Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

A COMPARATIVE-STUDY ON STM IMAGING AND ELECTROCATALYTIC ACTIVITY OF DIFFERENT SURFACES MODIFIED WITH FLAVIN ADENINE-DINUCLEOTIDE

Flavin adenine dinucleotide (FAD) was modified onto the highly oriented pyrolytic graphite (hopg) and glassy carbon electrode (gee) surfaces with three methods, respectively. Corresponding image analysis for FAD-modified hopg surfaces has been performed by scanning tunnelling microscope (STM) for the first time. The molecular resolution STM image of FAD adsorbed on the freshly-cleaved hopg was obtained, the quantitative size determination suggests that the FAD molecules adsorb side lying on the substrate surface. The anodization treatment of hopg surface yields many pits, which were clearly observed under STM. These pits provide active sites on the hopg surface for modification and the treated hopg can strongly adsorb FAD molecules, the latter exhibiting an irregular cluster structure on such a surface. When FAD was electrochemically deposited on the substrate surface, a chain structure was successfully observed. The adsorbed FAD on anodized glassy carbon electrode (gee) surface can effectively catalyze the reduction of glucose oxidase, hemoglobin and myoglobin, with a large decrease in the overvoltage, whereas the deposited FAD film exhibits excellent electrocatalysis towards dioxygen reduction.

ELECTROCATALYTIC OXIDATION OF REDUCED NICOTINAMIDE COENZYMES AT METHYLENE GREEN-MODIFIED ELECTRODES AND FABRICATION OF AMPEROMETRIC ALCOHOL BIOSENSORS

Chemically modified electrodes with Methylene Green adsorbed on the graphite surface and incorporated into carbon paste exhibit excellent electrocatalytic ability for oxidation of NADH. Alcohol dehydrogenase, nicotinamide adenine dinucleotide (NAD+) and m

ELECTROCATALYTIC REDUCTION OF DIOXYGEN BY AN ELECTROCHEMICALLY POLYMERIZED FLAVIN ADENINE-DINUCLEOTIDE FILM

An electrochemically polymerized flavin adenine dinucleotide (FAD) film electrode is reported for the first time. The polymerized film was prepared by a two-step method. The electrocatalytic reduction of dioxygen at a glassy carbon electrode (GCE) modifie

Riboflavin, Flavin Mononucleotide and Flavin Adenine Dinucleotide in Human Plasma and Erythrocytes at Baseline and after Low-Dose Riboflavin Supplementation.

Background: Vitamin B2 exists in blood as riboflavin and its cofactors, flavin mononucleotide (FMN) and FAD. The erythrocyte glutathione reductase activation coefficient (EGRAC) has traditionally been used to assess vitamin B2 status in humans. We investigated the relationships of EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD in elderly volunteers and their responses to riboflavin administration. Methods: EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD were determined in 124 healthy individuals with a mean age of 69 years. The same measurements were made in a subgroup of 46 individuals with EGRAC 1.20 who participated in a randomized double-blind 12-week intervention study and received riboflavin (1.6 mg/day; n = 23) or placebo (n = 23). Results: Median plasma concentrations were 10.5 nmol/L for riboflavin, 6.6 nmol/L for FMN, and 74 nmol/L for FAD. In erythrocytes, there were only trace amounts of riboflavin, whereas median FMN and FAD concentrations were 44 and 469 nmol/L, respectively. Erythrocyte FMN and FAD correlated with each other and with EGRAC and plasma riboflavin (P

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

Nicotinamide nucleotide and adenylate concentrations in mante, siphon and gill tissue of old and young Laternula elliptica individuals under control and experimental conditions

Future oceans are predicted to contain less oxygen than at present. This is because oxygen is less soluble in warmer water and predicted stratification will reduce mixing. Hypoxia in marine environments is thus likely to become more widespread in marine environments and understanding species-responses is important to predicting future impacts on biodiversity. This study used a tractable model, the Antarctic clam, Laternula elliptica, which can live for 36 years, and has a well-characterized ecology and physiology to understand responses to hypoxia and how the effect varied with age. Younger animals had a higher condition index, higher adenylate energy charge and transcriptional profiling indicated that they were physically active in their response to hypoxia, whereas older animals were more sedentary, with higher levels of oxidative damage and apoptosis in the gills. These effects could be attributed, in part, to age-related tissue scaling; older animals had proportionally less contractile muscle mass and smaller gills and foot compared with younger animals, with consequential effects on the whole-animal physiological response. The data here emphasize the importance of including age effects, as large mature individuals appear to be less able to resist hypoxic conditions and this is the size range that is the major contributor to future generations. Thus, the increased prevalence of hypoxia in future oceans may have marked effects on benthic organisms' abilities to persist and this is especially so for long-lived species when predicting responses to environmental perturbation.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

Higher plants share with animals a responsiveness to the Ca2+ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45Ca2+ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca2+ with a K1/2 = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca2+ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca2+ pathway distinguishes it from the InsP3- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca2+ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca2+ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca2+ release pathway is independent of cytosolic free Ca2+ and therefore incapable of operating Ca2+-induced Ca2+ release. In contrast to the sea urchin system, the NAADP-gated Ca2+ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca2+ mobilization pathways and Ca2+ release sites might contribute to the generation of stimulus-specific Ca2+ signals in plant cells.

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 muM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state. (C) 2004 Elsevier Ltd. Inc. All rights reserved.

Genome stability pathways in head and neck cancers

Genomic instability underlies the transformation of host cells toward malignancy, promotes development of invasion and metastasis and shapes the response of established cancer to treatment. In this review, we discuss recent advances in our understanding of genomic stability in squamous cell carcinoma of the head and neck (HNSCC), with an emphasis on DNA repair pathways. HNSCC is characterized by distinct profiles in genome stability between similarly staged cancers that are reflected in risk, treatment response and outcomes. Defective DNA repair generates chromosomal derangement that can cause subsequent alterations in gene expression, and is a hallmark of progression toward carcinoma. Variable functionality of an increasing spectrum of repair gene polymorphisms is associated with increased cancer risk, while aetiological factors such as human papillomavirus, tobacco and alcohol induce significantly different behaviour in induced malignancy, underpinned by differences in genomic stability. Targeted inhibition of signalling receptors has proven to be a clinically-validated therapy, and protein expression of other DNA repair and signalling molecules associated with cancer behaviour could potentially provide a more refined clinical model for prognosis and treatment prediction. Development and expansion of current genomic stability models is furthering our understanding of HNSCC pathophysiology and uncovering new, promising treatment strategies. © 2013 Glenn Jenkins et al.

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.