Melatonin prevents magnetic-field-induced neurite outgrowth in a subline of pheochromocytoma cells,PC12D

Exposure to extremely low frequency (ELF) magnetic fields (MFs) has been reported to affect several cellular processes, including cell growth and differentiation. Other research has demonstrated that the pineal gland and its hormone melatonin have a wide spectrum of effects in cells and organs and can exert modulatory actions on cell proliferation and cell differentiation. Since ELF electric and magnetic fields have been shown to influence pineal activity and melatonin synthesis and/or function, it has been suggested that some of the reported effects of ELF MFs could be a consequence of the direct action of these fields on the pineal gland and/or melatonin function. Possible interactions between MFs and melatonin effects are in an early stage of investigation. In this study, we have investigated the influence of melatonin on the in vitro response of a subline of pheochromocytoma cells, PC12D, to a MF. Cells were exposed to the combined action of a physiological concentration (10⁻¹⁰ M) of melatonin and a vertical, 50 Hz, 40 mG rms MF for 23 h. At the end of the treatment, the percentages of neurite-bearing cells were determined by microscopic examination and compared with those from samples treated with the field alone or with melatonin alone. MF exposure alone significantly increased the neurite outgrowth when compared with negative controls, supporting previous results by Blackman and coworkers; this effect was not observed when melatonin was present in the medium from the onset of the exposure. Although the mechanisms of action of melatonin and ELF MFs at the cellular level remain unknown, the present data suggest that physiological levels of melatonin can prevent PC12D cells from responding to the MF stimulus.     

Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by microdialysis-microbore liquid chromatography

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2–0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 μl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.     

Single nanocrystal arrays on patterned poly(ethylene glycol) copolymer microstructures using selective wetting and drying

Single nanocrystal arrays were fabricated on sub-microwells of poly(ethylene glycol) (PEG) copolymer using selective wetting on the hydrophilic regions of the exposed substrate surface and subsequent drying. Templates were produced by molding a thin film of a PEG-based random copolymer on hydrophilic substrates such as glass or silicon dioxide. The polymeric microstructures provide a topographical barrier around the well, which makes it possible to create nanocrystal arrays with controlled geometrical features. The size of the nanocrystal was found to decrease with decreasing well size and also decrease with decreasing topological height. A simple empirical equation was derived to predict the size of the crystal as a function of the pattern size and height, which is in good agreement with the experimental data.     

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells

Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.     

Changes in neurite outgrowth but not in cell division induced by low EMF exposure: influence of field strength and culture conditions on responses in rat PC12 pheochromocytoma cells

The effects of low electromagnetic field (EMF) exposure (4.5-15.8 microT, 50 Hz AC) on neurite outgrowth and cell division in rat PC12 pheochromocytoma cells were examined. The study involved two separate experimental series in which culture conditions during exposure to the magnetic fields differed. In series 1 (14 experiments in which culture conditions were not strongly conducive to cell differentiation [15% serum]), exposure to 4.5-8.25 microT for 23 h significantly inhibited neurite outgrowth by 21.5 +/- 1.3% (by Manova, p = 0.003). In contrast, in series 2 (12 experiments in which culture conditions promoted cellular differentiation [4% serum]), exposure to 4.35-8.25 microT for 23 h significantly stimulated neurite outgrowth by 16.9 +/- 1.1% (by Manova, p = 0.009). Thus, in both series, exposure to a narrow range of low EMF has significant, but opposite effects on neurite outgrowth. Exposure to higher fields, 8.25-12.5 microT (series 1) and 8.25-15.8 microT (series 2) had no significant effect on neurite outgrowth. These data, when considered with other reports, suggest that neuronal differentiation can be altered by low level EMF exposure. While this may not be detrimental, it merits further research. At present, the reasons for the significant changes in neurite outgrowth being confined to the same narrow field strength are unclear. As stated above, culture conditions in series 2 were more conducive to cell differentiation than those in series 1. This is reflected in the lower number of cells in control samples in series 2, at the end of the 23-h incubation, than in series 1 (- 16.9 +/- 1.7%, p = 0.003). As the same numbers were plated in both series, the medium used in series 1 allows more of the PC12 cells to divide; this is consistent with some cells reverting to a non-neuronal adrenal chromaffin phenotype [L. Greene, A. Tischler. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U. S. A., 73 (1976) 2424-2426]. Exposure to both ranges of magnetic fields (4.35-8.25 and 8.25-15.8 microT) has no effect on cell division. Thus, there is no evidence in this study that there is a mitogenic effect arising from low EMF exposure.     

Ordered arrays of semi-crown ligands on an Au(111) electrode surface: in situ STM study

Self-organized systems have attracted much at-tention due to their potential applications in nano- technology as a bottom-up?approach for the con-struction of molecule-scale devices and nanostruc-tures[1—4]. Beyond the self-assembly of small molecu-lar building blocks, Schnherr et al. recently suc-ceeded in arranging the rosette supramolecular nanos-tructures in two dimensions on HOPG[5,6]. Moreover, interest has tremendously increased in the su-pramolecular structures via coordination-dr…     

The role of chalcones: helichrysetin,xanthohumol, and flavokawin-C in promoting neurite outgrowth in PC12 Adh cells

Chalcones are a group of compounds widely distributed in plant kingdom. The aim of this study was to assess the neurite outgrowth stimulatory activity of selected chalcones, namely helichrysetin, xanthohumol and flavokawin-C. Using adherent rat pheochromocytoma (PC12 Adh) cells, the chalcones were subjected to neurite outgrowth assay and the extracellular nerve growth factor (NGF) levels were determined. Xanthohumol (10 μg/mL) displayed the highest (p < 0.05) percentage of neurite-bearing PC12 Adh cells and the highest (p < 0.05) NGF level in the culture medium of xanthohumol-treated cells. While, helichrysetin induced a moderately high numbers of neurite-bearing cells, flavokawin-C did not stimulate neurite outgrowth. This work supports the potential use of xanthohumol as a potential neuroactive compound to stimulate neurite outgrowth.     

Highly enhanced electrocatalytic oxidation of glucose on Cu(OH)2/CuO nanotube arrays modified copper electrode

A highly sensitive and fast-response biosensor based on cupric hydroxide/oxide (Cu(OH)₂/CuO) nanotube arrays (CNA) was successfully fabricated in this work. CNAs were prepared on copper electrode surface by simply immersing copper electrode in an aqueous solution of NaOH and (NH₄)₂S₂O₈. The morphology and the composition of the CNAs were characterized by scanning electron microscopy (SEM) and X-ray diffraction spectroscopy (XRD), respectively. The electrocatalytic activity of the CNA modified copper electrodes (CNA/Cu) towards glucose oxidation was investigated by cyclic voltammetry and amperometry. The CNA/Cu showed good non-enzymatic electrocatalytic responses to glucose in alkaline media and can be used for the development of enzyme-free glucose sensors.     

Recent advances on boron doped diamond (BDD) electrode as cathode in organic and inorganic preparative electrotransformations

Although anodic oxidation reactions mediated by boron-doped diamond (BDD) electrodes have been broadly studied, research on cathodic conversion remains still scarce despite the favorable characteristics of this electrode for such application. A detailed survey of the recent literature about processes in which boron-doped diamond electrode has been employed in cathodic reactions is presented, including inorganic species (O₂, CO₂, nitrate, metaborate) and organic compounds (carbonyl, nitro groups and halogenated compounds).     

Influence of molecular size,polarity and charge on the retention of organic molecules by nanofiltration

Because of the growing interest in nanofiltration for industrial use, a better insight in the retention mechanisms in nanofiltration is needed, which will make it possible to understand membrane performances for specific applications. In this paper, the retention of a series of organic molecules by four nanofiltration membranes was studied. The membranes that were used are NF70 (Dow/FilmTec), NTR 7450 (Nitto-Denko), UTC-20 (Toray Ind.) and Zirfon^® (VITO). In order to correlate the retention with the size of the molecule, which is the main factor that determines the retention, use was made of different parameters for the molecular size: molecular weight, the Stokes diameter, the equivalent molar diameter, and a diameter obtained with energy minimisation calculations. For each size parameter, the correlation with retention in nanofiltration experiments was calculated. For the Zirfon^® membrane, retentions were too low to obtain a good correlation. For the three other membranes, a good correlation with retention was found for each of the size parameters. Two other factors were found to have an influence on retention of organic molecules: the polarity of the molecule, and the charge of the molecule. The importance of these factors depends on the molecules as well as on the type of membrane.     

The kinetics and saturation of reversible adsorption on patterned (heterogeneous) surfaces

We analytically examine the time-dependent adsorption of analyte (solute) on a finite-sized adsorption region as a model for sensors utilizing patterned or heterogeneous surfaces. We account for both reversible adsorption (assuming first-order reaction) and saturation of the adsorption patch that may arise either from packing constraints (finite area) or because of a finite number of binding sites (ligands). Our main conclusions include the following: (1) Saturation effects, due to either finite patch size or finite number of binding sites, become significant at extremely short times. (2) Increasing the strength of binding between the analyte and the adsorption sites increases the adsorbed amount at short times, but, at long times, the mass adsorbed on a weakly binding patch is higher than that on a strongly binding one. (3) The sensitivity of detection, as defined by the adsorption of the minimal analyte mass required for signaling, over a fixed period of time, does not scale as 1/detection time. As a result, increasing the time over which adsorption occurs increases sensitivity, but not linearly. Sensitivity of detection also increases with increasing patch area and initial binding strength.     

Preparation and electrochemical behaviour of hydrophobic vitamin B(12) covalently immobilized onto platinum electrode

Hydrophobic vitamin B(12) was covalently immobilized onto a platinum electrode surface, and the immobilized complex exhibits Co(ii)/Co(i) redox couple and in situ the Co(i) species reacts with phenethyl bromide to form styrene under irradiation with visible light with a turnover number of over 6000 for 1 h.     

p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.     

Thermal decomposition of a series of tetranuclear carbonyl clusters Rh4(CO)12, Rh2Co2(CO)12, RhCo3(CO)12 and Co4(CO)12

Decarbonylation of the unsupported clusters Rh₄(CO)₁₂, Rh₂Co₂(CO)₁₂, RhCo₃(CO)₁₂ and Co₄(CO)₁₂ in a stream of hydrogen has been investigated by temperature programmed decomposition. Kinetic parameters for the thermal decomposition are presented, and the stabilities of the clusters are discussed. The profile for evolution of CO from Rh₄(CO)₁₂ indicates that a stable intermediate is formed. In all four cases methane is formed stepwise until most of the CO groups are evolved.     

Synthesis of 12(S),20-, 12(S),19(R)-, and 12(S),19(S)-dihydroxyeicosa-cis-5,8,14-trans-10-tetraenoic acids,metabolites of 12(S)-hete

Enantiospecific syntheses of the 20- and both 19-hydroxy metabolites of 12(S)-HETE were accomplished using readily available, chiral precursors.     

Dependence of patterned binary alkanethiolate self-assembled monolayers on "UV-photopatterning" conditions and evolution with time, terminal group, and methylene chain length

We have investigated the mechanism of UV photopatterning of binary alkanethiolate self-assembled monolayers (SAMs) adsorbed on Au(111) using time-of-flight secondary ion mass spectrometry. The SAMs were photopatterned using a 500 W Hg arc lamp. The patterning process is strongly dependent on the wavelength of light used. When an unfiltered arc lamp is employed, IR light impinges on the sample and causes considerable sample heating. Methyl-terminated SAMs with less than 14 carbons in the chain melt at the temperatures reached and become very disordered and so can be easily displaced by a second SAM. This leads to significant pattern degradation ("erosion"). SAMs with greater than 14 carbons undergo a transition to an incommensurate phase but remain stable on the surface, and the pattern is retained. When the IR light is filtered out, a different behavior is observed. UV-photopatterned methyl-terminated SAMs with 10 carbons in the chain are stable. Terminal group interactions, such as H-bonding, provide extra stabilization energy during photopatterning, so some patterns with shorter carbon chains may also be stable. The displacement of the photooxidized SAMs on the patterned surface follows kinetics similar to that of large-area SAM formation.     

Synthesis, Characterization, and Thermolysis of C(15)N(12)

Synthesis of 1-methyl-2-fluoro-4,5-dicyanoimidazole was done by halogen exchange between 1-methyl-2-bromo-4,5-dicyanoimidazole and potassium fluoride. Halogen exchange between 1-methyl-2-bromo-4,5-dicyanoimidazole and lithium chloride in N-methylpyrrolidinone at 150 degrees C yielded 1-methyl-2-chloro-4,5-dicyanoimidazole, and additional heating to 210 degrees C resulted in the demethylation to yield 2-chloro-4,5-dicyanoimidazole. Thermolyses of the 2-halo-4,5-dicyanoimidazole derivatives (F, Cl) and 1-iodo-2-halo-4,5-dicyanoimidazole derivatives (Cl, Br, I) between 100 and 290 degrees C were found to yield Tris(imidazo)[1,2-a:1,2-c:1,2-e]-1,3,5-triazine-2,3,5,6,8,9-hexacarbonitrile, or HTT, with (C(5)N(4))(3) composition. HTT has been characterized and purified and the crystal structure obtained. Thermolysis of HTT at 490-500 degrees C gives a material with C/N = 1.020. The thermal properties of HTT and its decomposition products show thermal stability to 350 degrees C.     

Theoretical study of isomerism, structure, and stability of dimers of C-doped aluminide clusters (C@Al12)2 and (C@Al12)(L@Al12) (L = Si, Ge)

The geometric, energetic, and vibrational characteristics of the dimers of carbon-doped aluminum clusters (C@Al₁₂)₂, (C@Al₁₂)(Si@Al₁₂), and (C@Al₁₂)(Ge@Al₁₂) have been calculated by ab initio density functional theory B3LYP method with the basis sets 6-31G* and 6-311+G*. As distinct from the silicon and germanium analogues (Si@Al₁₂)₂ and (Ge@Al₁₂)₂ with an AA lowest-lying structure, in which the Si@Al₁₂ and Ge@Al₁₂ blocks retain the shape of distorted icosahedra A, the symmetric AA structure is not typical of the (C@Al₁₂)₂ dimer (itcorresponds to a transition state) and the most favorable structures are two closely spaced BB and AD isomers, in which the C@Al₁₂ blocks have either a distorted icosahedral (A) or nonicosahedral geometry (B, a dicapped ten-vertex polyhedron; D, a marquee with a centered hexagonal base). The calculated energy of dissociation of the most favorable isomer AD into isolated C@Al₁₂ (I _h ) monomers is ～45 kcal/mol, which is 1.5–2 times as large as the corresponding dissociation energy of (Si@Al₁₂)₂ and (Ge@Al₁₂)₂. For mixed dimers of the (C@Al₁₂)(L@Al₁₂) type with L = Si or Ge, three types of low-lying isomers (AA, BA, and DA) are predicted in which the Si@Al₁₂ and Ge@Al₁₂ blocks retain their shape A and the C@Al₁₂ blocks have geometry A, B, and D, respectively. For all the isomers, the block-block bonds are comparable in length and energy to the bonds inside the blocks and have the same chemical nature. Specific features of the geometry, relative stability, ionization potentials, electron affinity, vibrational spectra, and other characteristics of doped aluminide dimers, which could be used for the identification of their isomers by spectroscopic methods, are discussed.     

General approach for the synthesis of 12-methoxy-substituted sarpagine indole alkaloids including (-)-12-methoxy-N(b)-methylvoachalotine, (+)-12-methoxy-N(a)-methylvellosimine, (+)-12-methoxyaffinisine, and (-)-fuchsiaefoline

[structures: see text] The enantiospecific synthesis of 7-methoxy-D-tryptophan ethyl ester was completed by combination of the Larock heteroannulation process with a Sch?llkopf-based chiral auxiliary in good yield. This ester was then employed in the first regiospecific, stereospecific total synthesis of (+)-12-methoxy-N(a)-methylvellosimine, (+)-12-methoxyaffinisine, (-)-fuchsiaefoline, and 12-methoxy-N(b)-methylvoachalotine in excellent overall yield. The asymmetric Pictet-Spengler reaction and enolate-driven palladium-catalyzed cross-coupling processes served as key steps. The quaternary center at C16 of 12-methoxy-N(b)-methylvoachalotine was established via the Tollens reaction between (+)-12-methoxy-N(a)-methylvellosimine and formaldehyde to form diol 17. The two prochiral primary alcohols in diol 17 were differentiated by the oxidative cyclization(DDQ) of the hydroxyl group at the axial position of 17 with the benzylic postion at [C6] to form a cyclic ether [C6-O17]. After oxidative formation of the alpha-ester at C16, the ether bond was reductively cleaved with TFA/Et3SiH in high yield. The DDQ-mediated oxidative cyclization and TFA/Et3SiH reductive cleavage served as protection/deprotection steps in order to provide a versatile entry into the voachalotine alkaloids.