAP surface in the presence of these peptides, which were efficient HAP crystallite dispersants.

Adsorption and mineralization effects of citrate and phosphocitrate on hydroxyapatite.

The adsorption of citrate and phosphocitrate ions by hydroxyapatite (HAP) surfaces and their influence on the constant composition growth kinetics of HAP have been investigated. Phosphocitrate was strongly adsorbed to HAP and inhibited crystal growth. When HAP surfaces containing preadsorbed citrate were exposed to phosphocitrate, the uptake of the latter markedly increased. The two additives behaved synergistically in their HAP crystal growth inhibition.

Physical chemical studies of calcium oxalate crystallization.

The physical chemical approach to the investigation of the calcium oxalate (CaOx) crystallization and urolith formation is the systematic examination of the various aspects of mineral precipitation and growth in pure solution, in the presence of individual urinary components, and in whole urine media. Recent experimental studies have indicated that while small urinary ions such as citrate, magnesium, and phosphocitrate retard the mineralization rate of CaOx, urinary macromolecules may act either as inhibitors of growth or promoters of nucleation. Some CaOx mineralization inhibitors have also been found to influence the growth mechanism of the phase and its flocculation properties. Therefore, urinary macromolecules that are adsorbed on the mineralizing crystals and incorporated into the developing stone may play a significant role in urolithiasis.

The effects of human salivary cystatins and statherin on hydroxyapatite crystallization.

The adsorption, at hydroxyapatite surfaces of neutral cystatin SN, acidic cystatin S and the phosphoserine-containing acidic cystatin S1 was compared to that of statherin. The effects of these adsorbed proteins on the constant-composition growth kinetics of hydroxyapatite were also studied. The neutral cystatin SN had a higher adsorption maximum than the acidic cystatins S and S1. Although the affinity of cystatin for hydroxyapatite surfaces was lower than that of statherin, their influence on the growth kinetics of hydroxyapatite was considerably greater, with the acidic cystatin S1 being the most active. At a surface concentration of 7.0 x 10(-8) mol m-2 hydroxyapatite, the cystatins decreased the rate of crystal growth by 80-95% as compared to that in the absence of protein. At this concentration, statherin showed a growth inhibition of 40%.