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PubMed2005

An amperometric hydrogen peroxide sensor based on immobilization of hemoglobin in poly(o-aminophenol) film at iron-cobalt hexacyanoferrate-modified gold electrode.

A series of hybrid iron-cobalt hexacyanoferrate (FeCoHCF) films were electrodeposited on gold electrodes from solutions containing 6mM Fe(CN)(6)(3-) with different concentrations of Co(2+) and Fe(3+). FeCoHCF films deposited from solutions with different molar ratios of iron were studied by cyclic voltammetry, and their solid states were characterized by Fourier transform infrared spectroscopy. The kind of FeCoHCF film that deposited from a solution with a molar ratio of iron of 0.4 showed the largest response current to H(2)O(2) and was characterized by energy-dispersive X-ray spectroscopy. Therefore, the optimized FeCoHCF film was combined with nonconducting poly(o-aminophenol) (POAP) film that entrapped the hemoglobin (Hb) to construct hydrogen peroxide biosensor. The response current of the Hb/POAP/FeCoHCF/Au electrode (29.8 nA) was nearly 40 and was 1.5 times that of the Hb/POAP/Au (0.7 nA) and POAP/FeCoHCF/Au (20 nA) electrodes, respectively. The Michaelis-Menten constant of Hb in the Hb/POAP/FeCoHCF/Au film was 9.31 mM. These results show that the immobilized Hb in the Hb/POAP/FeCoHCF/Au film exhibits higher catalytic activity and larger response current to H(2)O(2) by the mediation of FeCoHCF. In addition, effects of applied potential, solution pH, and electroactive interferent on the response current of the Hb/POAP/FeCoHCF/Au electrode were investigated in detail.

Aminophenols↗
PubMed1990

Analysis of the active site of the flavoprotein p-hydroxybenzoate hydroxylase and some ideas with respect to its reaction mechanism.

The flavoprotein p-hydroxybenzoate hydroxylase has been studied extensively by biochemical techniques by others and in our laboratory by X-ray crystallography. As a result of the latter investigations, well-refined crystal structures are known of the enzyme complexed (i) with its substrate p-hydroxybenzoate and (ii) with its reaction product 3,4-dihydroxybenzoate and (iii) the enzyme with reduced FAD. Knowledge of these structures and the availability of the three-dimensional structure of a model compound for the reactive flavin 4a-hydroperoxide intermediate has allowed a detailed analysis of the reaction with oxygen. In the model of this reaction intermediate, fitted to the active site of p-hydroxybenzoate hydroxylase, all possible positions of the distal oxygen were surveyed by rotating this oxygen about the single bond between the C4a and the proximal oxygen. It was found that the distal oxygen is free to sweep an arc of about 180 degrees in the active site. The flavin 4a-peroxide anion, which is formed after reaction of molecular oxygen with reduced FAD, might accept a proton from an active-site water molecule or from the hydroxyl group of the substrate. The position of the oxygen to be transferred with respect to the substrate appears to be almost ideal for nucleophilic attack of the substrate onto this oxygen. The oxygen is situated above the 3-position of the substrate where the substitution takes place, at an angle of about 60 degrees with the aromatic plane, allowing strong interactions with the pi electrons of the substrate. Polarization of the peroxide oxygen-oxygen bond by the enzyme may enhance the reactivity of flavin 4a-peroxide.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2004

(Fluoren-9-ylidene)methanedithiolato complexes of gold: synthesis, luminescence, and charge-transfer adducts.

Piperidinium 9H-fluorene-9-carbodithioate and its 2,7-di-tert-butyl-substituted analogue [(pipH)(S(2)CCH(C(12)H(6)R(2)-2,7)), R = H (1a), t-Bu (1b)] and 2,7-bis(octyloxy)-9H-fluorene-9-carbodithioic acid [HS(2)CCH(C(12)H(6)(OC(8)H(17))(2)-2,7), 2] and its tautomer [2,7-bis(octyloxy)fluoren-9-ylidene]methanedithiol [(HS)(2)C=C(C(12)H(6)(OC(8)H(17))(2)-2,7), 3] were employed for the preparation of gold complexes with the (fluoren-9-ylidene)methanedithiolato ligand and its substituted analogues. The gold(I) compounds Q(2)[Au(2)(mu-kappa(2)-S,S-S(2)C=C(C(12)H(6)R(2)-2,7))(2)], where Q(+) = PPN(+) or Pr(4)N(+) for R = H (Q(2)4a) or Q(+) = Pr(4)N(+) for R = OC(8)H(17) [(Pr(4)N)(2)4c], were synthesized by reacting Q[AuCl(2)] with 1a or 2 (1:1) and excess piperidine or diethylamine. Complexes of the type [(Au(PR'3))(2)(mu-kappa(2)-S,S-S(2)C=C(C(12)H(6)R(2)-2,7))(2)] with R = H and R' = Me (5a), Et (5b), Ph (5c), and Cy (5d) or R = t-Bu and R' = Me (5e), Et (5f), Ph (5g), and Cy (5h) were obtained by reacting [AuCl(PR'(3))] with 1a,b (1:2) and piperidine. The reactions of 1a,b or 2 with Q[AuCl(4)] (2:1) and piperidine or diethylamine gave Q[Au(kappa(2)-S,S-S(2)C=C(C(12)H(6)R(2)-2,7))(2)] with Q(+) = PPN(+) for R = H [(PPN)6a], Q(+) = PPN(+) or Bu(4)N(+) for R = t-Bu (Q6b), and Q(+) = Bu(4)N(+) for R = OC(8)H(17) [(Bu(4)N)6c]. Complexes Q6a-c reacted with excess triflic acid to give [Au(kappa(2)-S,S-S(2)C=C(C(12)H(6)R(2)-2,7))(kappa(2)-S,S-S(2)CCH(C(12)H(6)R(2)-2,7))] [R = H (7a), t-Bu (7b), OC(8)H(17) (7c)]. By reaction of (Bu(4)N)6b with PhICl(2) (1:1) the complex Bu(4)N[AuCl(2)(kappa(2)-S,S-S(2)C=C(C(12)H(6)(t-Bu)(2)-2,7))] [(Bu(4)N)8b] was obtained. The dithioato complexes [Au(SC(S)CH(C(12)H(8)))(PCy(3))] (9) and [Au(n)(S(2)CCH(C(12)H(8)))(n)] (10) were obtained from the reactions of 1a with [AuCl(PCy(3))] or [AuCl(SMe(2))], respectively (1:1), in the absence of a base. Charge-transfer adducts of general composition Q[Au(kappa(2)-S,S-S(2)C=C(C(12)H(6)R(2)-2,7))(2)].1.5TCNQ.xCH(2)Cl(2) [Q(+) = PPN(+), R = H, x = 0 (11a); Q(+) = PPN(+), R = t-Bu, x = 2 (11b); Q(+) = Bu(4)N(+), R = OC(8)H(17), x = 0 (11c)] were obtained from Q6a-c and TCNQ (1:2). The crystal structures of 5c.THF, 5e.(2)/(3)CH(2)Cl(2), 5g.CH(2)Cl(2), (PPN)6a.2Me(2)CO, and 11b were solved by X-ray diffraction studies. All the gold(I) complexes here described are photoluminescent at 77 K, and their emissions can be generally ascribed to LMMCT (Q(2)4a,c, 5a-h, 10) or LMCT (9) excited states.

Journal Article↗
PubMed2000

Neuroanatomical identification of mesencephalic premotor neurons coordinating eyelid with upgaze in the monkey and man.

Except during blinks, movements of the upper eyelid are tightly coupled to vertical eye movements. The premotor source for the coordination of lid and eye movements is unknown. The present paper provides the anatomical identification of a new premotor cell group in the rostral mesencephalon of the monkey and human, which lies in close proximity to the premotor center for vertical saccades and is thought to participate in lid-eye coordination. After injections of a retrograde transsynaptic tracer (tetanus toxin fragment C or BII(b)) into the levator palpebrae (LP), the superior rectus (SR), or the inferior oblique (IO) muscle of macaque monkeys, a small circumscribed group of premotor neurons was labeled in the central gray of the rostral mesencephalon, but not after superior oblique or inferior rectus muscle injections. This group lies immediately rostral to the interstitial nucleus of Cajal and medial to the rostral interstitial nucleus of the medial longitudinal fasciculus, each of which contain premotor neurons for vertical saccades, and was termed the M-group. Injections of tritiated leucine into the M-group led to afferent labeling primarily over LP motoneurons. In addition, label was present over the SR- and IO-motoneuron subgroups in the oculomotor nucleus and frontalis muscle motoneurons in the facial nucleus. This projection pattern of the M-group suggests a role in the coordination of the upper eyelid and eyes during upgaze. Double-labeling experiments in macaque monkeys revealed that the M-group is strongly parvalbumin immunoreactive and contains high levels of cytochrome oxidase activity. With these two histochemical markers, the homologue of the M-group was identified in the human brain as well.

Animals↗
PubMed1996

Interaction between parasympathetic and sympathetic nerves in prevertebral ganglia: morphological evidence for vagal efferent innervation of ganglion cells in the rat.

Vagal efferent preganglionic neurons were anterogradely labeled by injecting either DiI or DiA, fluorescent lipophilic carbocyanine dyes, into the dorsal motor nucleus of the vagus of the rat. All neurons of the peripheral nervous system (outside the blood-brain barrier) were then fluorescently counterstained in vivo by injecting Fluorogold (Fluorochrome, Inc., Englewood, CO) intraperitoneally. The upper abdominal prevertebral ganglia, including the numerous microganglia associated with the periarterial plexuses of the celiac and superior mesenteric arteries, were identified and dissected in formalin-fixed tissue under ultraviolet light and stereomicroscopic guidance. In 14 of 15 animals analyzed (93%), labeled vagal efferent fibers were found to penetrate into both the left and right celiac ganglia and the superior mesenteric ganglion, as well as into some of the associated microganglia. These projections formed varicose terminal-like structures, highly suggestive of synaptic contacts surrounding individual ganglion cells. In about half the animals, such vagal innervation was also seen in the left and right suprarenal ganglia. The specificity of vagal efferent labeling was confirmed by control experiments, which included injections in vagotomized animals and direct selective labeling of vagal afferents from the nodose ganglia. It is concluded that vagal efferent preganglionics innervate principal ganglion cells of prevertebral ganglia. These vagal contacts may either directly modulate the postganglionic outflow or else gate some or all of the potential modulatory inputs to these postganglionic neurons, thus allowing the vagal system to exert a more selective influence on sympathetic outflow. Finally, the use of laser scanning confocal microscopy and the in toto Fluorogold staining method for investigations of the peripheral nervous system are discussed.

Animals↗
PubMed1998

XPS and STM studies of pseudo-AgX monolayers organized on Ag(111) and Au/Ag(111) surfaces.

Simple and well-defined model AgX surfaces are useful to illuminate a variety of surface-related phenomena associated with AgX microcrystals in a different perspective with fewer experimental impediments. From this viewpoint, we discuss a couple of pseudo-AgX monolayers that can be organized on a highly planar sputter-grown Ag(111) or ultrathin-Au-covered Ag(111) film. The monolayer structures have been satisfactorily characterized by combination of XPS and STM methods. In particular, the monolayer AgX formed on Au/Ag(111) proved to have an almost identical structure with the bilayer AgX(111) atomic configuration, thus serving as a superior monolayer model of AgX or pseudo 2D ionic crystal for deeper understanding of the processes involving the AgX surfaces.

Gold Compounds↗
PubMed1996

Ultrastructural localization of the intercellular adhesion molecule (ICAM-1) on the cell surface of high endothelial venules in lymph nodes.

BACKGROUND: The high endothelial venules (HEV) in the lymph nodes are essential for lymphocyte recirculation. As a first step, the HEV surface interacts with lymphocytes through adhesion molecules. It is important to know where adhesion molecules are expressed on the surface ultrastructure and how these structures interact with lymphocytes. METHODS: To demonstrate the ultrastructural mechanism of interaction between the HEV surface and lymphocytes through the intercellular adhesion molecule (ICAM-1), rat mesenteric lymph nodes were perfused through the superior mesenteric artery with the primary antibody (antirat ICAM-1 antibody) and secondary antibody (antimouse IgG coupled to 15 nm gold particles), which were diluted with hypothermic University of Wisconsin (UW) solution. After the immunoreaction, we analyzed the HEV three-dimensionally and quantitatively using immunoscanning electron microscopy (ISEM) combined with transmission electron microscopy (TEM). RESULTS: HEV expressed ICAM-1 in a 5-30-fold higher concentration than other vessels. Its distribution was extensive over the luminal surface of the cell down to the junctional area. The endothelial surface of HEV undulated to form branched microfolds, along which ICAM-1 was expressed. Cytoplasmic processes of lymphocytes were seen in microfurrows between microfolds and adhered to the sides of the folds. CONCLUSIONS: These observations imply that the microfolds expressing ICAM-1 and microfurrows are specific ultrastructural features for trapping lymphocytes, thus initiating lymphocyte emigration into the lymph node parenchyma.

Animals↗
PubMed1999

Abdominal peritoneum as a defense organ: analysis of ICAM-1 expression in the LPS-stimulated rat.

To investigate the immune environment of the peritoneal cavity, ICAM-1 (intercellular adhesion molecule) expression on the apical surface of the hepatic peritoneum of LPS (lipopolysaccharide) stimulated rats was analyzed ultrastructurally and chronologically with immunoTEM&SEM. ICAM-1 expression was restricted to the side of microvilli of the mesothelial cells. Microvilli demonstrated bulbous tips and included fuzzy coats and strands. Bulbous tips sometimes expressed the antigen, but fuzzy coats and strands did not. Intervillar cell surfaces lacked its expression. Although ICAM-1 expression increased eightfold 24 hr after stimulation, the selective expression remained unchanged. These results suggest that microvilli are closely associated with cell migration in the peritoneal cavity through adhesion molecules that establish a road for migration.

Animals↗
PubMed2000

Naked Au55 clusters: dramatic effect of a thiol-terminated dendrimer

Reaction of the thiol-terminated fourth-generation dendrimer 2-G4 (96 SH groups) with the gold cluster compound Au55(PPh3)12Cl6 in a 3:1 molar ratio in dichloromethane results in the formation of bare Au55 clusters. The cuboctahedrally shaped Au55 particles coalesce to well-formed microcrystals (Au55) infinity. The role of the dendrimer is not only to remove the phosphine and chlorine ligands but also to act as an ideal matrix for perfect crystal growth. Transmission electron microscopy (TEM), small- and wide-angle X-ray diffraction (SAXRD and WAXRD) measurements indicate a structure where rows of edge-linked Au55 building blocks form a distorted cubic lattice. The X-ray data fit best if a 5% reduction of the Au-Au bond length in the Au55 clusters is assumed, in agreement with previous extended X-ray absorption fine structure (EXAFS) measurements. Energy-dispersive X-ray spectroscopy (EDX) analyses and IR investigations show the absence of PPh3 and Cl in the microcrystals.

Journal Article↗
PubMed2001

Gold is a T cell polyclonal activator in BN and LEW rats but favors IL-4 expression only in autoimmune prone BN rats.

Gold salts are beneficial in the treatment of rheumatoid arthritis but may induce immune-mediated disorders in predisposed patients. Gold salts induce Th2-dependent autoimmunity in Brown-Norway (BN) rats but not in Lewis (LEW) rats. The aim of this study was to define molecular targets of gold salts and to approach why LEW rats are resistant. Gold salts act on early steps of transduction in T cells from BN and LEW rats since they trigger tyrosine phosphorylation of numerous proteins including p56(lck) and a calcium signal which results in IL-4 and IFN-gamma expression by BN and LEW T cells. However, the IL-4 response was favored in BN spleen cells in vitro and in vivo. IFN-gamma, produced in part by CD8(+) cells, contributes to the resistance of LEW rats since gold salt-injected LEW rats receiving anti-CD8 or anti-IFN-gamma mAb displayed the parameters characteristics of gold salt-induced Th2 autoimmunity although to a lesser extent than in BN rats. Gold salts transduce a signal in BN and LEW spleen cells resulting in IL-4 and IFN-gamma gene transcription with a preferential IL-4 response in BN rats, a Th2-prone strain, while IFN-gamma contributes to the resistance of LEW rats.

Animals↗
PubMed2004

Vitamin A distribution and content in tissues of the lamprey, Lampetra japonica.

Vitamin A (retinol and retinyl ester) distribution and content in tissues of a lamprey (Lampetra japonica) were analyzed by morphological methods, namely, gold chloride staining, fluorescence microscopy to detect specific vitamin A autofluorescence, and electron microscopy, as well as high-performance liquid chromatography (HPLC). Hepatic stellate cells showed an abundance of vitamin A stored in lipid droplets in their cytoplasm. Similar cells storing vitamin A were present in the intestine, kidney, gill, and heart in both female and male lampreys. Morphological data obtained by gold chloride staining method, fluorescence microscopy, transmission electron microscopy, and HPLC quantification of retinol were consistent. The highest level of total retinol measured by HPLC was found in the intestine. The second and third highest concentrations of vitamin A were found in the liver and the kidney, respectively. These vitamin A-storing cells were not epithelial cells, but mesoderm-derived cells. We propose as a hypothesis that these cells belong to the stellate cell system (family) that stores vitamin A and regulates homeostasis of the vitamin in the whole body in the lamprey. Fibroblastic cells in the skin and somatic muscle stored little vitamin A. These results indicate that there is difference in the vitamin A-storing capacity between the splanchnic and intermediate mesoderm-derived cells (stellate cells) and somatic and dorsal mesoderm-derived cells (fibroblasts) in the lamprey. Stellate cells derived from the splanchnic and intermediate mesoderm have high capacity and fibroblasts derived from the somatic and dorsal mesoderm have low capacity for the storage of vitamin A in the lamprey.

Animals↗
PubMed1994

Morphology and distribution of astrocytes in the molecular layer of the dentate gyrus in NZB/BlNJ, Dreher, and C57BL/6J mice.

Numerous ectopic granule cells are found in the molecular layer of the dentate gyrus in NZB/BlNJ and dreher mutant mice. These ectopic neurons occur either singly or in small clusters. In contrast, few ectopic granule cells are seen in the dentate molecular layer of C57BL/6J mice and dreher control littermates. In this investigation we have examined the morphology, number, and distribution of molecular layer astrocytes in NZB/BlNJ, dreher homozygotes, dreher littermate controls, and C57BL/6J mice to determine the effect of the presence of ectopic granule cells. In the molecular layer of C57BL/6J mice and dreher control littermates, astrocytes have a typical stellate appearance with processes emanating in all directions. The arborization of astrocytes in areas devoid of ectopic granule cells in NZB/BlNJ mice and dreher homozygotes was similar to that in C57BL/6J mice and dreher control littermates. In contrast, the morphology of astrocytes in the immediate vicinity of ectopic granule cells or ectopic clusters was distinctly non-stellate. Furthermore, the somata and processes of these astrocytes occasionally appeared to make intimate contact with the ectopic granule cells. A quantitative analysis of the number and distribution of astrocytes in NZB/BlNJ vs C57BL/6J mice and dreher vs. control littermates indicated that these parameters were not altered by the presence of the ectopic neurons. We conclude that the trophic effects of ectopic neurons on glial cells can affect the growth and orientation of astrocytic processes without a concomitant effect on glial cell number.

Animals↗