A light and electron microscopic study of NADPH-diaphorase-,calretinin- and parvalbumin-containing neurons in the rat nucleus accumbens

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.

Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolus aureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 104 times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with nH values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme. H4folate; (±)-L-tetrahydrofolate

Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via

Oxalate, formate, formamide, and methanol metabolism in Thiobacillus novellus.

Thiobacillus novellus was able to grow with oxalate, formate, formamide, and methanol as sole sources of carbon and energy. Extensive growth on methanol required yeast extract or vitamins. Glyoxylate carboligase was detected in extracts of oxalate-grown cells. Ribulose bisphosphate carboxylase was found in extracts of cells grown on formate, formamide, and thiosulfate. These data indicate that oxalate is utilized heterotrophically in the glycerate pathway, and formate and formamide are utilized autotrophically in the ribulose bisphosphate pathway. Nicotinamide adenine dinucleotide-linked formate dehydrogenase was present in extracts of oxalate-, formate-, formamide-, and methanol-grown cells but was absent in thiosulfate- and acetate-grown cells.

Electrochemical Functionalization of a Gold Electrode with Redox-Active Self-Assembled Monolayer for Electroanalytical Application

The electrochemical functionalization of a Au electrode with a redox-active monolayer and the electroanalytical applications of the functionalized electrode are described. Reaction of the electrochemically derived o-quinone on the self-assembled monolayer (SAM) of 6-mercaptopurine (MPU) on a Au electrode gives a redox-active 4-(6-mercapto-purin-9-yl)benzene-1,2-diol (MPBD) self-assembly under optimized conditions. Electrochemical quartz crystal microbalance technique has been employed to follow the functionalization of the electrode in real time. Electrochemically derived o-quinone reacts at the N(9) position of the self-assembled MPU in neutral pH. Raman spectral measurement confirms the reaction of o-quinone on MPU self-assembly. MPBD shows a well-defined reversible redox response, characteristic of a surface-confined redox mediator at 0.21 V in neutral pH. The anodic peak potential (Epa) of MPBD shifts by −60 mV while changing the solution pH by 1 unit, indicating that the redox reaction involves two electrons and two protons. The surface coverage (Γ) of MPBD was 7.2 ± 0.3 × 10-12 mol/cm2. The apparent heterogeneous rate constant (ksapp) for MPBD was 268 ± 6 s-1. MPBD efficiently mediates the oxidation of nicotinamide adenine dinucleotide (NADH) and ascorbate (AA). A large decrease in the overpotential and significant increase in the peak current with respect to the unmodified electrode has been observed. Surface-confined MPBD has been successfully used for the amperometric sensing of NADH and AA in neutral pH at the nanomolar level.

Biosynthesis of isoleucine and valine in Mycobacterium tuberculosis H37 Rv

The enzymes involved in the biosynthesis of isoleucine and valine have been shown to be present in cell-free extracts of Mycobacterium tuberculosis H37Rv. In addition to the known enzymes of the pathway, cell-free extracts of this organism contain a new enzyme. When cell-free extracts were incubated with acetolactate and Image -ascorbic acid, without reduced nicotinamide adenine dinucleotide phosphate, the isomer of acetolactate, viz., α-keto-β-hydroxyisovalerate, was found to accumulate and was identified by different methods. The reaction is enzymic, and Image -ascorbic acid cannot be replaced by other reducing agents such as hydroquinone, 2,6-dichlorophenol indophenol, or glutathione; by derivatives of Image -ascorbic acid such as dehydroascorbic acid or dimethyl ascorbic acid; or by cobamide coenzyme. Since the extracts also isomerize α-acetohydroxybutyrate to α-keto-β-hydroxy-β-methylvalerate, the enzyme catalyzing the reaction has been termed “acetohydroxy acid isomerase.” This is the first time that the presence of acetohydroxy acid isomerase has been reported in any biological system and that a specific metabolic role has been assigned for Image -ascorbic acid. The extract also possesses reductase activity to convert α-keto-β-hydroxyisovalerate to α,β-dihydroxyisovalerate in the presence of reduced nicotinamide adenine dinucleotide phosphate.

Enzyme induction in Neurospora crassa

I. Studies on Nicotinamide Adenine Dinucleotide Glycohydrase (NADase)

NADase, like tyrosinase and L-amino acid oxidase, is not present in two day old cultures of wild type Neurospora, but it is coinduced with those two enzymes during starvation in phosphate buffer. The induction of NADase, like tyrosinase, is inhibited by puromycin. The induction of all three enzymes is inhibited by actinomycin D. These results suggest that NADase is synthesized de novo during induction as has been shown directly for tyrosinase. NADase induction differs in being inhibited by certain amino acids.

The tyrosinaseless mutant ty-1 contains a non-dialyzable, heat labile inhibitor of NADase. A new mutant, P110A, synthesizes NADase and L-amino acid oxidase while growing. A second strain, pe, fl;cot, makes NADase while growing. Both strains can be induced to make the other enzymes. These two strains prove that the control of these three enzymes is divisible. The strain P110A makes NADase even when grown in the presence of Tween 80. The synthesis of both NADase and L-amino acid oxidase by P110A is suppressed by complete medium. The theory of control of the synthesis of the enzymes is discussed.

II. Studies with EDTA

Neurospora tyrosinase contains copper but, unlike other phenol oxidases, this copper has never been removed reversibly. It was thought that the apo-enzyme might be made in vivo in the absence of copper. Therefore cultures were treated with EDTA to remove copper before the enzyme was induced. Although no apo-tyrosinase was detected, new information on the induction process was obtained.

A treatment of Neurospora with 0.5% EDTA pH 7, inhibits the subsequent induction during starvation in phosphate buffer of tyrosinase, L-amino acid oxidase and NADase. The inhibition of tyrosinase and L-amino acid oxidase induction is completely reversed by adding 5 x 10^-5M CaCl₂, 5 x 10^-4M CuSO₄, and a mixture of L-amino acids (2 x 10^-3M each) to the buffer. Tyrosinase induction is also fully restored by 5 x 10^-4M CaCl₂ and amino acids. As yet NADase has been only partially restored.

The copper probably acts by sequestering EDTA left in the mycelium and may be replaced by nickel. The EDTA apparently removes some calcium from the mycelium, which the added calcium replaces. Magnesium cannot replace calcium. The amino acids probably replace endogenous amino acids lost to the buffer after the EDTA treatment.

The EDTA treatment also increases permeability, thereby increasing the sensitivity of induction to inhibition by actinomycin D and allowing cell contents to be lost to the induction buffer. EDTA treatment also inhibits the uptake of exogenous amino acids and their incorporation into proteins.

The lag period that precedes the first appearance of tyrosinase is demonstrated to be a separate dynamic phase of induction. It requires oxygen. It is inhibited by EDTA, but can be completed after EDTA treatment in the presence of 5 x 10^-5M CaCl₂ alone, although no tyrosinase is synthesized under these conditions.

The time course of induction has an early exponential phase suggesting an autocatalytic mechanism of induction.

The mode of action of EDTA, the process of induction and the kinetics of induction are discussed.

Low potential detection of glutamate based on the electrocatalytic oxidation of NADH at thionine/single-walled carbon nanotubes composite modified electrode

A glutamate biosensor based on the electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH), which was generated by the enzymatic reaction, was developed via employing a single-walled carbon nanotubes/thionine (Th-SWNTs) nanocomposite as a mediator and an enzyme immobilization matrix. The biosensor, which was fabricated by immobilizing glutamate dehydrogenase (GIDH) on the surface of Th-SWNTs, exhibited a rapid response (ca. 5 s), a low detection limit (0.1 mu M), a wide and useful linear range (0.5-400 mu M), high sensitivity (137.3 +/- 15.7) mu A mM(-1) cm(-2), higher biological affinity, as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, uric acid, and 4-acetamidophenol, did not cause any interference due to the use of a low operating potential (190 mV vs. NHE). The biosensor can be used to quantify the concentration of glutamate in the physiological level. The Th-SWNTs system represents a simple and effective approach to the integration of dehydrogenase and electrodes, which can provide analytical access to a large group of enzymes for wide range of bioelectrochemical applications including biosensors and biofuel cells.

Highly ordered mesoporous carbons as electrode material for the construction of electrochemical dehydrogenase- and oxidase-based biosensors

In this work, the excel lent catalytic activity of highly ordered mesoporous carbons (OMCs) to the electrooxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H2O2) was described for the construction of electrochemical alcohol dehydrogenase (ADH) and glucose oxidase (GOD)-based biosensors.

Electrochemical Sensing and Biosensing Platform Based on Chemically Reduced Graphene Oxide

In this paper, the characterization and application of a chemically reduced graphene oxide modified glassy carbon (CR-GO/GC) electrode, a novel electrode system, for the preparation of electrochemical sensing and biosensing platform are proposed. Different kinds of important inorganic and organic electroactive compounds (i.e., probe molecule (potassium ferricyanide), free bases of DNA (guanine (G), adenine (A), thymine (T), and cytosine (C)), oxidase/dehydrogenase-related molecules (hydrogen peroxide (H2O2/beta-nicotinamide adenine dinucleotide (NADH)), neurotransmitters (dopamine (DA)), and other biological molecules (ascorbic acid (AA), uric acid (UA), and acetaminophen (APAP)) were employed to study their electrochemical responses at the CR-GO/GC electrode, which shows more favorable electron transfer kinetics than graphite modified glassy carbon (graphite/GC) and glassy carbon (GC) electrodes.

A simple route to incorporate redox mediator into carbon nanotubes/Nafion composite film and its application to determine NADH at low potential

Through a new and simple ion-exchange route, two-electron redox mediator thionine has been deliberately incorporated into the carbon nanotubes (CNTs)/Nafion composite film due to the fact that there is strong interaction between any of two among the three materials (ion-exchange process between thionine and Nafion, strong adsorption of thionine by CNTs, and wrapping and solubilizing of CNTs with Nation). The good homogenization of electron conductor CNTs in the integrated films provides the possibility of three-dimensional electron conductive network. The resulting integrated films exhibited high and stable electrocatalytic activity toward NADH oxidation with the significant decrease of high overpotential, which responds more sensitively more than those modified by thioine or CNTs alone. Such high electrocatalytic activity facilitated the low potential determination of NADH (as low as -0.1 V), which eliminated the interferences from other easily oxidizable species. In a word, the immobilization approach is very simple, timesaving and effective, which could be extended to the immobilization of other cationic redox mediators into the CNTs/Nafion composite film. And these features may offer potential promise for the design of amperometric biosensors.

Electrostatic assembly of polyaniline and platinum-poly(amidoamine) dendrimers hybrid nanocomposite multilayer, and its electrocatalysis towards CO and O-2

The electrostatic layer-by-layer assembly method was successfully used in a multilayer buildup of polyaniline (PANT) and platinum nanocrystals encapsulated in the carboxyl-terminated poly(amidoamine) dendrimers (generation 4.5 G4.5COOH) (Pt-G4.5COOH NPs) on solid substrates. Multilayer growth was monitored by ultraviolet-visible (UV-vis) absorption spectroscopy. The AFM observation revealed a molecularly smooth (PANI/Pt-G4.5COOH NPs) multilayer film which is rougher and thicker than the multilayer of PANT and G4.5COOH (G4.5COOH/PANI)(m). The PANI/Pt-G4.5COOH NPs multilayers show a fast surface-confined electron-exchange process at the Au electrode in an acid solution, and remains stable, reversible and electroactive, even in neutral solution. Furthermore, the multilayers show a strong elect rocatalytic response towards CO oxidation and O-2 reduction, and the catalytic capability can be easily tuned by the control of multilayer thickness.

One-step synthesis of gold-polyaniline core-shell particles

A one-step method has been developed for synthesizing gold-polyaniline (Au@PANI) core-shell particles by using chlorauric acid (HAuCl4) to oxidize aniline in the presence of acetic acid and Tween 40 at room temperature. SEM images indicated that the resulting core-shell particles were composed of submicrometre-scale Au particles and PANI shells with an average thickness of 25 nm. Furthermore, a possible mechanism concerning the growth of Au@PANI particles was also proposed based on the results of control experiments.

The modification of screen-printed carbon electrodes with amino group and its application to construct a H2O2 biosensor

Electrooxidation of thionine on screen-printed carbon electrode gives rise to the modification of the surface with amino groups for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The biosensor was constructed using multilayer enzymes which covalently immobilized onto the surface of amino groups modified screen-printed carbon electrode using glutaraldehyde as a bifunctional reagent. The multilayer assemble of HRP has been characterized with the cyclic voltammetry and the faradaic impedance spectroscopy. The H2O2 biosensor exhibited a fast response (2 s) and low detection limit (0.5 muM).

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 muM.