Adenosine and adenine nucleotides as regulators of cerebral blood flow: roles of acidosis, cell swelling, and KATP channels.

PubMed

Phillis, John W

2004-01-01

A considerable volume of evidence implicates the purine adenosine in the regulation of cerebral blood flow during states such as hypotension, neural activation, hypoxia/ischemia, and hypercapnia/acidosis. The aim of this review is to describe developments in our understanding of the roles that adenosine and the adenine nucleotides play in cerebral blood flow control, with some comparisons to coronary blood flow. The first part of the review focuses on the categorization of receptors for adenosine (A1, A2A, A2B, and A3) and the adenine nucleotides, ATP and ADP (P2X and P2Y). Frequently used agonists and antagonists for these different receptors are mentioned. A description follows of the distribution of these different receptors in cerebral arterioles. The second part of the review initially deals with the literature on the release of adenosine and adenine nucleotides into the extracellular space of the brain, describing the various techniques used to make these measurements and assessing the pitfalls associated with their use. This is followed by a discussion of the factors affecting purine release, which include cell swelling and acidosis. The third section evaluates the role of smooth muscle potassium channels in controlling arteriolar diameter. There is evidence for an important role of KATP and KCa channels, but less is known about the contributions of voltage-dependent (KV) and inwardly rectifying (KIR) channels. This section ends with a discussion on the reported inhibitory effect of nitric oxide synthase inhibitors on the KATP channel and the consequences of such an action for the interpretation of much of the published work on nitric oxide as a regulator of cerebral blood flow. The fourth section evaluates the data supporting a role of adenosine and ATP in the regulation of cerebral blood flow during autoregulation, hypotension, neural activity, hypoxia/ ischemia, and hypercapnia. Studies using antagonists and potentiators of adenosine's actions have led to

Adenine nucleotide translocator promotes oxidative phosphorylation and mild uncoupling in mitochondria after dexamethasone treatment.

PubMed

Arvier, Matthieu; Lagoutte, Laëtitia; Johnson, Gyasi; Dumas, Jean-François; Sion, Benoit; Grizard, Genevieve; Malthièry, Yves; Simard, Gilles; Ritz, Patrick

2007-11-01

The composition of the mitochondrial inner membrane and uncoupling protein [such as adenine nucleotide translocator (ANT)] contents are the main factors involved in the energy-wasting proton leak. This leak is increased by glucocorticoid treatment under nonphosphorylating conditions. The aim of this study was to investigate mechanisms involved in glucocorticoid-induced proton leak and to evaluate the consequences in more physiological conditions (between states 4 and 3). Isolated liver mitochondria, obtained from dexamethasone-treated rats (1.5 mg.kg(-1).day(-1)), were studied by polarography, Western blotting, and high-performance thin-layer chromatography. We confirmed that dexamethasone treatment in rats induces a proton leak in state 4 that is associated with an increased ANT content, although without any change in membrane surface or lipid composition. Between states 4 and 3, dexamethasone stimulates ATP synthesis by increasing both the mitochondrial ANT and F1-F0 ATP synthase content. In conclusion, dexamethasone increases mitochondrial capacity to generate ATP by modifying ANT and ATP synthase. The side effect is an increased leak in nonphosphorylating conditions.

Impaired oxidative metabolism increases adenine nucleotide breakdown in McArdle's disease.

PubMed

Sahlin, K; Areskog, N H; Haller, R G; Henriksson, K G; Jorfeldt, L; Lewis, S F

1990-10-01

Two patients with muscle phosphorylase deficiency [McArdle's disease (McA)] were studied during bicycle exercise at 40 (n = 2) and 60 W (n = 1). Peak heart rate was 170 and 162 beats/min, corresponding to approximately 90% of estimated maximal heart rate. Muscle samples were taken at rest and immediately after exercise from the quadriceps femoris. Lactate content remained low in both muscle and blood. Acetylcarnitine, which constitutes a readily available form of acetyl units and thus a substrate for the tricarboxylic acid cycle, was very low in McA patients both at rest and during exercise, corresponding to approximately 17 and 11%, respectively, of that in healthy subjects. Muscle NADH was unchanged during exercise in McA patients in contrast to healthy subjects, in whom NADH increases markedly at high exercise intensities. Despite low lactate levels, arterial plasma NH3 and muscle inosine 5'-monophosphate increased more steeply relative to work load in McA patients than in healthy subjects. The low postexercise levels of lactate, acetylcarnitine, and NADH in McA patients support the idea that exercise performance is limited by the availability of oxidative fuels. Increases in muscle inosine 5'-monophosphate and plasma NH3 indicate that lack of glycogen as an oxidative fuel is associated with adenine nucleotide breakdown and increased deamination of AMP. It is suggested that the early onset of fatigue in McA patients is caused by an insufficient rate of ADP phosphorylation, resulting in transient increases in ADP.

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

PubMed Central

Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

2016-01-01

Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass.

PubMed

Davidson, Jesse A; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan; Wischmeyer, Paul E; Klawitter, Jelena

2016-01-01

Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

PubMed

Kondo, Jiro; Westhof, Eric

2011-10-01

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

Development of a method for the analysis of nucleotides from the mantle tissue of the mussel Mytilus galloprovincialis.

PubMed

Blanco López, S L; Moal, J; San Juan Serrano, F

2000-09-01

Reversed-phase HPLC was applied to obtain a sensitive and efficient means for quantitating nucleotides in the mussel Mytilus galloprovincialis. We obtained a good separation of adenylic, guanylic, uridylic and cytidylic nucleotides. Adenine nucleotides play a critical role in the regulation and integration of cellular metabolism; particularly in the mantle tissue in the mussel, they are involved in the regulation of the enzyme glycogen phosphorylase, a key enzyme in the transfer of bioenergetic reserves (glycogen) to gametogenic development; it is of great importance to have a measure of the concentrations in vivo during the reproductive cycle of the organism. Different elution conditions were tested: isocratic versus step gradient elution, different mobile phase pH and the type and proportion of ion-pairing agent added to the mobile phase. The best method was selected and the separation and accurate determination of adenine, citidine, guanine and uridine nucleotides was accomplished within a 20-min run, with UV-Vis detection (254 nm).

Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on cape cod

USGS Publications Warehouse

Metge, D.W.; Brooks, M.H.; Smith, R.L.; Harvey, R.W.

1993-01-01

Changes in adenylate energy charge (EC(A)) and in total adenine nucleotides (A(T)) and DNA content (both normalized to the abundance of free- living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. EC(A)s (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated EC(A)s (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although EC(A) and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture. These data suggest that the DNA content of groundwater bacteria may be more sensitive than their A(T) to the degree of carbon loading, which may have significant ramifications in the use of nucleic acids and adenine nucleotides for estimating the metabolic status of bacterial communities within more highly contaminated aquifers.

The adsorption and reaction of adenine nucleotides on montmorillonite

NASA Technical Reports Server (NTRS)

Ferris, James P.; Hagan, William J., Jr.

1986-01-01

The binding of AMP to Zn(2+)-montmorillonite is investigated in the presence of salts and Good's zwitterion buffers, PIPES and MES. The initial concentrations of nucleotide and the percent adsorbtion are used to calculate the adsorption isotherms, and the Langmuir adsorption equation is used for the analysis of data. The adsorption coefficient was found to be three times greater in the presence of 0.2 M PIPES than in its absence. In addition, basal spacings measured by X-ray diffraction were increased by the buffer. These results are interpreted in terms of a model in which the adsorption of AMP is mediated by a Zn(2+) complex of PIPES in different orientations in the interlamellar region of the montmorillonite. Mixed ligand complexes of this type are reminiscent of the complexes observed between metal ions and biological molecules in living systems.

Molecular Dynamics Simulations of Creatine Kinase and Adenine Nucleotide Translocase in Mitochondrial Membrane Patch*

PubMed Central

Karo, Jaanus; Peterson, Pearu; Vendelin, Marko

2012-01-01

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies. PMID:22241474

Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions.

PubMed

Vaartjes, W J; den Breejen, J N; Geelen, M J; van den Bergh, S G

1980-08-05

1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, cannot account for the observed changes in the rate of pyruvate transport. 4. It is proposed that the pretreatment of the mitochondria directly modulates the activity of the mitochondrial pyruvate carrier. The possible regulatory role of such a modulation system is discussed.

PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

PubMed Central

Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

2015-01-01

Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

Silver-induced reconstruction of an adeninate-based metal-organic framework for encapsulation of luminescent adenine-stabilized silver clusters.

PubMed

Jonckheere, Dries; Coutino-Gonzalez, Eduardo; Baekelant, Wouter; Bueken, Bart; Reinsch, Helge; Stassen, Ivo; Fenwick, Oliver; Richard, Fanny; Samorì, Paolo; Ameloot, Rob; Hofkens, Johan; Roeffaers, Maarten B J; De Vos, Dirk E

2016-05-21

Bright luminescent silver-adenine species were successfully stabilized in the pores of the MOF-69A (zinc biphenyldicarboxylate) metal-organic framework, starting from the intrinsically blue luminescent bio-MOF-1 (zinc adeninate 4,4'-biphenyldicarboxylate). Bio-MOF-1 is transformed to the MOF-69A framework by selectively leaching structural adenine linkers from the original framework using silver nitrate solutions in aqueous ethanol. Simultaneously, bright blue-green luminescent silver-adenine clusters are formed inside the pores of the recrystallized MOF-69A matrix in high local concentrations. The structural transition and concurrent changes in optical properties were characterized using a range of structural, physicochemical and spectroscopic techniques (steady-state and time-resolved luminescence, quantum yield determination, fluorescence microscopy). The presented results open new avenues for exploring the use of MOFs containing luminescent silver clusters for solid-state lighting and sensor applications.

Effects of chlorogenic acid on adenine nucleotides hydrolyzing enzyme activities and expression in platelets of rats experimentally demyelinated with ethidium bromide.

PubMed

Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; Beckmann, Diego V; Castilhos, Lívia G; Thorstenberg, Maria L P; Jaques, Jeandre A Dos S; Souza, Viviane do C G; Farias, Júlia G; Martins, Caroline C; Schetinger, Maria R C

2016-07-01

The effects of chlorogenic acid (one of the major phenolic acid found in human diets) were investigated on the adenine nucleotides hydrolyzing enzymes; ecto-nucleotide pyrophosphatase/phophodiesterase (E-NPP), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'- nucleotidase and ecto-adenosine deaminase (E-ADA) activities and expression in platelets of rats experimentally demyelinated with ethidium bromide. Rats were divided into four groups of eight animals each. Group I rats were control rats; injected with saline (CT), group II rats were injected with saline and treated with chlorogenic acid (AC), group III rats were injected with 0.1% ethidium bromide (EB) and group IV rats were injected with 0.1% EB and treated with chlorogenic acid (EB+AC). The activities of the enzymes were analyzed using colorimetric methods, and the gene expression of NTPDase 1, 2 and 3 were analyzed using the polymerase chain reaction (PCR). The results revealed that there was a significant (P<0.01) reduction in E-NPP activity in EB group (1.63±0.10nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). However, treatment with chlorogenic acid significantly (P<0.05) increased E-NPP activity in EB group. Furthermore, no significant (P>0.05) change was observed in the E-NPP activity of EB+AC group (2.19±0.08nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). In addition, there was a significant (P<0.05) increase in AMP hydrolysis in EB rat group when compared to CT group. No significant (P>0.05) difference was observed in AMP hydrolysis between AC, AC+EB and CT groups. Conversely, there were no significant (P>0.05) differences in ATP and ADP hydrolyses between all the groups (AC, EB, AC+EB and CT groups). Likewise, there were no significant (P>0.05) changes in E-ADA activity and percentage platelet aggregation among all groups

Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

PubMed Central

Kekenes-Huskey, Peter M.; Liao, Tao; Gillette, Andrew K.; Hake, Johan E.; Zhang, Yongjie; Michailova, Anushka P.; McCulloch, Andrew D.; McCammon, J. Andrew

2013-01-01

Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions. PMID:24209858

Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

PubMed

Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

2015-09-01

The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

PubMed

Oza, Mihir D; Prasad, Kamalesh; Siddhanta, A K

2012-08-01

New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications. Copyright © 2012. Published by Elsevier Ltd.

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

PubMed

Zutz, Christoph; Chiang, Yi Ming; Faehnrich, Bettina; Bacher, Markus; Hellinger, Roland; Kluger, Bernhard; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin

2017-04-01

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives. Copyright © 2017 Elsevier GmbH. All rights reserved.

Molecular recognition at adenine nucleotide (P2) receptors in platelets.

PubMed

Jacobson, Kenneth A; Mamedova, Liaman; Joshi, Bhalchandra V; Besada, Pedro; Costanzi, Stefano

2005-04-01

Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.

Seasonal changes of nucleotides in mussel (Mytilus galloprovincialis) mantle tissue.

PubMed

Blanco, S L; Suárez, M P; San Juan, F

2006-03-01

Seasonal variations of nucleotides in Mytilus galloprovincialis mantle tissue were analyzed. Separation and quantification was achieved by reversed-phase high-performance liquid chromatography. Total nucleotides show a pronounced seasonal variation with maximum and minimum values in autumn and spring, respectively. Adenine nucleotides accounted for the major part in spring and summer, guanosine and cytidine nucleotides in winter; uridine nucleotides were relatively constant throughout the year. Their inverse variation suggests inter-conversion among them and the maintenance of the potential cell energy in winter by other triphosphate nucleotides different from ATP. These results reflect environmental and nutritional conditions, and also the reserves and gametogenic cycles taking place in M. galloprovincialis mantle tissue.

Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.

1990-06-01

High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubatedmore » under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.« less

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes.

PubMed

Cardouat, G; Duparc, T; Fried, S; Perret, B; Najib, S; Martinez, L O

2017-09-01

Ecto-F 1 -ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F 1 -ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y 13 -mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F 1 -ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F 1 -ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F 1 -ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis. Copyright © 2017. Published by Elsevier B.V.

Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

PubMed

Sallin, Olivier; Reymond, Luc; Gondrand, Corentin; Raith, Fabio; Koch, Birgit; Johnsson, Kai

2018-05-29

We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD + ) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP + ) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP + are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP + ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action. © 2018, Sallin et al.

A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.

PubMed

Esipov, R S; Abramchik, Yu A; Fateev, I V; Konstantinova, I D; Kostromina, M A; Muravyova, T I; Artemova, K G; Miroshnikov, A I

2016-01-01

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D -pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp . 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D -pentoses into 5-phosphates that were further converted into 5-phospho-α- D -pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D -ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

NASA Astrophysics Data System (ADS)

Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

2015-01-01

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Adenine nucleotide translocase-1 induces cardiomyocyte death through upregulation of the pro-apoptotic protein Bax.

PubMed

Baines, Christopher P; Molkentin, Jeffery D

2009-06-01

Overexpression of the adenine nucleotide translocase (ANT) has been shown to be cytotoxic in several cell types. Although ANT was originally proposed to be a critical component of the mitochondrial permeability transition (MPT) pore, recent data have suggested that this may not be the case. We therefore hypothesized that the cytotoxic actions of ANT are through an alternative mechanism, independent of the MPT pore. Infection of cultured neonatal cardiomyocytes with an ANT1-encoding adenovirus induced a gene dosage-dependent increase in cell death. However, ANT1 overexpression failed to induce MPT, and neither pharmacological nor genetic inhibition of the MPT pore was able to prevent ANT1-induced cell death. These data suggested that ANT1-induced death progressed through an MPT pore-independent pathway. Somewhat surprisingly, we observed that protein levels of Bax, a pro-apoptotic Bcl protein, were consistently elevated in ANT1-infected cardiomyocytes. Membranes isolated from ANT1-infected myocytes exhibited significantly increased amounts of membrane-inserted Bax, and immunocytochemistry revealed increased Bax activation in ANT1-infected myocytes. Co-expression with the Bax antagonist Bcl2 was able to greatly reduce the degree of ANT1-induced cell death. Furthermore, Bax/Bak-deficient fibroblasts were resistant to the cytotoxic effects of ANT1 overexpression. Interestingly, ANT1 overexpression was also associated with enhanced production of reactive oxygen species (ROS), and the antioxidant MnTBAP was able to significantly attenuate both the ANT1-induced upregulation of Bax and cell death. Taken together, these data indicate that ANT mediates cell death, not through the MPT pore, but rather via a ROS-dependent upregulation and activation of Bax.

Dietary nucleotide supplementation raises erythrocyte 2, 3-diphosphoglycerate concentration in neonatal rats.

PubMed

Scopesi, F; Verkeste, C M; Paola, D; Gazzolo, D; Pronzato, M A; Bruschettini, P L; Marinari, U M

1999-03-01

The present study was designed to test if dietary intake of nucleotides increases erythrocyte 2,3-diphosphoglycerate (2,3-DPG) in neonatal rats. To this end, rat pups were fed a nucleotide-supplemented formula (S, n = 14) from d 9 until d 16 after birth. The results were compared with those obtained from a group of breast-fed pups (C, n = 14) and a group of pups artificially fed with nucleotide-free formula (NS, n = 14). Neonatal weight, 2,3-DPG concentration, hematocrit (Hct) and hemoglobin concentration (Hb) were determined before the experiment (d 9) and after 7 d of treatment (d 16). In all groups, 2,3-DPG concentration was greater at d 16 than d 9, and the increase was greater in the S group than in the NS group. Alterations in neonatal weight, Hct and Hb concentration did not differ among the groups. On d 16 the 2, 3-DPG/Hb ratio, reflecting the affinity of hemoglobin for oxygen, was significantly higher in the C and S groups than in the NS group. We conclude that in neonatal rats, dietary nucleotides increase erythrocyte 2,3-DPG concentration. Studies need to be conducted in humans to assess the effect of this increase on both neonatal peripheral hemodynamics and metabolism in this species.

Cardiolipin content is involved in liver mitochondrial energy wasting associated with cancer-induced cachexia without the involvement of adenine nucleotide translocase.

PubMed

Julienne, Cloé Mimsy; Tardieu, Marine; Chevalier, Stéphan; Pinault, Michelle; Bougnoux, Philippe; Labarthe, François; Couet, Charles; Servais, Stéphane; Dumas, Jean-François

2014-05-01

Cancer-induced cachexia describes the progressive skeletal muscle wasting associated with many cancers leading to shortened survival time in cancer patients. We previously reported that cardiolipin content and energy-wasting processes were both increased in liver mitochondria in a rat model of peritoneal carcinosis (PC)-induced cachexia. To increase the understanding of the cellular biology of cancer cachexia, we investigated the involvement of adenine nucleotide translocator (ANT) in mitochondrial energy-wasting processes in liver mitochondria of PC and pair-fed control rats and its interactions with cardiolipin in isolated liver mitochondria from healthy rats exposed to cardiolipin-enriched liposomes. We showed in this study that functional ANT content was decreased in liver mitochondria from PC rats but without any effects on the efficiency of ATP synthesis. Moreover, non-phosphorylating energy wasting was not affected by saturating concentrations of carboxyatractylate (CAT), a potent inhibitor of ANT, in liver mitochondria from PC rats. Decreased efficiency of ATP synthesis was found in normal liver mitochondria exposed to cardiolipin-enriched liposomes, with increased non-phosphorylating energy wasting, thus mimicking mitochondria from PC rats. However, the functional ANT content in these cardiolipin-enriched mitochondria was unchanged, although non-phosphorylating energy wasting was reduced by CAT-induced inhibition of ANT. Finally, non-phosphorylating energy wasting was increased in cardiolipin-enriched mitochondria with substrates for complexes 1 and 2, but not for complex 4. In conclusion, increased energy wasting measured in liver mitochondria from rats with cancer cachexia is dependent on cardiolipin but independent of ANT. Interactions between ANT and cardiolipin are modified when cancer cachexia occurs. Copyright © 2014 Elsevier B.V. All rights reserved.

Two Adenine Nucleotide Translocase Paralogues Involved in Cell Proliferation and Spermatogenesis in the Silkworm Bombyx mori

PubMed Central

Sugahara, Ryohei; Jouraku, Akiya; Nakakura, Takayo; Kusakabe, Takahiro; Yamamoto, Takenori; Shinohara, Yasuo; Miyoshi, Hideto; Shiotsuki, Takahiro

2015-01-01

Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4. PMID:25742135

Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes

PubMed Central

2016-01-01

The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein–protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid–protein interactions. PMID:27786441

Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes.

PubMed

Hedger, George; Rouse, Sarah L; Domański, Jan; Chavent, Matthieu; Koldsø, Heidi; Sansom, Mark S P

2016-11-15

The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein-protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid-protein interactions.

Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

2015-11-01

Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes

PubMed Central

Kondo, Jiro; Westhof, Eric

2011-01-01

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide–protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson–Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson–Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues. PMID:21737431

Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation: Enhanced Stability and Potency as P2Y1 Receptor Agonists

PubMed Central

Ravi, R. Gnana; Kim, Hak Sung; Servos, Jörg; Zimmermann, Herbert; Lee, Kyeong; Maddileti, Savitri; Boyer, José L.; Harden, T. Kendall; Jacobson, Kenneth A.

2016-01-01

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y1, P2Y2, P2Y4, and P2Y11 receptors, but not P2Y6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208–218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y1 or human P2Y1 and P2Y2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC50 at the hP2Y1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl–(N)-methanocarba-ATP and its N6-Me analogue were also highly selective, full agonists at P2Y1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y1 receptor agonist with an EC50 of 160 nM and was selective versus hP2Y2 and hP2Y4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of

Updating Our View of Organelle Genome Nucleotide Landscape

PubMed Central

Smith, David Roy

2012-01-01

Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits. PMID:22973299

Adenine nucleotide transport in sonic submitochondrial particles. Kinetic properties and binding of specific inhibitors.

PubMed

Lauquin, G J; Villiers, C; Michejda, J W; Hryniewiecka, L V; Vignais, P V

1977-05-11

1. A procedure for preparation of sonic submitochondrial particles competent for adenine nucleotide transport is described. ADP or ATP transport was assayed, in the presence of oligomycin, in a saline medium made of 0.125 M KCl, 1 mM EDTA, 10 mM 4-morpholinopropane sulfonic acid buffer, pH 6.5. 2. Sonic particles transport ADP and ATP by an exchange diffusion process. Externally added ADP (or ATP) is exchanged with internal ADP and ATP with a stoichiometry of one to one. The V value for ADP transport 5 degrees C was between 2 and 3 nmol/min per mg protein. 3. The transport system in sonic particles is specific for ADP and ATP. It is strongly dependent on temperature. The activation energy between 0 and 9 degrees C is approx. 35 kcal/mol. The optimum pH is 6.5, 4, Like in intact mitochondria, externally added ADP is transported into sonic particles faster at a given concentration than externally added ATP. The V value for ADP transport is 1.5-2 times higher than the V value for ATP transport. 5. The transition from the energized to the deenergized state in sonic particles results in a decrease of the pH gradient across the membrane (internal pH less than external pH) and in a 2-4 fold increase in the Km value for ATP. This latter effect is opposite that found for transport of added ATP in intact mitochondria (Souverijn, J.H.M., Huisman, L.A., Rosing J. and Kemp, Jr., A. (1973) Biochim. Biophys. Acta 305, 185-198). Energization has no effect on the V value of ATP transport in sonic particles. 6. In contrast to intact mitochondria, inhibition of ADP transport in sonic particles by bongkrekic acid does not have any lag-time and does not depend on pH. The inhibition caused by bongkrekic acid is a mixed type inhibition with a Ki value of 1.2 micronM. Atractyloside and carboxyatractyloside do not inhibit ADP transport in sonic particles, unless the particles have been preloaded with these inhibitors during the sonication. 7. Palmityl-CoA added to sonic particles inhibits

The initial uridine of primary piRNAs does not create the tenth adenine that Is the hallmark of secondary piRNAs.

PubMed

Wang, Wei; Yoshikawa, Mayu; Han, Bo W; Izumi, Natsuko; Tomari, Yukihide; Weng, Zhiping; Zamore, Phillip D

2014-12-04

PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. We find that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3. Copyright © 2014 Elsevier Inc. All rights reserved.

Intramolecular interactions in aminoacyl nucleotides: Implications regarding the origin of genetic coding and protein synthesis

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.; Watkins, C. L.; Hall, L. M.

1986-01-01

Cellular organisms store information as sequences of nucleotides in double stranded DNA. This information is useless unless it can be converted into the active molecular species, protein. This is done in contemporary creatures first by transcription of one strand to give a complementary strand of mRNA. The sequence of nucleotides is then translated into a specific sequence of amino acids in a protein. Translation is made possible by a genetic coding system in which a sequence of three nucleotides codes for a specific amino acid. The origin and evolution of any chemical system can be understood through elucidation of the properties of the chemical entities which make up the system. There is an underlying logic to the coding system revealed by a correlation of the hydrophobicities of amino acids and their anticodonic nucleotides (i.e., the complement of the codon). Its importance lies in the fact that every amino acid going into protein synthesis must first be activated. This is universally accomplished with ATP. Past studies have concentrated on the chemistry of the adenylates, but more recently we have found, through the use of NMR, that we can observe intramolecular interactions even at low concentrations, between amino acid side chains and nucleotide base rings in these adenylates. The use of this type of compound thus affords a novel way of elucidating the manner in which amino acids and nucleotides interact with each other. In aqueous solution, when a hydrophobic amino acid is attached to the most hydrophobic nucleotide, AMP, a hydrophobic interaction takes place between the amino acid side chain and the adenine ring. The studies to be reported concern these hydrophobic interactions.

Adenine specific DNA chemical sequencing reaction.

PubMed Central

Iverson, B L; Dervan, P B

1987-01-01

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions. Images PMID:3671067

The in vivo effects of adenine-induced chronic kidney disease on some renal and hepatic function and CYP450 metabolizing enzymes.

PubMed

Al Za'abi, M; Shalaby, A; Manoj, P; Ali, B H

2017-05-04

Adenine-induced model of chronic kidney disease (CKD) is a widely used model especially in studies testing novel nephroprotective agents. We investigated the effects of adenine-induced CKD in rats on the activities of some xenobiotic metabolizing enzymes in liver and kidneys, and on some in vivo indicators of drug metabolism (viz pentobarbitone sleeping time, and plasma concentration of theophylline 90 min post administration). CKD was induced by orally feeding adenine (0.25 % w/w) for 35 days. Adenine induced all the characteristics of CKD, which was confirmed by biochemical and histological findings. Glutathione concentration and activities of some enzymes involved in its metabolism were reduced in kidneys and livers of rats with CKD. Renal CYP450 1A1 activity was significantly inhibited by adenine, but other measured isoenzymes (1A2, 3A4 and 2E1) were not significantly affected. Adenine significantly prolonged pentobarbitone-sleeping time and increased plasma theophylline concentration 90 min post administration. Adenine also induced a moderate degree of hepatic damages as indicated histologically and by significant elevations in some plasma enzymes. The results suggest that adenine-induced CKD is associated with significant in vivo inhibitory activities on some drug-metabolizing enzymes, with most of the effect on the kidneys rather than the liver.

Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism.

PubMed

Eltzschig, Holger K; Thompson, Linda F; Karhausen, Jorn; Cotta, Richard J; Ibla, Juan C; Robson, Simon C; Colgan, Sean P

2004-12-15

Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.

Selectivity and activity of adenine dinucleotides at recombinant P2X2 and P2Y1 purinoceptors.

PubMed Central

Pintor, J.; King, B. F.; Miras-Portugal, M. T.; Burnstock, G.

1996-01-01

1. Adenine dinucleotides (Ap3A, x = 2-6) are naturally-occurring polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. The selectivity and activity of adenine dinucleotides for neuronally-derived recombinant P2 purinoceptors were studied using P2X2 and P2Y1 subtypes expressed in Xenopus oocytes. 2. For the P2Y1 subtype derived from chick brain, Ap3A was equipotent and as active as ATP (EC50 values: 375 +/- 86 nM and 334 +/- 25 nM, respectively). Ap4A was a weak partial agonist and other dinucleotides were inactive as agonists. None of the inactive dinucleotides were antagonists nor modulated the activity of Ap3A and ATP. 3. For the P2X2 subtype derived from rat PC12 cells, Ap4A was as active as ATP but less potent (EC50 values: 15.2 +/- 1 microM and 3.7 +/- 0.7 microM, respectively). Other adenosine dinucleotides were inactive as either agonists or antagonists. 4. Ap5A (1-100 nM) potentiated ATP-responses at the P2X2 subtype, showing an EC50 of 2.95 +/- 0.7 nM for this modulatory effect. Ap5A (10 nM) shifted the concentration-response curves for ATP to the left by one-half log10 unit but did not alter the Hill co-efficient for ATP (nH = 2.1 +/- 0.1). Ap5A (10 nM) failed to potentiate Ap4A-responses but did enhance the efficacy of the P2 purinoceptor antagonist, suramin, by 12 fold at the P2X2 subtype. 5. In conclusion, the results show that ionotropic (P2X2) and metabotropic (P2Y1) ATP receptors which occur in the CNS are activated selectively by naturally-occurring adenine dinucleotides which are known to be released with nucleotides from storage vesicles. The observed potentiation of P2X2-responses by Ap5A, where co-released with ATP by brain synaptosomes, may have a functional bearing in purinergic signalling in the CNS. PMID:8922753

Radioresistance of Adenine to Cosmic Rays.

PubMed

Vignoli Muniz, Gabriel S; Mejía, Christian F; Martinez, Rafael; Auge, Basile; Rothard, Hermann; Domaracka, Alicja; Boduch, Philippe

2017-04-01

The presence of nucleobases in carbonaceous meteorites on Earth is an indication of the existence of this class of molecules in outer space. However, space is permeated by ionizing radiation, which can have damaging effects on these molecules. Adenine is a purine nucleobase that amalgamates important biomolecules such as DNA, RNA, and ATP. Adenine has a unique importance in biochemistry and therefore life. The aim of this work was to study the effects of cosmic ray analogues on solid adenine and estimate its survival when exposed to corpuscular radiation. Adenine films were irradiated at GANIL (Caen, France) and GSI (Darmstadt, Germany) by 820 MeV Kr 33+ , 190 MeV Ca 10+ , 92 MeV Xe 23+ , and 12 MeV C 4+ ion beams at low temperature. The evolution of adenine molecules under heavy ion irradiation was studied by IR absorption spectroscopy as a function of projectile fluence. It was found that the adenine destruction cross section (σ d ) follows an electronic stopping power (S e ) power law under the form: CS e n ; C is a constant, and the exponential n is a dimensionless quantity. Using the equation above to fit our results, we determined σ d  = 4 × 10 -17 S e 1.17 , with S e in kiloelectronvolts per micrometer (keV μm -1 ). New IR absorption bands arise under irradiation of adenine and can be attributed to HCN, CN - , C 2 H 4 N 4 , CH 3 CN, and (CH 3 ) 3 CNC. These findings may help to understand the stability and chemistry related to complex organic molecules in space. The half-life of solid adenine exposed to the simulated interstellar medium cosmic ray flux was estimated as (10 ± 8) × 10 6 years. Key Words: Heavy ions-Infrared spectroscopy-Astrochemistry-Cosmic rays-Nucleobases-Adenine. Astrobiology 17, 298-308.

Radioresistance of Adenine to Cosmic Rays

NASA Astrophysics Data System (ADS)

Vignoli Muniz, Gabriel S.; Mejía, Christian F.; Martinez, Rafael; Auge, Basile; Rothard, Hermann; Domaracka, Alicja; Boduch, Philippe

2017-04-01

The presence of nucleobases in carbonaceous meteorites on Earth is an indication of the existence of this class of molecules in outer space. However, space is permeated by ionizing radiation, which can have damaging effects on these molecules. Adenine is a purine nucleobase that amalgamates important biomolecules such as DNA, RNA, and ATP. Adenine has a unique importance in biochemistry and therefore life. The aim of this work was to study the effects of cosmic ray analogues on solid adenine and estimate its survival when exposed to corpuscular radiation. Adenine films were irradiated at GANIL (Caen, France) and GSI (Darmstadt, Germany) by 820 MeV Kr33+, 190 MeV Ca10+, 92 MeV Xe23+, and 12 MeV C4+ ion beams at low temperature. The evolution of adenine molecules under heavy ion irradiation was studied by IR absorption spectroscopy as a function of projectile fluence. It was found that the adenine destruction cross section (σd) follows an electronic stopping power (Se) power law under the form: CSen; C is a constant, and the exponential n is a dimensionless quantity. Using the equation above to fit our results, we determined σd = 4 × 10-17 Se1.17, with Se in kiloelectronvolts per micrometer (keV μm-1). New IR absorption bands arise under irradiation of adenine and can be attributed to HCN, CN-, C2H4N4, CH3CN, and (CH3)3CNC. These findings may help to understand the stability and chemistry related to complex organic molecules in space. The half-life of solid adenine exposed to the simulated interstellar medium cosmic ray flux was estimated as (10 ± 8) × 106 years.

Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seongmin; Verdine, Gregory L.; Harvard)

2010-01-14

Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases havemore » been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.« less

Crystallization and preliminary X-ray diffraction study of recombinant adenine phosphoribosyltransferase from the thermophilic bacterium Thermus thermophilus strain HB27

NASA Astrophysics Data System (ADS)

Sinitsyna, E. V.; Timofeev, V. I.; Tuzova, E. S.; Kostromina, M. A.; Murav'eva, T. I.; Esipov, R. S.; Kuranova, I. P.

2017-07-01

Adenine phosphoribosyltransferase (APRT) belongs to the type I phosphoribosyltransferase family and catalyzes the formation of adenosine monophosphate via transfer of the 5-phosphoribosyl group from phosphoribosyl pyrophosphate to the nitrogen atom N9 of the adenine base. Proteins of this family are involved in a salvage pathway of nucleotide synthesis, thus providing purine base utilization and maintaining the optimal level of purine bases in the body. Adenine phosphoribosyltransferase from the extremely thermophilic Thermus thermophilus strain HB27 was produced using a highly efficient E. coli producer strain and was then purified by affinity and gel-filtration chromatography. This enzyme was successfully employed as a catalyst for the cascade biosynthesis of biologically important nucleotides. The screening of crystallization conditions for recombinant APRT from T. thermophilus HB27 was performed in order to determine the enzyme structure by X-ray diffraction. The crystallization conditions, which were found by the vapor-diffusion technique, were then optimized to apply the counter-diffusion technique. The crystals of the enzyme were grown by the capillary counter-diffusion method. The crystals belong to sp. gr. P1211 and have the following unitcell parameters: a = 69.86 Å, b = 82.16 Å, c = 91.39 Å, α = γ = 90°, β = 102.58°. The X-ray diffraction data set suitable for the determination of the APRT structure at 2.6 Å resolution was collected from the crystals at the SPring-8 synchrotron facility (Japan).

Hydrothermal stability of adenine under controlled fugacities of N2, CO2 and H2.

PubMed

Franiatte, Michael; Richard, Laurent; Elie, Marcel; Nguyen-Trung, Chinh; Perfetti, Erwan; LaRowe, Douglas E

2008-04-01

An experimental study has been carried out on the stability of adenine (one of the five nucleic acid bases) under hydrothermal conditions. The experiments were performed in sealed autoclaves at 300 degrees C under fugacities of CO(2), N(2) and H(2) supposedly representative of those in marine hydrothermal systems on the early Earth. The composition of the gas phase was obtained from the degradation of oxalic acid, sodium nitrite and ammonium chloride, and the oxidation of metallic iron. The results of the experiments indicate that after 200 h, adenine is still present in detectable concentration in the aqueous phase. In fact, the concentration of adenine does not seem to be decreasing after approximately 24 h, which suggests that an equilibrium state may have been established with the inorganic constituents of the hydrothermal fluid. Such a conclusion is corroborated by independent thermodynamic calculations.

The Relationship of Mucus Concentration (Hydration) to Mucus Osmotic Pressure and Transport in Chronic Bronchitis

PubMed Central

Coakley, Raymond D.; Button, Brian; Henderson, Ashley G.; Zeman, Kirby L.; Alexis, Neil E.; Peden, David B.; Lazarowski, Eduardo R.; Davis, C. William; Bailey, Summer; Fuller, Fred; Almond, Martha; Qaqish, Bahjat; Bordonali, Elena; Rubinstein, Michael; Bennett, William D.; Kesimer, Mehmet; Boucher, Richard C.

2015-01-01

Rationale: Chronic bronchitis (CB) is characterized by persistent cough and sputum production. Studies were performed to test whether mucus hyperconcentration and increased partial osmotic pressure, in part caused by abnormal purine nucleotide regulation of ion transport, contribute to the pathogenesis of CB. Objectives: We tested the hypothesis that CB is characterized by mucus hyperconcentration, increased mucus partial osmotic pressures, and reduced mucus clearance. Methods: We measured in subjects with CB as compared with normal and asymptomatic smoking control subjects indices of mucus concentration (hydration; i.e., percentage solids) and sputum adenine nucleotide/nucleoside concentrations. In addition, sputum partial osmotic pressures and mucus transport rates were measured in subjects with CB. Measurements and Results: CB secretions were hyperconcentrated as indexed by an increase in percentage solids and total mucins, in part reflecting decreased extracellular nucleotide/nucleoside concentrations. CB mucus generated concentration-dependent increases in partial osmotic pressures into ranges predicted to reduce mucus transport. Mucociliary clearance (MCC) in subjects with CB was negatively correlated with mucus concentration (percentage solids). As a test of relationships between mucus concentration and disease, mucus concentrations and MCC were compared with FEV1, and both were significantly correlated. Conclusions: Abnormal regulation of airway surface hydration may slow MCC in CB and contribute to disease pathogenesis. PMID:25909230

The Role of the Plant Hormone Benzyl Adenine to Promote Growth for the Diatom Thalassiosira pseudonana

NASA Astrophysics Data System (ADS)

Gutierrez Franco, D.; Vernet, M.; Walters, R. J.; Tan, M.

2016-02-01

This study was inspired by the establishment of autoinduction in the model diatom Thalassiosira pseudonana, and the identification of the cytokinin plant hormone benzyl adenine (BA) as a potential autoinducer in this species via comparative genome studies. The effects of a wide range (0.0017518 mg/L-500 mg/L) of concentrations of benzyl adenine on the growth dynamics of T. pseudonana have been explored. The results suggest that a concentration of 5 mg BA/L has the highest positive effect on the growth rate of T. pseudonana batch cultures, compared to the other concentrations tested. Furthermore, concentrations of >100 mg BA/L were lethal. No marked effects on the lag phase length were observed. However, it is possible that some trade-offs between growth rate and lag phase length exist as a result of benzyl adenine. For instance, the BA concentration that exhibited the highest growth rate (5mg BA/L; µ=1.06 d-1) had a negative effect on the lag phase length (6 days), as compared to our control (lag phase length = 5 d; µ=0.81 d-1). On the other hand, at 10 mg BA/L, a slightly smaller growth rate of 1.01 d-1 was observed, with a shorter lag phase length of 4 days, suggesting that benzyl adenine may not have a positive effect on all growth parameters at once. These results provide insight into the physiological and biochemical mechanisms of cell-to-cell communication employed by diatoms, and supports the hypothesis that hormones may play an important role in bloom development.

Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

PubMed

Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

2016-01-05

A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

Acute Knockdown of Uncoupling Protein-2 Increases Uncoupling via the Adenine Nucleotide Transporter and Decreases Oxidative Stress in Diabetic Kidneys

PubMed Central

Friederich-Persson, Malou; Aslam, Shakil; Nordquist, Lina; Welch, William J.; Wilcox, Christopher S.; Palm, Fredrik

2012-01-01

Increased O2 metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O2 consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (−30–50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT. PMID:22768304

Lack of effect of dietary nucleotide supplementation on erythrocyte 2,3-diphosphoglycerate concentration. A study on preterm neonates.

PubMed

Scopesi, Fabio; Canini, Silvana; Arioni, Cesare; Mazzella, Massimo; Gazzolo, Diego; Lantieri, Pasquale B; Bonacci, Wanda; Serra, Giovanni

2006-06-01

Recently we demonstrated an increased 2,3-diphosphoglycerate (2,3-DPG) erythrocyte concentration in rat pups subjected to nucleotide-enriched artificial feeding. The present study was carried out to test the hypothesis that a possible increase in 2,3-DPG concentration can also be obtained in human neonates who are fed nucleotide-enriched formula. Preterm neonates born or referred to the neonatal intensive care unit of the G. Gaslini Hospital, Genoa University, with a gestational age >30 weeks and <37 weeks were enrolled in our randomized trial. Recruitment took place within 48-72 hours from birth. Only newborns of mothers deciding not to breast-feed were eligible to be randomized for the supplemented group (FN) or non-supplemented group (RF). Breast-fed newborns were considered the control group (C). The study window (for supplementation and blood samples) was restricted to the first two weeks following birth (from the 2nd (t1) to the 16th (t2) day of life). At the end of our study, only 21 neonates were eligible for statistical analysis. The stimulating action of dietary nucleotides on 2,3-DPG concentration failed to be demonstrated; increases in 2,3-DPG concentration that were observed in newborns fed with nucleotide supplemented formula (FN) were comparable to those observed in newborns fed with regular formula (RF) and breast-fed newborns. The EC recommendation for the amount of nucleotides allowed in formula milk does not seem to be high enough to have positive effects on 2,3-DPG synthesis. Whether this possible 'pharmacological' effect can be achieved by a higher intake of ingested nucleotides and/or a change in the proportions of single nucleotides contained in milk formulas remain interesting end points to be elucidated.

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

PubMed Central

Burton, K

1977-01-01

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases. PMID:413544

Molecular recognition of modified adenine nucleotides by the P2Y(1)-receptor. 1. A synthetic, biochemical, and NMR approach.

PubMed

Halbfinger, E; Major, D T; Ritzmann, M; Ubl, J; Reiser, G; Boyer, J L; Harden, K T; Fischer, B

1999-12-30

The remarkably high potencies of 2-thioether-adenine nucleotides regarding the activation of the P2Y(1)-receptor (P2Y(1)-R) in turkey erythrocyte membranes represent some of the largest substitution-promoted increases in potencies over that of a natural receptor ligand. This paper describes the investigation regarding the origin of the high potency of these P2Y(1)-R ligands over that of ATP. For this study, an integrated approach was employed combining the synthesis of new ATP analogues, their biochemical evaluation, and their SAR analysis involving NMR experiments and theoretical calculations. These experiments and calculations were performed to elucidate the conformation and to evaluate the electronic nature of the investigated P2Y(1)-R ligands. ATP analogues synthesized included derivatives where C2 or C8 positions were substituted with electron-donating groups such as ethers, thioethers, or amines. The compounds were tested for their potency to induce P2Y(1)-R-mediated activation of phospholipase C in turkey erythrocytes and Ca(2+) response in rat astrocytes. 8-Substituted ATP and AMP derivatives had little or no effect on phospholipase C or on calcium levels, whereas the corresponding 2-substituted ATP analogues potently increased the levels of inositol phosphates and ¿Ca(2+)(i). AMP analogues were ineffective except for 2-butylthio-AMP which induced a small Ca(2+) response. P2Y(1)-R activity of these compounds was demonstrated by testing these ligands also on NG108-15 neuroblastoma x glioma hybrid cells. NMR data together with theoretical calculations imply that steric, rather than electronic, effects play a major role in ligand binding to the P2Y(1)-R. Hydrophobic interactions and H-bonds of the C2 substituent appear to be important determinants of a P2Y(1)-R ligand affinity.

Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart.

PubMed Central

Nichols, B J; Rigoulet, M; Denton, R M

1994-01-01

The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available. PMID:7980405

Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog

NASA Technical Reports Server (NTRS)

Yamauchi, Keiko; Hales, Nathan W.; Robinson, Sandra M.; Niehoff, Michael L.; Ramesh, Vani; Pellis, Neal R.; Kulkarni, Anil D.

2002-01-01

Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.

Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum

NASA Technical Reports Server (NTRS)

Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

1992-01-01

The membrane ATPase from Halobacterium saccharovorum was purified as described by Hochstein et al. (1987) and was incubated with C-14 labeled N-ethylmaleimide (NEM), with and without adenine nucleotides, to determine the effect of nucleotides on the enzyme labeling. It was found that NEM incorporates into the 87,000-Da subunit (subunit I) of the enzyme and that the conditions for enzyme modification are similar to those which result in the inhibition of the enzyme activity. The presence of ATP, ADP, and AMP was found to reduce both the inhibitor incorporation and enzyme inhibition. It was shown that the reaction involves a modification of thiol groups.

Effect of atracylodes rhizome polysaccharide in rats with adenine-induced chronic renal failure.

PubMed

Yang, C; Liu, C; Zhou, Q; Xie, Y C; Qiu, X M; Feng, X

2015-01-01

The aim of the study was to elucidate the therapeutic effects of Atracylodes rhizome polysaccharide on adenine-induced chronic renal failure in rats. Fifty male Sprague Dawley rats were selected and randomly divided in to 5 groups (n=10 rats per group): The normal control group, the chronic renal failure pathological control group, the dexamethasone treatment group and two Atracylodes rhizome polysaccharide treatment groups, treated with two different concentrations of the polysaccharide, the Atracylodes rhizome polysaccharide high group and the Atracylodes rhizome polysaccharide low group. All the rats, except those in the normal control group were fed adenine-enriched diets, containing 10 g adenine per kg food for 3 weeks. After being fed with adenine, the dexamethasone treatment group, Atracylodes rhizome polysaccharide high group and Atracylodes rhizome polysaccharide low group rats were administered the drug orally for 2 weeks. On day 35, the kidney coefficient of the rats and the serum levels of creatinine, blood urea nitrogen, total protein and hemalbumin were determined. Subsequent to experimentation on a model of chronic renal failure in rats, the preparation was proven to be able to reduce serum levels of creatinine, blood urea nitrogen and hemalbumin levels (P<0.05) and improve renal function. Atracylodes rhizome polysaccharide had reversed the majority of the indices of chronic renal failure in rats.

N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

PubMed

Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

1982-10-25

When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

PubMed Central

Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

1982-01-01

When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand

NASA Astrophysics Data System (ADS)

Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao

2017-05-01

A novel adenine (Ad) fluorescence probe (EuIII-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the EuIII-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the EuIII-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the EuIII-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using EuIII-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of EuIII-dtpa-bis(guanine) at 570 nm as a function of adenine (Ad) concentration in the range of 0.00-5.00 × 10- 5 mol L- 1 was observed. The detection limit is about 4.70 × 10- 7 mol L- 1.

Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification.

PubMed

Afsari, Sepideh; Korshoj, Lee E; Abel, Gary R; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-11-28

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

DOE PAGES

Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; ...

2014-11-17

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2’-deoxyguanosine found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate E. coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerase discriminates between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities,more » nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine (Cy) and 8-oxodGTP(syn) utilizes its Hoogsteen edge to base pair with adenine (Ad). Here in this paper we utilized time-lapse crystallography to follow 8-oxo-dGTP insertion opposite Ad or Cy with human DNA pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxodGTP utilizes charge modulation during insertion that can lead to a blocked DNA repair intermediate.« less

Synthesis of adenine-modified reduced graphene oxide nanosheets.

PubMed

Cao, Huaqiang; Wu, Xiaoming; Yin, Gui; Warner, Jamie H

2012-03-05

We report here a facile strategy to synthesize the nanocomposite of adenine-modified reduced graphene oxide (AMG) via reaction between adenine and GOCl which is generated from SOCl(2) reacted with graphite oxide (GO). The as-synthesized AMG was characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), UV-vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), and galvanostatic discharge analysis. The AMG owns about one adenine group per 53 carbon atoms on a graphene sheet, which improves electronic conductivity compared with reduced graphene oxide (RGO). The AMG displays enhanced supercapacitor performance compared with RGO accompanying good stability and good cycling behavior in the supercapacitor.

Metabolic adaptation to long term changes in gravity environment

NASA Astrophysics Data System (ADS)

Slenzka, K.; Appel, R.; Rahmann, H.

Biochemical analyses of the brain of Cichlid fish larvae, exposed during their very early development for 7 days to an increased acceleration of 3g (hyper-gravity), revealed a decrease in brain nucleoside diphosphate kinase (NDPK) as well as creatine kinase (BB-CK) activity. Using high performance liquid chromatography (HPLC) the concentrations of adenine nucleotides (AMP, ADP, ATP), phosphocreatine (CP), as well as of nicotineamide adenine dinucleotides (NAD, NADP) were analyzed in the brain of hyper-g exposed larvae vs. 1g controls. A slight reduction in the total adenine nucleotides (TAN) as well as the adenylate energy charge (AEC) was found. In parallel a significant increase in the NAD concentration and a corresponding decrease in NADP concentration occurred in larva's hyper-g brains vs. 1g controls. These results give further evidence for an influence of gravity on cellular level and furthermore contribute to a clarification of the cellular signal-response chain for gravity perception.

Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

PubMed

Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2014-01-01

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

Different Effects of Guanine Nucleotides (GDP and GTP) on Protein-Mediated Mitochondrial Proton Leak

PubMed Central

Woyda-Ploszczyca, Andrzej M.; Jarmuszkiewicz, Wieslawa

2014-01-01

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP. PMID:24904988

Mung bean nuclease: mode of action and specificity vs synthetic esters of 3′-nucleotides

PubMed Central

Kole, R.; Sierakowska, Halina; Szemplińska, Halina; Shugar, D.

1974-01-01

Mung bean nuclease hydrolyzes synthetic esters of 3′-nucleotides to nucleosides and phosphate esters; esters of 2′-nucleotides, and 2′→ 5′ internucleotide linkages, are resistant. Esters of ribonucleotides are cleaved at 100-fold the rate for deoxyribonucleotides, the increased rate being due to presence of the 2′-hydroxyl and not to differences in conformation. Introduction of a 5′-substituent leads to a 3-fold increase in rate. The rates of hydrolysis vary up to 10-fold with the nature of the base, in the order adenine > hypoxanthine > uracil; and up to 6-fold with the nature of the ester radical. This form of cleavage of esters of 3′-nucleotides is also characteristic for nuclease-3′-nucleotidase activities from potato tubers and wheat, suggesting that one type of enzyme is responsible for all these activities. PMID:10793750

Adenine and 2-aminopurine: paradigms of modern theoretical photochemistry.

PubMed

Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio C

2006-06-06

Distinct photophysical behavior of nucleobase adenine and its constitutional isomer, 2-aminopurine, has been studied by using quantum chemical methods, in particular an accurate ab initio multiconfigurational second-order perturbation theory. After light irradiation, the efficient, ultrafast energy dissipation observed for nonfluorescent 9H-adenine is explained here by the nonradiative internal conversion process taking place along a barrierless reaction path from the initially populated 1(pipi* La) excited state toward a low-lying conical intersection (CI) connected with the ground state. In contrast, the strong fluorescence recorded for 2-aminopurine at 4.0 eV with large decay lifetime is interpreted by the presence of a minimum in the 1(pipi* La) hypersurface lying below the lowest CI and the subsequent potential energy barrier required to reach the funnel to the ground state. Secondary deactivation channels were found in the two systems related to additional CIs involving the 1(pipi* Lb) and 1(npi*) states. Although in 9H-adenine a population switch between both states is proposed, in 7H-adenine this may be perturbed by a relatively larger barrier to access the 1(npi*) state, and, therefore, the 1(pipi* Lb) state becomes responsible for the weak fluorescence measured in aqueous adenine at approximately 4.5 eV. In contrast to previous models that explained fluorescence quenching in adenine, unlike in 2-aminopurine, on the basis of the vibronic coupling of the nearby 1(pipi*) and 1(npi*) states, the present results indicate that the 1(npi*) state does not contribute to the leading photophysical event and establish the prevalence of a model based on the CI concept in modern photochemistry.

Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model

NASA Astrophysics Data System (ADS)

Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang

2016-07-01

Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

Adenine and 2-aminopurine: Paradigms of modern theoretical photochemistry

PubMed Central

Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio C.

2006-01-01

Distinct photophysical behavior of nucleobase adenine and its constitutional isomer, 2-aminopurine, has been studied by using quantum chemical methods, in particular an accurate ab initio multiconfigurational second-order perturbation theory. After light irradiation, the efficient, ultrafast energy dissipation observed for nonfluorescent 9H-adenine is explained here by the nonradiative internal conversion process taking place along a barrierless reaction path from the initially populated 1(ππ* La) excited state toward a low-lying conical intersection (CI) connected with the ground state. In contrast, the strong fluorescence recorded for 2-aminopurine at 4.0 eV with large decay lifetime is interpreted by the presence of a minimum in the 1(ππ* La) hypersurface lying below the lowest CI and the subsequent potential energy barrier required to reach the funnel to the ground state. Secondary deactivation channels were found in the two systems related to additional CIs involving the 1(ππ* Lb) and 1(nπ*) states. Although in 9H-adenine a population switch between both states is proposed, in 7H-adenine this may be perturbed by a relatively larger barrier to access the 1(nπ*) state, and, therefore, the 1(ππ* Lb) state becomes responsible for the weak fluorescence measured in aqueous adenine at ≈4.5 eV. In contrast to previous models that explained fluorescence quenching in adenine, unlike in 2-aminopurine, on the basis of the vibronic coupling of the nearby 1(ππ*) and 1(nπ*) states, the present results indicate that the 1(nπ*) state does not contribute to the leading photophysical event and establish the prevalence of a model based on the CI concept in modern photochemistry. PMID:16731617

The catalase activity of diiron adenine deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.

2011-12-01

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometrymore » and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.« less

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamat, S.S.; Swaminathan, S.; Bagaria, A.

2011-03-22

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. Themore » apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism

Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Kamat; A Bagaria; D Kumaran

2011-12-31

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{supmore » -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical

The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet-dense granules

PubMed Central

Chintala, Sreenivasulu; Tan, Jian; Gautam, Rashi; Rusiniak, Michael E.; Guo, Xiaoli; Li, Wei; Gahl, William A.; Huizing, Marjan; Spritz, Richard A.; Hutton, Saunie; Novak, Edward K.; Swank, Richard T.

2007-01-01

Platelet dense granules are lysosome-related organelles which contain high concentrations of several biologically important low-molecular-weight molecules. These include calcium, serotonin, adenine nucleotides, pyrophosphate, and polyphosphate, which are necessary for normal blood hemostasis. The synthesis of dense granules and other lysosome-related organelles is defective in inherited diseases such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS). HPS and CHS mutations in 8 human and at least 16 murine genes have been identified. Previous studies produced contradictory findings for the function of the murine ashen (Rab27a) gene in platelet-dense granules. We have used a positional cloning approach with one line of ashen mutants to establish that a new mutation in a second gene, Slc35d3, on mouse chromosome 10 is the basis of this discrepancy. The platelet-dense granule defect is rescued in BAC transgenic mice containing the normal Slc35d3 gene. Thus, Slc35d3, an orphan member of a nucleotide sugar transporter family, specifically regulates the contents of platelet-dense granules. Unlike HPS or CHS genes, it has no apparent effect on other lysosome-related organelles such as melanosomes or lysosomes. The ash-Roswell mouse mutant is an appropriate model for human congenital-isolated delta-storage pool deficiency. PMID:17062724

Silver-induced reconstruction of an adeninate-based metal–organic framework for encapsulation of luminescent adenine-stabilized silver clusters† †Electronic supplementary information (ESI) available: Experimental details and additional structural, physicochemical and optical characterisation. See DOI: 10.1039/c6tc00260a Click here for additional data file.

PubMed Central

Jonckheere, Dries; Coutino-Gonzalez, Eduardo; Baekelant, Wouter; Bueken, Bart; Reinsch, Helge; Stassen, Ivo; Fenwick, Oliver; Richard, Fanny; Samorì, Paolo; Ameloot, Rob; Hofkens, Johan

2016-01-01

Bright luminescent silver-adenine species were successfully stabilized in the pores of the MOF-69A (zinc biphenyldicarboxylate) metal–organic framework, starting from the intrinsically blue luminescent bio-MOF-1 (zinc adeninate 4,4′-biphenyldicarboxylate). Bio-MOF-1 is transformed to the MOF-69A framework by selectively leaching structural adenine linkers from the original framework using silver nitrate solutions in aqueous ethanol. Simultaneously, bright blue-green luminescent silver-adenine clusters are formed inside the pores of the recrystallized MOF-69A matrix in high local concentrations. The structural transition and concurrent changes in optical properties were characterized using a range of structural, physicochemical and spectroscopic techniques (steady-state and time-resolved luminescence, quantum yield determination, fluorescence microscopy). The presented results open new avenues for exploring the use of MOFs containing luminescent silver clusters for solid-state lighting and sensor applications. PMID:28496980

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

PubMed Central

Rutter, G A; Denton, R M

1988-01-01

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system. PMID:3421900

Adenine alleviates iron overload by cAMP/PKA mediated hepatic hepcidin in mice.

PubMed

Zhang, Yingqi; Wang, Xudong; Wu, Qian; Wang, Hao; Zhao, Lu; Wang, Xinhui; Mu, Mingdao; Xie, Enjun; He, Xuyan; Shao, Dandan; Shang, Yanna; Lai, Yongrong; Ginzburg, Yelena; Min, Junxia; Wang, Fudi

2018-03-30

Hemochromatosis is prevalent and often associated with high rates of morbidity and mortality worldwide. The safe alternative iron-reducing approaches are urgently needed in order to better control iron overload. Our unbiased vitamin screen for modulators of hepcidin, a master iron regulatory hormone, identifies adenine (vitamin B4) as a potent hepcidin agonist. Adenine significantly induced hepcidin mRNA level and promoter activity activation in human cell lines, possibly through BMP/SMAD pathway. Further studies in mice validated the effect of adenine on hepcidin upregulation. Consistently, adenine dietary supplement in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD pathway. Notably, adenine-rich diet significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe -/- mice, a murine model of hereditary hemochromatosis. To further validate this finding, we selected pharmacological inhibitors against BMP (LDN193189). We found LDN193189 strongly blocked the hepcidin induction by adenine. Moreover, we uncovered an essential role of cAMP/PKA-dependent axis in triggering adenine-induced hepcidin expression in primary hepatocytes by using 8 br cAMP, a cAMP analog, and H89, a potent inhibitor for PKA signaling. These findings suggest a potential therapeutic role of adenine for hereditary hemochromatosis. © 2018 Wiley Periodicals, Inc.

Preparation and evaluation of molecularly imprinted polymers based on 9-ethyladenine for the recognition of nucleotide bases in capillary electrochromatography.

PubMed

Huang, Yi-Chen; Lin, Chun-Chi; Liu, Chuen-Ying

2004-02-01

A molecularly imprinted polymer (MIP) comprising 9-ethyladenine was polymerized in situ inside the capillary for the electrochromatographic separation of nucleotide bases. The capillary wall was first functionalized with 3-trimethoxysilylpropyl methacrylate (10% v/v) and 1,1-diphenyl-2-picrylhydrazyl (0.01% w/v) in toluene. Following this treatment, the capillary was filled with acetonitrile containing 9-ethyladenine, methacrylic acid, ethylene glycol dimethacrylate, and initiator. After polymerization, the MIP was shrunk into a film against the inner wall of the capillary with the syringe pump. The template was then removed with methanol under nitrogen flow. For evaluation the feasibility of the MIP column for the separation of nucleotide bases, some parameters including the pH, concentration of the background electrolyte, the applied voltage as well as the effect of organic modifier were studied. The migration behavior of nucleotide bases on the MIP column was also compared with that on the bare fused-silica column. The results indicated that the MIP columns demonstrated better recognition properties at a pH range of 6-8. The efficiency (plates/m) at pH 8 for the nonimprinted analyte was 75,300 for cytosine, 50,200 for thymine, and 14,800 for guanine. However, the efficiency for the imprinted analyte, adenine, was quite low. This was evidenced by the broad peak, yielding only 2600 plates/m.

Effects of Nicotinamide Adenine Dinucleotide (NAD(+)) and Diadenosine Tetraphosphate (Ap4A) on Electrical Activity of Working and Pacemaker Atrial Myocardium in Guinea Pigs.

PubMed

Pustovit, K B; Abramochkin, D V

2016-04-01

Effects of nucleotide polyphosphate compounds (nicotinamide adenine dinucleotide, NAD(+); diadenosine tetraphosphate, Ap4A) on the confi guration of action potentials were studied in isolated preparations of guinea pig sinoatrial node and right atrial appendage (auricle). In the working myocardium, NAD(+) and Ap4A in concentrations of 10(-5) and 10(-4) M had no effect on resting potential, but significantly reduced the duration of action potentials; the most pronounced decrease was found at 25% repolarization. In the primary pacemaker of the sinoatrial node, both concentrations of NAD(+) and Ap4A induced hyperpolarization and reduction in the rate of slow diastolic depolarization, but significant slowing of the sinus rhythm was produced by these substances only in the concentration of 10(-4) M. Moreover, AP shortening and marked acceleration of AP upstroke were observed in the pacemaker myocardium after application of polyphosphates. Comparative analysis of the effects of NAD(+) and Ap4A in the working and pacemaker myocardium drove us to a hypothesis on inhibitory effects of these substances on L-type calcium current accompanied by stimulation of one or several potassium currents, which induce enhancement of repolarization and hyperpolarization of membranes probably mediated by the activation of purine receptors.

Prebiotic stereoselective synthesis of purine and noncanonical pyrimidine nucleotide from nucleobases and phosphorylated carbohydrates.

PubMed

Kim, Hyo-Joong; Benner, Steven A

2017-10-24

According to a current "RNA first" model for the origin of life, RNA emerged in some form on early Earth to become the first biopolymer to support Darwinism here. Threose nucleic acid (TNA) and other polyelectrolytes are also considered as the possible first Darwinian biopolymer(s). This model is being developed by research pursuing a "Discontinuous Synthesis Model" (DSM) for the formation of RNA and/or TNA from precursor molecules that might have been available on early Earth from prebiotic reactions, with the goal of making the model less discontinuous. In general, this is done by examining the reactivity of isolated products from proposed steps that generate those products, with increasing complexity of the reaction mixtures in the proposed mineralogical environments. Here, we report that adenine, diaminopurine, and hypoxanthine nucleoside phosphates and a noncanonical pyrimidine nucleoside (zebularine) phosphate can be formed from the direct coupling reaction of cyclic carbohydrate phosphates with the free nucleobases. The reaction is stereoselective, giving only the β-anomer of the nucleotides within detectable limits. For purines, the coupling is also regioselective, giving the N -9 nucleotide for adenine as a major product. In the DSM, phosphorylated carbohydrates are presumed to have been available via reactions explored previously [Krishnamurthy R, Guntha S, Eschenmoser A (2000) Angew Chem Int Ed 39:2281-2285], while nucleobases are presumed to have been available from hydrogen cyanide and other nitrogenous species formed in Earth's primitive atmosphere. Published under the PNAS license.

Absence of uncoupling protein-3 leads to greater activation of an adenine nucleotide translocase-mediated proton conductance in skeletal muscle mitochondria from calorie restricted mice.

PubMed

Bevilacqua, Lisa; Seifert, Erin L; Estey, Carmen; Gerrits, Martin F; Harper, Mary-Ellen

2010-08-01

Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated 'energy-wasting' process during CR. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Dissociative Excitation of Adenine by Electron Impact

NASA Astrophysics Data System (ADS)

McConkey, J. William; Trocchi, Joshuah; Dech, Jeffery; Kedzierski, Wladek

2017-04-01

Dissociative excitation of adenine (C6H5NH2) into excited atomic fragments has been studied in the electron impact energy range from threshold to 300 eV. A crossed beam system coupled to a vacuum ultraviolet (VUV) monochromator is used to study emissions in the wavelength range from 110 to 200 nm. The beam of adenine vapor from a stainless steel oven is crossed at right angles by the electron beam and the resultant UV radiation is detected in a mutually orthogonal direction. The strongest feature in the spectrum is H Lyman- α. Financial support from NSERC and CFI, Canada, is gratefully acknowledged.

Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

NASA Astrophysics Data System (ADS)

Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

2015-12-01

Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process.

Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

PubMed Central

Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

2015-01-01

Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process. PMID:26643504

Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

PubMed Central

Hatanaka, M; Del Giudice, R; Long, C

1975-01-01

Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

Glycogen synthase activation by sugars in isolated hepatocytes.

PubMed

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

Ameliorative effect of ursolic acid on renal fibrosis in adenine-induced chronic kidney disease in rats.

PubMed

Thakur, Richa; Sharma, Anshuk; Lingaraju, Madhu C; Begum, Jubeda; Kumar, Dhirendra; Mathesh, Karikalan; Kumar, Pawan; Singh, Thakur Uttam; Kumar, Dinesh

2018-05-01

Ursolic acid (UA), an ursane-type pentacyclic triterpenoid commonly found in apple peels and holy basil has been shown to possess many beneficial effects. Renal fibrosis is a complication of kidney injury and associated with increased risk of morbidity and mortality. In our previous investigation, a lupane-type pentacyclic triterpenoid, betulinic acid (BA) was found to have protective effect on chronic kidney disease (CKD) and renal fibrosis. This prompted us to explore the therapeutic value of UA, a chemically related compound to BA in CKD. CKD was induced by feeding adenine with the feed at a concentration of 0.75% for 28 days. UA at the dose rate of 30 mg/kg in 0.5% carboxy methyl cellulose (CMC) was administered by oral route, simultaneously with adenine feeding for 28 days. Adenine feeding increased the kidney weight to body weight index, decreased the kidney function due to injury as indicated by increased markers like serum urea, uric acid, creatinine, cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) and initiated the fibrotic response in kidney by increasing the profibrotic proteins viz. transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), fibronectin and collagen. However, treatment with UA reversed the damage induced by adenine as shown by reduced kidney injury and fibrosis markers which was further clearly evident in histological picture indicating the suitability of UA for use in CKD. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of aqueous extract and anthocyanins of calyces of Hibiscus sabdariffa (Malvaceae) in rats with adenine-induced chronic kidney disease.

PubMed

Ali, Badreldin H; Cahliková, Lucie; Opletal, Lubomir; Karaca, Turan; Manoj, Priyadarsini; Ramkumar, Aishwarya; Al Suleimani, Yousuf M; Al Za'abi, Mohammed; Nemmar, Abderrahim; Chocholousova-Havlikova, Lucie; Locarek, Miroslav; Siatka, Tomas; Blunden, Gerald

2017-09-01

The aim of this work was to assess the possible beneficial effects of aqueous extracts of Hibiscus sabdariffa L. calyces and anthocyanins isolated therefrom in an adenine-induced chronic kidney disease (CKD) model. Rats were orally given, for 28 consecutive days, either adenine alone or together with either aqueous extract of H. sabdariffa calyces (5 and 10%) or anthocyanins (50, 100 and 200 mg/kg of anthocyanin concentrate). For comparative purposes, two groups of rats were given lisinopril (10 mg/kg). When either H. sabdariffa aqueous extract or the anthocyanins isolated from it was administered along with adenine, the adverse effects of adenine-induced CKD were significantly lessened, mostly in a dose-dependent manner. The positive effects were similar to those obtained by administration of lisinopril. The results obtained show that both H. sabdariffa and its anthocyanins could be considered as possible promising safe dietary agents that could be used to attenuate the progression of human CKD. This could have added significance as H. sabdariffa tea is widely consumed in many parts of Africa and Asia and is thus readily available. © 2017 Royal Pharmaceutical Society.

Was adenine the first purine?

NASA Technical Reports Server (NTRS)

Schwartz, Alan W.; Bakker, C. G.

1989-01-01

Oligomerization of HCN (1 molar) in the presence of added formaldehyde (0.5 molar) produced an order of magnitude more 8-hydroxymethyladenine than adenine or any other biologically significant purine. This result suggests that on the prebiotic earth, nucleoside analogs may have been synthesized directly in more complex mixtures of HCN with other aldehydes.

The rate of the AMP/adenosine substrate cycle in concanavalin-A-stimulated rat lymphocytes.

PubMed Central

Szondy, Z; Newsholme, E A

1989-01-01

The effect of adenosine on the metabolism of prelabelled adenine nucleotides was investigated in concanavalin-A-stimulated rat lymphocytes. Adenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin, caused a 2-fold increase in the ATP concentration. This effect was, in part, countereacted by an increased rate of adenine nucleotide catabolism, which could be explained by a stimulation of AMP deaminase (EC 3.5.4.6). At the same time a continuous rate of labelled adenosine production was found, which was not affected by the increased ATP concentration and which could only be detected by the trapping effect of a high concentration of added unlabelled adenosine. It is concluded that the rate of the substrate cycle between AMP and adenosine is low (1.9 +/- 0.2 nmol/h per 10(7) cells) in comparison to the rate of AMP deamination. PMID:2552990

Sequencing of adenine in DNA by scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Tanaka, Hiroyuki; Taniguchi, Masateru

2017-08-01

The development of DNA sequencing technology utilizing the detection of a tunnel current is important for next-generation sequencer technologies based on single-molecule analysis technology. Using a scanning tunneling microscope, we previously reported that dI/dV measurements and dI/dV mapping revealed that the guanine base (purine base) of DNA adsorbed onto the Cu(111) surface has a characteristic peak at V s = -1.6 V. If, in addition to guanine, the other purine base of DNA, namely, adenine, can be distinguished, then by reading all the purine bases of each single strand of a DNA double helix, the entire base sequence of the original double helix can be determined due to the complementarity of the DNA base pair. Therefore, the ability to read adenine is important from the viewpoint of sequencing. Here, we report on the identification of adenine by STM topographic and spectroscopic measurements using a synthetic DNA oligomer and viral DNA.

Novel biocatalytic systems for maintaining the nucleotide balance based on adenylate kinase immobilized on carbon nanostructures.

PubMed

Hetmann, Anna; Wujak, Magdalena; Bolibok, Paulina; Zięba, Wojciech; Wiśniewski, Marek; Roszek, Katarzyna

2018-07-01

In this study graphene oxide (GO), carbon quantum dots (CQD) and carbon nanoonions (CNO) have been characterized and applied for the first time as a matrix for recombinant adenylate kinase (AK, EC 2.7.4.3) immobilization. AK is an enzyme fulfilling a key role in metabolic processes. This phosphotransferase catalyzes the interconversion of adenine nucleotides (ATP, ADP and AMP) and thereby participates in nucleotide homeostasis, monitors a cellular energy charge as well as acts as a component of purinergic signaling system. The AK activity in all obtained biocatalytic systems was higher as compared to the free enzyme. We have found that the immobilization on carbon nanostructures increased both activity and stability of AK. Moreover, the biocatalytic systems consisting of AK immobilized on carbon nanostructures can be easily and efficiently lyophilized without risk of desorption or decrease in the catalytic activity of the investigated enzyme. The positive action of AK-GO biocatalytic system in maintaining the nucleotide balance in in vitro cell culture was proved. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiler, Monica; Schmetzer, Helga; German Research Center for Environmental Health, Munich

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP andmore » GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.« less

Detection of Guanine and Adenine Using an Aminated Reduced Graphene Oxide Functional Membrane-Modified Glassy Carbon Electrode

PubMed Central

Li, Di; Yang, Xiao-Lu; Xiao, Bao-Lin; Geng, Fang-Yong; Hong, Jun; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

2017-01-01

A new electrochemical sensor based on a Nafion, aminated reduced graphene oxide and chitosan functional membrane-modified glassy carbon electrode was proposed for the simultaneous detection of adenine and guanine. Fourier transform-infrared spectrometry (FTIR), transmission electron microscopy (TEM), and electrochemical methods were utilized for the additional characterization of the membrane materials. The prepared electrode was utilized for the detection of guanine (G) and adenine (A). The anodic peak currents to G and A were linear in the concentrations ranging from 0.1 to 120 μM and 0.2 to 110 μM, respectively. The detection limits were found to be 0.1 μM and 0.2 μM, respectively. Moreover, the modified electrode could also be used to determine G and A in calf thymus DNA. PMID:28718793

Electron transfer driven decomposition of adenine and selected analogs as probed by experimental and theoretical methods

NASA Astrophysics Data System (ADS)

Cunha, T.; Mendes, M.; Ferreira da Silva, F.; Eden, S.; García, G.; Bacchus-Montabonel, M.-C.; Limão-Vieira, P.

2018-04-01

We report on a combined experimental and theoretical study of electron-transfer-induced decomposition of adenine (Ad) and a selection of analog molecules in collisions with potassium (K) atoms. Time-of-flight negative ion mass spectra have been obtained in a wide collision energy range (6-68 eV in the centre-of-mass frame), providing a comprehensive investigation of the fragmentation patterns of purine (Pu), adenine (Ad), 9-methyl adenine (9-mAd), 6-dimethyl adenine (6-dimAd), and 2-D adenine (2-DAd). Following our recent communication about selective hydrogen loss from the transient negative ions (TNIs) produced in these collisions [T. Cunha et al., J. Chem. Phys. 148, 021101 (2018)], this work focuses on the production of smaller fragment anions. In the low-energy part of the present range, several dissociation channels that are accessible in free electron attachment experiments are absent from the present mass spectra, notably NH2 loss from adenine and 9-methyl adenine. This can be understood in terms of a relatively long transit time of the K+ cation in the vicinity of the TNI tending to enhance the likelihood of intramolecular electron transfer. In this case, the excess energy can be redistributed through the available degrees of freedom inhibiting fragmentation pathways. Ab initio theoretical calculations were performed for 9-methyl adenine (9-mAd) and adenine (Ad) in the presence of a potassium atom and provided a strong basis for the assignment of the lowest unoccupied molecular orbitals accessed in the collision process.

Transformation by Complementation of an Adenine Auxotroph of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

PubMed Central

Alic, Margaret; Kornegay, Janet R.; Pribnow, David; Gold, Michael H.

1989-01-01

Swollen basidiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per μg of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies. Analysis of conidia and basidiospores from transformants demonstrated that the transforming character was mitotically and meiotically stable on both selective and nonselective media. Genetic crosses between double mutants transformed for adenine prototrophy and other auxotrophic strains yielded Ade− progeny, which indicated that integration occurred at a site(s) other than the resident adenine biosynthetic gene. Images PMID:16347848

Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.).

PubMed

Katahira, Riko; Ashihara, Hiroshi

2009-12-01

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --> nicotinamide mononucleotide --> nicotinamide riboside --> nicotinic acid riboside --> nicotinic acid mononucleotide --> nicotinic acid adenine dinucleotide --> NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.

Transformation by complementation of an adenine auxotroph of the lignin-degrading basidiomycete Phanerochaete chrysosporium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alic, M.; Kornegay, J.R.; Pribnow, D.

1989-02-01

Swollen basiodiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per {mu}g of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies. Analysis of conidia and basiodiospores from transformants demonstrated that the transforming character was mitotically and meiotically stable on both selective and nonselective media. Genetic crosses between double mutants transformed for adenine prototrophy and othermore » auxotrophic strains yielded Ade{sup {minus}} progeny, which indicated that integration occurred at a site(s) other than the resident adenine biosynthetic gene.« less

Purine metabolism in Toxoplasma gondii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krug, E.C.; Marr, J.J.; Berens, R.L.

1989-06-25

We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the nextmore » most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.« less

Relative similarity within purine nucleotide and ligand structures operating on nitric oxide synthetase, guanylyl cyclase and potassium (K ATP, BK Ca) channels.

PubMed

Williams, W Robert

2011-01-01

Purine nucleotides play a central role in signal transduction events initiated at the cell membrane. The NO-cGMP-cGK pathway, in particular, mediates events involving NOS and some classes of K(+) ion channel. The aim of this study is to investigate relative molecular similarity within the ligands binding to NOS, K(ATP), BK(Ca) channels and regulatory nucleotides. Minimum energy conformers of the ligand structures were superimposed and fitted to L-arginine and the nucleotides of adenine and guanine using a computational program. Distinctive patterns were evident in the fitting of NOS isoform antagonists to L-arginine. K(ATP) channel openers and antagonists superimposed on the glycosidic linkage and imidazole ring of the purine nucleotides, and guanidinium and ribose groups of GTP in the case of glibenclamide. The fits of BK(Ca) channel openers and antagonists to cGMP were characterized by the linear dimensions of their structures; distances between terminal oxy groups in respect of dexamethasone and aldosterone. The findings provide structural evidence for the functional interaction between K(+) channel openers/antagonists and the regulatory nucleotides. Use of the purine nucleotide template systematizes the considerable heterogeneity evident within the structures of ligands operating on K(+) ion channels. © 2010 The Author. JPP © 2010 Royal Pharmaceutical Society.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization*

PubMed Central

Wang, Weiyi; Jiang, Qiyang; Argentini, Manuela; Cornu, David; Gigant, Benoît; Knossow, Marcel; Wang, Chunguang

2012-01-01

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension. PMID:22403406

Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA.

PubMed

Huang, Ke-Jing; Niu, De-Jun; Sun, Jun-Yong; Han, Cong-Hui; Wu, Zhi-Wei; Li, Yan-Li; Xiong, Xiao-Qin

2011-02-01

A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334V. The detection limit for individual determination of guanine and adenine was 5.0×10(-8)M and 2.5×10(-8)M (S/N=3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G+C)/(A+T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability. Copyright © 2010 Elsevier B.V. All rights reserved.

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

PubMed

Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

Advances in targeting cyclic nucleotide phosphodiesterases

PubMed Central

Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

2014-01-01

Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

Binding of p-mercaptobenzoic acid and adenine to gold-coated electroless etched silicon nanowires studied by surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Mohaček-Grošev, Vlasta; Gebavi, Hrvoje; Bonifacio, Alois; Sergo, Valter; Daković, Marko; Bajuk-Bogdanović, Danica

2018-07-01

Modern diagnostic tools ever aim to reduce the amount of analyte and the time needed for obtaining the result. Surface-enhanced Raman spectroscopy is a method that could satisfy both of these requirements, provided that for each analyte an adequate substrate is found. Here we demonstrate the ability of gold-sputtered silicon nanowires (SiNW) to bind p-mercaptobenzoic acid in 10-3, 10-4 and 10-5 M and adenine in 30 and 100 μM concentrations. Based on the normal mode analysis, presented here for the first time, the binding of p-mercaptobenzoic acid is deduced. The intensity enhancement of the 1106 cm-1 band is explained by involvement of the Csbnd S stretching deformation, and the appearance of the broad 300 cm-1 band attributed to Ssbnd Au stretching mode. Adenine SERS spectra demonstrate the existence of the 7H tautomer since the strongest band observed is at 736 cm-1. The adenine binding is likely to occur in several ways, because the number of observed bands in the 1200-1600 cm-1 interval exceeds the number of observed bands in the normal Raman spectrum of the free molecule.

Zhen-wu-tang ameliorates adenine-induced chronic renal failure in rats: regulation of the canonical Wnt4/beta-catenin signaling in the kidneys.

PubMed

La, Lei; Wang, Lili; Qin, Fei; Jiang, Jian; He, Songqi; Wang, Chunxia; Li, Yuhao

2018-06-12

Zhen-wu-tang (ZWT), composed of Radix Aconiti lateralis, Rhizoma Atractylodis macrocephalae, Poria, Radix Paeoniae alba and ginger, is a classic Chinese herbal formula for the treatment of chronic kidney diseases that may cause chronic renal failure (CRF). To better understand its clinical use, this study investigated the effects and underlying mechanisms of action of ZWT on CRF. CRF was induced by adenine. ZWT was given via an oral gavage method. The serum biochemical parameters were measured enzymatically or by ELISA. The kidneys were examined pathohistologically. The gene expression was analyzed by real time PCR and Western blot. Similar to the positive control losartan, ZWT extract inhibited adenine-induced increase in serum concentrations of creatinine, BUN and advanced oxidation protein products in rats. These effects were accompanied by attenuation of proteinuria and renal pathological changes and suppression of renal mRNA and protein overexpression of Collagen IV and fibronectin, two of the key components of fibrosis. Mechanistically, renal mRNA and protein expression of Wnt4, a Wnt signaling ligand, was increased in the adenine-treated group, compared to the vehicle-treated control. Consistently, Wnt4 downstream genes beta-catenin and Axin were also overexpressed. Treatment with ZWT extract and losartan suppressed adenine-stimulated overexpression of these mRNAs and proteins. The present results demonstrate that ZWT extract ameliorates adenine-induced CRF in rats by regulation of the canonical Wnt4/beta-catenin signaling in the kidneys. Our findings provide new insight into the underlying renoprotective mechanisms of the ancient formula. Copyright © 2017. Published by Elsevier B.V.

Prolonged Pulmonary Exposure to Diesel Exhaust Particles Exacerbates Renal Oxidative Stress, Inflammation and DNA Damage in Mice with Adenine-Induced Chronic Renal Failure.

PubMed

Nemmar, Abderrahim; Karaca, Turan; Beegam, Sumaya; Yuvaraju, Priya; Yasin, Javed; Hamadi, Naserddine Kamel; Ali, Badreldin H

2016-01-01

Epidemiological evidence indicates that patients with chronic kidney diseases have increased susceptibility to adverse outcomes related to long-term exposure to particulate air pollution. However, mechanisms underlying these effects are not fully understood. Presently, we assessed the effect of prolonged exposure to diesel exhaust particles (DEP) on chronic renal failure induced by adenine (0.25% w/w in feed for 4 weeks), which is known to involve inflammation and oxidative stress. DEP (0.5m/kg) was intratracheally (i.t.) instilled every 4th day for 4 weeks (7 i.t. instillation). Four days following the last exposure to either DEP or saline (control), various renal endpoints were measured. While body weight was decreased, kidney weight increased in DEP+adenine versus saline+adenine or DEP. Water intake, urine volume, relative kidney weight were significantly increased in adenine+DEP versus DEP and adenine+saline versus saline. Plasma creatinine and urea increased and creatinine clearance decreased in adenine+DEP versus DEP and adenine+saline versus saline. Tumor necrosis factor α, lipid peroxidation and reactive oxygen species were significantly increased in adenine+DEP compared with either DEP or adenine+saline. The antioxidant calase was significantly decreased in adenine+DEP compared with either adenine+saline or DEP. Notably, renal DNA damage was significantly potentiated in adenine+DEP compared with either adenine+saline or DEP. Similarly, systolic blood pressure was increased in adenine+DEP versus adenine+saline or DEP, and in DEP versus saline. Histological evaluation revealed more collagen deposition, higher number of necrotic cell counts and dilated tubules, cast formation and collapsing glomeruli in adenine+DEP versus adenine+saline or DEP. Prolonged pulmonary exposure to diesel exhaust particles worsen renal oxidative stress, inflammation and DNA damage in mice with adenine-induced chronic renal failure. Our data provide biological plausibility that air

Communication: Site-selective bond excision of adenine upon electron transfer

NASA Astrophysics Data System (ADS)

Cunha, T.; Mendes, M.; Ferreira da Silva, F.; Eden, S.; García, G.; Limão-Vieira, P.

2018-01-01

This work demonstrates that selective excision of hydrogen atoms at a particular site of the DNA base adenine can be achieved in collisions with electronegative atoms by controlling the impact energy. The result is based on analysing the time-of-flight mass spectra yields of potassium collisions with a series of labeled adenine derivatives. The production of dehydrogenated parent anions is consistent with neutral H loss either from selective breaking of C-H or N-H bonds. These unprecedented results open up a new methodology in charge transfer collisions that can initiate selective reactivity as a key process in chemical reactions that are dominant in different areas of science and technology.

Single nucleotide polymorphism discrimination with and without an ethidium bromide intercalator.

PubMed

Fenati, Renzo A; Connolly, Ashley R; Ellis, Amanda V

2017-02-15

Single nucleotide polymorphism (SNP) genotyping is an important aspect in understanding genetic variations. Here, we discriminate SNPs using toe-hold mediated displacement reactions. The biological target is an 80 nucleotide long double-stranded-DNA from the mtDNA HV1 region, associated with maternal ancestry. This target has been specially designed with a pendant toehold and a cationic fluorophore, ATTO 647N, as a reporter, produced in a polymerase chain reaction. Rates of reaction for the toehold-polymerase chain reaction products (TPPs) with their corresponding complementary displacing sequences, labelled with a Black Hole Quencher 1, followed the order TPP-Cytosine > TPP-Thymine > TPP-Adenine ≥ TPP-Guanine. Non-complementary rates were the slowest with mismatches involving cytosine. These reactions, operating in a static/or contact mode, gave averaged readouts between SNPs within 15 min (with 80-90% quenching), compared to 25-30 min in previous studies involving fluorescence resonance energy transfer. Addition of an intercalating agent, ethidium bromide, retarded the rate of reaction in which cytosine was involved, presumably through stabilization of the base pairing, which resulted in markedly improved discrimination of cytosine containing SNPs. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-dimensional network stability of nucleobases and amino acids on graphite under ambient conditions: adenine, L-serine and L-tyrosine.

PubMed

Bald, Ilko; Weigelt, Sigrid; Ma, Xiaojing; Xie, Pengyang; Subramani, Ramesh; Dong, Mingdong; Wang, Chen; Mamdouh, Wael; Wang, Jianguo; Besenbacher, Flemming

2010-04-14

We have investigated the stability of two-dimensional self-assembled molecular networks formed upon co-adsorption of the DNA base, adenine, with each of the amino acids, L-serine and L-tyrosine, on a highly oriented pyrolytic graphite (HOPG) surface by drop-casting from a water solution. L-serine and L-tyrosine were chosen as model systems due to their different interaction with the solvent molecules and the graphite substrate, which is reflected in a high and low solubility in water, respectively, compared with adenine. Combined scanning tunneling microscopy (STM) measurements and density functional theory (DFT) calculations show that the self-assembly process is mainly driven by the formation of strong adenine-adenine hydrogen bonds. We find that pure adenine networks are energetically more stable than networks built up of either pure L-serine, pure L-tyrosine or combinations of adenine with L-serine or L-tyrosine, and that only pure adenine networks are stable enough to be observable by STM under ambient conditions.

ON THE INTERACTION OF ADENINE WITH IONIZING RADIATION: MECHANISTICAL STUDIES AND ASTROBIOLOGICAL IMPLICATIONS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, Nicholas L.; Ullrich, Susanne; Bennett, Chris J.

2011-04-01

The molecular inventory available on the prebiotic Earth was likely derived from both terrestrial and extraterrestrial sources. A complete description of which extraterrestrial molecules may have seeded early Earth is therefore necessary to fully understand the prebiotic evolution which led to life. Galactic cosmic rays (GCRs) are expected to cause both the formation and destruction of important biomolecules-including nucleic acid bases such as adenine-in the interstellar medium within the ices condensed on interstellar grains. The interstellar ultraviolet (UV) component is expected to photochemically degrade gas-phase adenine on a short timescale of only several years. However, the destruction rate is expectedmore » to be significantly reduced when adenine is shielded in dense molecular clouds or even within the ices of interstellar grains. Here, biomolecule destruction by the energetic charged particle component of the GCR becomes important as it is not fully attenuated. Presented here are results on the destruction rate of the nucleobase adenine in the solid state at 10 K by energetic electrons, as generated in the track of cosmic ray particles as they penetrate ices. When both UV and energetic charged particle destructive processes are taken into account, the half-life of adenine within dense interstellar clouds is found to be {approx}6 Myr, which is on the order of a star-forming molecular cloud. We also discuss chemical reaction pathways within the ices to explain the production of observed species, including the formation of nitriles (R-C{identical_to}N), epoxides (C-O-C), and carbonyl functions (R-C=O).« less

Adenine Inhibits TNF-α Signaling in Intestinal Epithelial Cells and Reduces Mucosal Inflammation in a Dextran Sodium Sulfate-Induced Colitis Mouse Model.

PubMed

Fukuda, Toshihiko; Majumder, Kaustav; Zhang, Hua; Turner, Patricia V; Matsui, Toshiro; Mine, Yoshinori

2016-06-01

Adenine (6-amino-6H-purine), found in molokheiya (Corchorus olitorius L.), has exerted vasorelaxation effects in the thoracic aorta. However, the mode of action of the anti-inflammatory effect of adenine is unclear. Thus, we investigated to clarify the effect of adenine on chronic inflammation of the gastrointestinal tract. In intestinal epithelial cells, adenine significantly inhibited tumor necrosis factor-α-induced interleukin-8 secretion. The inhibition of adenine was abolished under the treatment of inhibitors of adenyl cyclase (AC) and protein kinase A (PKA), indicating the effect of adenine was mediated through the AC/PKA pathway. Adenine (5, 10, and 50 mg/kg BW/day) was administered orally for 14 days to female BALB/c mice, and then 5% dextran sodium sulfate (DSS) was given to induce colitis. Adenine (5 mg/kg BW/day) significantly prevented DSS-induced colon shortening, expression of pro-inflammatory cytokines, and histological damage in the colon. These results suggest that adenine can be a promising nutraceutical for the prevention of intestinal inflammation.

Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

PubMed

Usha, S; Selvaraj, S

2015-01-01

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.

Dietary L-lysine prevents arterial calcification in adenine-induced uremic rats.

PubMed

Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Isaka, Yoshitaka; Rakugi, Hiromi

2014-09-01

Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. Copyright © 2014 by the American Society of Nephrology.

Dietary l-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

PubMed Central

Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Rakugi, Hiromi

2014-01-01

Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. PMID:24652795

Characterization of a dam Mutant of Serratia marcescens and Nucleotide Sequence of the dam Region

PubMed Central

Ostendorf, Tammo; Cherepanov, Peter; de Vries, Johann; Wackernagel, Wilfried

1999-01-01

The DNA of Serratia marcescens has N6-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from S. marcescens Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (dam) coding for the adenine methyltransferase (Dam enzyme) of S. marcescens was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a dam-13::Tn9 mutant of Escherichia coli. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of E. coli. The dam gene is located between flanking genes which are similar to those found to the sides of the E. coli dam gene. The results of complementation studies indicated that like Dam of E. coli and unlike Dam of Vibrio cholerae, the Dam enzyme of S. marcescens plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors. PMID:10383952

Probing the ATP site of GRP78 with nucleotide triphosphate analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun

GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the

Probing the ATP site of GRP78 with nucleotide triphosphate analogs

DOE PAGES

Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; ...

2016-05-04

GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the

Brain purine metabolism and xanthine dehydrogenase/oxidase conversion in hyperammonemia are under control of NMDA receptors and nitric oxide.

PubMed

Kaminsky, Yury; Kosenko, Elena

2009-10-19

In hyperammonemia, a decrease in brain ATP can be a result of adenine nucleotide catabolism. Xanthine dehydrogenase (XD) and xanthine oxidase (XO) are the end steps in the purine catabolic pathway and directly involved in depletion of the adenylate pool in the cell. Besides, XD can easily be converted to XO to produce reactive oxygen species in the cell. In this study, the effects of acute ammonia intoxication in vivo on brain adenine nucleotide pool and xanthine and hypoxanthine, the end degradation products of adenine nucleotides, during the conversion of XD to XO were studied. Injection of rats with ammonium acetate was shown to lead to the dramatic decrease in the ATP level, adenine nucleotide pool size and adenylate energy charge and to the great increase in hypoxanthine and xanthine 11 min after the lethal dose indicating rapid degradation of adenylates. Conversion of XD to XO in hyperammonemic rat brain was evidenced by elevated XO/XD activity ratio. Injection of MK-801, a NMDA receptor blocker, prevented ammonia-induced catabolism of adenine nucleotides and conversion of XD to XO suggesting that in vivo these processes are mediated by activation of NMDA receptors. The in vitro dose-dependent effects of sodium nitroprusside, a NO donor, on XD and XO activities are indicative of the direct modification of the enzymes by nitric oxide. This is the first report evidencing the increase in brain xanthine and hypoxanthine levels and adenine nucleotide breakdown in acute ammonia intoxication and NMDA receptor-mediated prevention of these alterations.

Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraser, Marie E., E-mail: frasm@ucalgary.ca; Cherney, Maia M.; Marcato, Paola

2006-07-01

Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active asmore » an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.« less

Quercetin Attenuates Vascular Calcification through Suppressed Oxidative Stress in Adenine-Induced Chronic Renal Failure Rats.

PubMed

Chang, Xue-Ying; Cui, Lei; Wang, Xing-Zhi; Zhang, Lei; Zhu, Dan; Zhou, Xiao-Rong; Hao, Li-Rong

2017-01-01

This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism. Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta ( P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway.

Quercetin Attenuates Vascular Calcification through Suppressed Oxidative Stress in Adenine-Induced Chronic Renal Failure Rats

PubMed Central

Chang, Xue-ying; Cui, Lei; Wang, Xing-zhi; Zhang, Lei; Zhu, Dan

2017-01-01

Background This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. Methods 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism. Results Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta (P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. Conclusions Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway. PMID:28691026

Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration

PubMed Central

Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W.; Davis, James G.; Agarwal, Beamon; Baur, Joseph A.

2016-01-01

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing Nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nampt overexpressing mice were mildly hyperglycemic at baseline and, similarly to the mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking Nampt in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. Conclusion NAD availability is limiting during liver regeneration and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. PMID:27809334

Spectroscopic investigation on cocrystal formation between adenine and fumaric acid based on infrared and Raman techniques

NASA Astrophysics Data System (ADS)

Du, Yong; Fang, Hong Xia; Zhang, Qi; Zhang, Hui Li; Hong, Zhi

2016-01-01

As an important component of double-stranded DNA, adenine has powerful hydrogen-bond capability, due to rich hydrogen bond donors and acceptors existing within its molecular structure. Therefore, it is easy to form cocrystal between adenine and other small molecules with intermolecular hydrogen-bond effect. In this work, cocrystal of adenine and fumaric acid has been characterized as model system by FT-IR and FT-Raman spectral techniques. The experimental results show that the cocrystal formed between adenine and fumaric acid possesses unique spectroscopical characteristic compared with that of starting materials. Density functional theory (DFT) calculation has been performed to optimize the molecular structures and simulate vibrational modes of adenine, fumaric acid and the corresponding cocrystal. Combining the theoretical and experimental vibrational results, the characteristic bands corresponding to bending and stretching vibrations of amino and carbonyl groups within cocrystal are shifted into lower frequencies upon cocrystal formation, and the corresponding bond lengths show some increase due to the effect of intermolecular hydrogen bonding. Different vibrational modes shown in the experimental spectra have been assigned based on the simulation DFT results. The study could provide experimental and theoretical benchmarks to characterize cocrystal formed between active ingredients and cocrystal formers and also the intermolecular hydrogen-bond effect within cocrystal formation process by vibrational spectroscopic techniques.

Synthesis, spectroscopic, structural and thermal characterizations of vanadyl(IV) adenine complex prospective as antidiabetic drug agent

NASA Astrophysics Data System (ADS)

El-Megharbel, Samy M.; Hamza, Reham Z.; Refat, Moamen S.

2015-01-01

The vanadyl(IV) adenine complex; [VO(Adn)2]ṡSO4; was synthesized and characterized. The molar conductivity of this complex was measured in DMSO solution that showed an electrolyte nature. Spectroscopic investigation of the green solid complex studied here indicate that the adenine acts as a bidentate ligand, coordinated to vanadyl(IV) ions through the nitrogen atoms N7 and nitrogen atom of amino group. Thus, from the results presented the vanadyl(IV) complex has square pyramid geometry. Further characterizations using thermal analyses and scanning electron techniques was useful. The aim of this paper was to introduce a new drug model for the diabetic complications by synthesized a novel mononuclear vanadyl(IV) adenine complex to mimic insulin action and reducing blood sugar level. The antidiabetic ability of this complex was investigated in STZ-induced diabetic mice. The results suggested that VO(IV)/adenine complex has antidiabetic activity, it improved the lipid profile, it improved liver and kidney functions, also it ameliorated insulin hormone and blood glucose levels. The vanadyl(IV) complex possesses an antioxidant activity and this was clear through studying SOD, CAT, MDA, GSH and methionine synthase. The current results support the therapeutic potentiality of vanadyl(IV)/adenine complex for the management and treatment of diabetes.

Supramolecular polymeric chemosensor for biomedical applications: design and synthesis of a luminescent zinc metallopolymer as a chemosensor for adenine detection.

PubMed

Chow, Cheuk-Fai

2012-11-01

Adenine is an important bio-molecule that plays many crucial roles in food safety and biomedical diagnostics. Differentiating adenine from a mixture of adenosine and other nucleic bases (guanine, thymine, cytosine, and uracil) is particularly important for both biological and clinical applications. A neutral Zn(II) metallosupramolecular polymer based on acyl hydrazone derived coordination centres (P1) were generated through self-assembly polymerization. It is a linear coordination polymer that behaves like self-standing film. The synthesis, (1)H-NMR characterization, and spectroscopic properties of this supramolecular material are reported. P1 was found to be a chemosensor specific to adenine, with a luminescent enhancement. The binding properties of P1 with common nucleic bases and nucleosides reveal that this supramolecular polymer is very selective to adenine molecules (~20 to 420 times more selectivity than other nucleic bases). The formation constant (K) of P1 to adenine was found to be log K = 4.10 ± 0.02. This polymeric chemosensor produces a specific response to adenine down to 90 ppb. Spectrofluorimetric and (1)H-NMR titration studies showed that the P1 polymer allows each Zn(II) coordination centre to bind to two adenine molecules through hydrogen bonding with their imine and hydrazone protons.

Changes in calmodulin concentration and cyclic 3',5'-nucleotide phosphodiesterase activity in skeletal muscle of hyper- and hypothyroid rats.

PubMed

Mano, T; Iwase, K; Yoshimochi, I; Sawai, Y; Oda, N; Nishida, Y; Mokuno, T; Kotake, M; Nakai, A; Hayakawa, N

1995-08-01

Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.

Heptacopper(II) and dicopper(II)-adenine complexes: synthesis, structural characterization, and magnetic properties

DOE PAGES

Leite Ferreira, B. J. M.; Brandão, Paula; Dos Santos, A. M.; ...

2015-07-13

The syntheses, crystal structures, and magnetic properties of two new copper(II) complexes with molecular formulas [Cu 7(μ 2-OH 2) 6(μ 3-O) 6(adenine) 6(NO 3) 26H 2O (1) and [Cu 2(μ 2-H 2O) 2(adenine) 2(H 2O) 4](NO 3) 42H 2O (2) are reported. We composed the heptanuclear compound of a central octahedral CuO 6 core sharing edges with six adjacent copper octahedra. In 2, the copper octahedra shares one equatorial edge. In both compounds, these basic copper cluster units are further linked by water bridges and bridging adenine ligands through N3 and N9 donors. All copper(II) centers exhibit Jahn-Teller distorted octahedralmore » coordination characteristic of a d 9 center. Our study of the magnetic properties of the heptacopper complex revealed a dominant ferromagnetic intra-cluster interaction, while the dicopper complex exhibits antiferromagnetic intra-dimer interactions with weakly ferromagnetic inter-dimer interaction.« less

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

PubMed

El Bissati, Kamal; Downie, Megan J; Kim, Seong-Kyoun; Horowitz, Michael; Carter, Nicola; Ullman, Buddy; Ben Mamoun, Choukri

2008-10-01

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.

Nonselective enrichment for yeast adenine mutants by flow cytometry

NASA Technical Reports Server (NTRS)

Bruschi, C. V.; Chuba, P. J.

1988-01-01

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

Spectroscopic investigation on cocrystal formation between adenine and fumaric acid based on infrared and Raman techniques.

PubMed

Du, Yong; Fang, Hong Xia; Zhang, Qi; Zhang, Hui Li; Hong, Zhi

2016-01-15

As an important component of double-stranded DNA, adenine has powerful hydrogen-bond capability, due to rich hydrogen bond donors and acceptors existing within its molecular structure. Therefore, it is easy to form cocrystal between adenine and other small molecules with intermolecular hydrogen-bond effect. In this work, cocrystal of adenine and fumaric acid has been characterized as model system by FT-IR and FT-Raman spectral techniques. The experimental results show that the cocrystal formed between adenine and fumaric acid possesses unique spectroscopical characteristic compared with that of starting materials. Density functional theory (DFT) calculation has been performed to optimize the molecular structures and simulate vibrational modes of adenine, fumaric acid and the corresponding cocrystal. Combining the theoretical and experimental vibrational results, the characteristic bands corresponding to bending and stretching vibrations of amino and carbonyl groups within cocrystal are shifted into lower frequencies upon cocrystal formation, and the corresponding bond lengths show some increase due to the effect of intermolecular hydrogen bonding. Different vibrational modes shown in the experimental spectra have been assigned based on the simulation DFT results. The study could provide experimental and theoretical benchmarks to characterize cocrystal formed between active ingredients and cocrystal formers and also the intermolecular hydrogen-bond effect within cocrystal formation process by vibrational spectroscopic techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

Some reactions of the hydroxyl adduct of adenine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanhemmen, J.J.

1975-01-01

The chemical reactions of purine derivatives resulting from pulse radiolysis were studied. Some reactions of the hydroxyl adduct of adenine are described and one of these reactions was compared with similar reactions of hydroxyl adducts of other purine derivatives. Evidence is given that in various purines opening of the imidazole ring is due to unimolecular rearrangements of the hydroxyl adducts. (GRA)

Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

PubMed

Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W; Davis, James G; Agarwal, Beamon; Baur, Joseph A

2017-02-01

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630). © 2016 by the American Association for the Study of Liver Diseases.

Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid).

PubMed

Yamamoto, Tetsuya; Moriwaki, Yuji; Takahashi, Sumio

2005-06-01

There are many factors that contribute to hyperuricemia, including obesity, insulin resistance, alcohol consumption, diuretic use, hypertension, renal insufficiency, genetic makeup, etc. Of these, alcohol (ethanol) is the most important. Ethanol enhances adenine nucleotide degradation and increases lactic acid level in blood, leading to hyperuricemia. In beer, purines also contribute to an increase in plasma uric acid. Although rare, dehydration and ketoacidosis (due to ethanol ingestion) are associated with the ethanol-induced increase in serum uric acid levels. Ethanol also increases the plasma concentrations and urinary excretion of hypoxanthine and xanthine via the acceleration of adenine nucleotide degradation and a possible weak inhibition of xanthine dehydrogenase activity. Since many factors such as the ALDH2*1 gene and ADH2*2 gene, daily drinking habits, exercise, and dehydration enhance the increase in plasma concentration of uric acid induced by ethanol, it is important to pay attention to these factors, as well as ingested ethanol volume, type of alcoholic beverage, and the administration of anti-hyperuricemic agents, to prevent and treat ethanol-induced hyperuricemia.

Photoinitiator Nucleotide for Quantifying Nucleic Acid Hybridization

PubMed Central

Johnson, Leah M.; Hansen, Ryan R.; Urban, Milan; Kuchta, Robert D.; Bowman, Christopher N.

2010-01-01

This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′-deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm2. Film heights of 10–20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm2 while below the detection limit of ~10 EITC-nucleotides/μm2, no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges. PMID:20337438

An experimental and theoretical vibrational study of interaction of adenine and thymine with artificial seawaters: A prebiotic chemistry experiment.

PubMed

Anizelli, Pedro R; Baú, João P T; Nabeshima, Henrique S; da Costa, Marcello F; de Santana, Henrique; Zaia, Dimas A M

2014-05-21

Nucleic acid bases play important roles in living beings. Thus, their interaction with salts the prebiotic Earth could be an important issue for the understanding of origin of life. In this study, the effect of pH and artificial seawaters on the structure of adenine and thymine was studied via parallel determinations using FT-IR, Raman spectroscopy and theoretical calculations. Thymine and adenine lyophilized in solutions at basic and acidic conditions showed characteristic bands of the enol-imino tautomer due to the deprotonation and the hydrochloride form due to protonation, respectively. The interaction of thymine and adenine with different seawaters representative of different geological periods on Earth was also studied. In the case of thymine a strong interaction with Sr(2+) promoted changes in the Raman and infrared spectra. For adenine changes in infrared and Raman spectra were observed in the presence of salts from all seawaters tested. The experimental results were compared to theoretical calculations, which showed structural changes due to the presence of ions Na(+), Mg(2+), Ca(2+) and Sr(2+) of artificial seawaters. For thymine the bands arising from C4=C5 and C6=O stretching were shifted to lower values, and for adenine, a new band at 1310cm(-1) was observed. The reactivity of adenine and thymine was studied by comparing changes in nucleophilicity and energy of the HOMO orbital. Copyright © 2014 Elsevier B.V. All rights reserved.

Randomised double-blind trial of acyclovir (Zovirax) and adenine arabinoside in herpes simplex amoeboid corneal ulceration.

PubMed

Collum, L M; Logan, P; McAuliffe-Curtin, D; Hung, S O; Patterson, A; Rees, P J

1985-11-01

Fifty-one patients were treated in a dual-centre, double-blind comparison of acyclovir and adenine arabinoside in herpetic amoeboid (geographic) corneal ulceration. Twenty-four of the 25 patients receiving acyclovir healed in a mean time of 12.2 days, while 24 of the 26 patients treated with adenine arabinoside healed in a mean time of 11.0 days. There was no statistically significant difference between the two groups in terms of healing. A second analysis, excluding any patients who had received antiviral treatment immediately prior to entry into the study, showed that 18 of the 19 who received acyclovir healed in an average of 11.7 days and 18 of the 19 recipients of adenine arabinoside healed in a mean time of 11.2 days. Again the difference was not statistically significant.

Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase.

PubMed

Harris, Lawrence D; Harijan, Rajesh K; Ducati, Rodrigo G; Evans, Gary B; Hirsch, Brett M; Schramm, Vern L

2018-01-19

Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid, and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocation-like transition states with a partial positive charge residing on the pentose ring. Core chemistry for synthesis of transition state analogues related to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) reactant of these enzymes could be developed by stereospecific placement of bis-phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and the bis-phosphates anchor both the 1- and 5-phosphate binding sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone redox methodology was applied to the stereocontrolled synthesis of three stereoisomers of a selectively monoprotected diol relevant to the synthesis of transition-state analogue inhibitors. These polyhydroxylated pyrrolidine natural product analogues were bis-phosphorylated to generate analogues of the ribocationic form of 5-phosphoribosyl 1-phosphate. A safe, high yielding synthesis of the key intermediate represents a new route to these transition state mimics. An enantiomeric pair of iminoaltritol bis-phosphates (L-DIAB and D-DIAB) was prepared and shown to display inhibition of Plasmodium falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers by altered ring geometry and bis-phosphate catalytic site contacts.

Nucleotide variability in the 5-enolpyruvylshikimate-3-phosphate synthase gene from Eleusine indica (L.) Gaertn.

PubMed

Chong, J L; Wickneswari, R; Ismail, B S; Salmijah, S

2008-02-01

This study reports the results of the partial DNA sequence analysis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant (R) and glyphosate-susceptible (S) biotypes of Eleusine indica (L.) Gaertn from Peninsular Malaysia. Sequencing results revealed point mutation at nucleotide position 875 in the R biotypes of Bidor, Chaah and Temerloh. In the Chaah R population, substitution of cytosine (C) to adenine (A) resulted in the change of threonine (Thr106) to proline (Pro106) and from C to thymidine (T) in the Bidor R population, leading to serine (Ser106) from Pro106. As for the Temerloh R, C was substituted by T resulting in the change of Pro106 to Ser106. A new mutation previously undetected in the Temerloh R was revealed with C being substituted with A, resulting in the change of Pro106 to Thr106 indicating multiple founding events rather than to the spread of a single resistant allele. There was no point mutation recorded at nucleotide position 875 previously demonstrated to play a pivotal role in conferring glyphosate resistance to E. indica for the Lenggeng, Kuala Selangor, Melaka R populations. Thus, there may be another resistance mechanism yet undiscovered in the resistant Lenggeng, Kuala Selangor and Melaka populations.

Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

PubMed

Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

2016-05-01

Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

Absorption by DNA single strands of adenine isolated in vacuo: The role of multiple chromophores

NASA Astrophysics Data System (ADS)

Nielsen, Lisbeth Munksgaard; Pedersen, Sara Øvad; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted

2012-02-01

The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (˜3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.

Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors.

PubMed

Rode, W; Jastreboff, M M

1984-01-01

Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.

Structural energetics of the adenine tract from an intrinsic transcription terminator.

PubMed

Huang, Yuegao; Weng, Xiaoli; Russu, Irina M

2010-04-02

Intrinsic transcription termination sites generally contain a tract of adenines in the DNA template that yields a tract of uracils at the 3' end of the nascent RNA. To understand how this base sequence contributes to termination of transcription, we have investigated two nucleic acid structures. The first is the RNA-DNA hybrid that contains the uracil tract 5'-rUUUUUAU-3' from the tR2 intrinsic terminator of bacteriophage lambda. The second is the homologous DNA-DNA duplex that contains the adenine tract 5'-dATAAAAA-3'. This duplex is present at the tR2 site when the DNA is not transcribed. The opening and the stability of each rU-dA/dT-dA base pair in the two structures are characterized by imino proton exchange and nuclear magnetic resonance spectroscopy. The results reveal concerted opening of the central rU-dA base pairs in the RNA-DNA hybrid. Furthermore, the stability profile of the adenine tract in the RNA-DNA hybrid is very different from that of the tract in the template DNA-DNA duplex. In the RNA-DNA hybrid, the stabilities of rU-dA base pairs range from 4.3 to 6.5 kcal/mol (at 10 degrees C). The sites of lowest stability are identified at the central positions of the tract. In the template DNA-DNA duplex, the dT-dA base pairs are more stable than the corresponding rU-dA base pairs in the hybrid by 0.9 to 4.6 kcal/mol and, in contrast to the RNA-DNA hybrid, the central base pairs have the highest stability. These results suggest that the central rU-dA/dT-dA base pairs in the adenine tract make the largest energetic contributions to transcription termination by promoting both the dissociation of the RNA transcript and the closing of the transcription bubble. The results also suggest that the high stability of dT-dA base pairs in the DNA provides a signal for the pausing of RNA polymerase at the termination site. Copyright 2010 Elsevier Ltd. All rights reserved.

Studies on the inhibition of Moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds.

PubMed

Hawtrey, Arthur; Pieterse, Anton; van Zyl, Johann; Van der Bijl, Pieter; Van der Merwe, Marichen; Nel, William; Ariatti, Mario

2008-09-01

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 '-phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5'-AMP inhibited the enzyme partially using poly (rA).oligo d(pT)(16-18) as template-primer with [(3)H]dTTP. In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m, and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above template-primer system. The compounds all inhibited the reverse transcriptase with IC(50) values of approximately 60-80 microM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the nucleotide 8-(6-aminohexyl) amino-adenosine-5'-phosphate, the inhibitory activity of this compound increased significantly (IC(50) = 5 microM).

Reaction of. beta. -propiolactone with derivatives of adenine and with DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, R.; Mieyal, J.J.; Goldthwait, D.A.

1982-01-01

The reaction of deoxyadenosine with ..beta..-propiolactone produces two derivatives. One is 1-(2-carboxyethyl)-2-deoxyadenosine (CEdA). The proposed structure for the other is 3-(..beta..-D-2-deoxyribosyl)-7,8-dihydropyrimido-(2,l-i)purine-9-one (dDPP). Spectral characteristics of both compounds are presented. These include u.v. spectra of each in acidic, neutral and alkaline solutions, i.r. spectra, fluorescence spectra, and n.m.r. spectra. The dDPP can be converted to CEdA by mild acid hydrolysis, and the CEdA can be converted to dDPP by reaction with a carbodiimide derivative. When poly A was reacted with ..beta..-propiolactone, the yield of dDPP in the polymer was 7-9%. When double-stranded DNA was alkylated by (/sup 3/H)..beta..-propiolactone at relatively highmore » concentrations and then acid hydrolyzed to separate 1-(2-carboxyethyl)adenine (CEA) and 7-(2-carboxyethyl)guanine (CEG), and CEA to CEG ratio of up to 0.62 was obtained. With relatively low concentrations of (/sup 3/H)..beta..-propiolactone, the yield of CEA was low with double-stranded DNA but was 5-6 fold greater with single-stranded DNA.« less

Adsorption of nucleotides onto Fe-Mg-Al rich swelling clays

NASA Astrophysics Data System (ADS)

Feuillie, Cécile; Daniel, Isabelle; Michot, Laurent J.; Pedreira-Segade, Ulysse

2013-11-01

Mineral surfaces may have played a role in the origin of the first biopolymers, by concentrating organic monomers from a dilute ocean. Swelling clays provide a high surface area for the concentration of prebiotic monomers, and have therefore been the subject of numerous investigations. In that context, montmorillonite, the most abundant swelling clay in modern environments, has been extensively studied with regard to adsorption and polymerization of nucleic acids. However, montmorillonite was probably rather marginal on the primitive ocean floor compared to iron-magnesium rich phyllosilicates such as nontronite that results from the hydrothermal alteration of a mafic or ultramafic oceanic crust. In the present paper, we study the adsorption of nucleotides on montmorillonite and nontronite, at various pH and ionic strength conditions plausible for Archean sea-water. A thorough characterization of the mineral surfaces shows that nucleotide adsorb mainly on the edge faces of the smectites by ligand exchange between the phosphate groups of the nucleotides and the -OH groups from the edge sites over a wide pH range (4-10). Nontronite is more reactive than montmorillonite. At low pH, additional ion exchange may play a role as the nucleotides become positively charged.

DNA Three-Way Junction for Differentiation of Single-Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles.

PubMed

Sun, Feifei; You, Ying; Liu, Jie; Song, Quanwei; Shen, Xiaotong; Na, Na; Ouyang, Jin

2017-05-23

A label- and enzyme-free fluorescent sensor for the detection of single-nucleotide polymorphisms (SNPs) at room temperature is proposed, using new copper nanoparticles (CuNPs) as fluorescent reporters. The CuNPs were constructed by using a DNA three-way junction (3WJ) template. In this assay, two complementary adenine/thymine-rich probes can hybridize with the wild-type target simultaneously to construct a 3WJ structure, serving as an efficient scaffold for the generation of CuNPs. However, the CuNPs produce weak fluorescence when the probes bind with a mutant-type target. SNPs can be identified by the difference in fluorescence intensity of the CuNPs. This SNPs detection strategy is straightforward, cost-effective, and avoids the complicated procedures of labeling or enzymatic reactions. The fluorescent sensor is versatile and can be applied to all types of mutation because the probes are programmable. Moreover, the sensor exhibits good detection performance in biological samples. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Caffeic acid treatment alters the extracellular adenine nucleotide hydrolysis in platelets and lymphocytes of adult rats.

PubMed

Anwar, Javed; Spanevello, Roselia Maria; Pimentel, Victor Camera; Gutierres, Jessié; Thomé, Gustavo; Cardoso, Andreia; Zanini, Daniela; Martins, Caroline; Palma, Heloisa Einloft; Bagatini, Margarete Dulce; Baldissarelli, Jucimara; Schmatz, Roberta; Leal, Cláudio Alberto Martins; da Costa, Pauline; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

2013-06-01

This study evaluated the effects of caffeic acid on ectonucleotidase activities such as NTPDase (nucleoside triphosphate diphosphohydrolase), Ecto-NPP (nucleotide pyrophosphatase/phosphodiesterase), 5'-nucleotidase and adenosine deaminase (ADA) in platelets and lymphocytes of rats, as well as in the profile of platelet aggregation. Animals were divided into five groups: I (control); II (oil); III (caffeic acid 10 mg/kg); IV (caffeic acid 50 mg/kg); and V (caffeic acid 100 mg/kg). Animals were treated with caffeic acid diluted in oil for 30 days. In platelets, caffeic acid decreased the ATP hydrolysis and increased ADP hydrolysis in groups III, IV and V when compared to control (P<0.05). The 5'-nucleotidase activity was decreased, while E-NPP and ADA activities were increased in platelets of rats of groups III, IV and V (P<0.05). Caffeic acid reduced significantly the platelet aggregation in the animals of groups III, IV and V in relation to group I (P<0.05). In lymphocytes, the NTPDase and ADA activities were increased in all groups treated with caffeic acid when compared to control (P<0.05). These findings demonstrated that the enzymes were altered in tissues by caffeic acid and this compound decreased the platelet aggregation suggesting that caffeic acid should be considered a potentially therapeutic agent in disorders related to the purinergic system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

NASA Astrophysics Data System (ADS)

Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

2015-10-01

This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

Ab initio electron propagator calculations of transverse conduction through DNA nucleotide bases in 1-nm nanopore corroborate third generation sequencing.

PubMed

Kletsov, Aleksey A; Glukhovskoy, Evgeny G; Chumakov, Aleksey S; Ortiz, Joseph V

2016-01-01

The conduction properties of DNA molecule, particularly its transverse conductance (electron transfer through nucleotide bridges), represent a point of interest for DNA chemistry community, especially for DNA sequencing. However, there is no fully developed first-principles theory for molecular conductance and current that allows one to analyze the transverse flow of electrical charge through a nucleotide base. We theoretically investigate the transverse electron transport through all four DNA nucleotide bases by implementing an unbiased ab initio theoretical approach, namely, the electron propagator theory. The electrical conductance and current through DNA nucleobases (guanine [G], cytosine [C], adenine [A] and thymine [T]) inserted into a model 1-nm Ag-Ag nanogap are calculated. The magnitudes of the calculated conductance and current are ordered in the following hierarchies: gA>gG>gC>gT and IG>IA>IT>IC correspondingly. The new distinguishing parameter for the nucleobase identification is proposed, namely, the onset bias magnitude. Nucleobases exhibit the following hierarchy with respect to this parameter: Vonset(A)

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

PubMed

Burton, Peter; Adams, David R; Abraham, Achamma; Allcock, Robert W; Jiang, Zhong; McCahill, Angela; Gilmour, Jane; McAbney, John; Kaupisch, Alexandra; Kane, Nicole M; Baillie, George S; Baker, Andrew H; Milligan, Graeme; Houslay, Miles D; Mountford, Joanne C

2010-12-15

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington's disease related cardiomyopathy.

PubMed

Toczek, Marta; Zielonka, Daniel; Zukowska, Paulina; Marcinkowski, Jerzy T; Slominska, Ewa; Isalan, Mark; Smolenski, Ryszard T; Mielcarek, Michal

2016-11-01

Huntington's disease (HD) is mainly thought of as a neurological disease, but multiple epidemiological studies have demonstrated a number of cardiovascular events leading to heart failure in HD patients. Our recent studies showed an increased risk of heart contractile dysfunction and dilated cardiomyopathy in HD pre-clinical models. This could potentially involve metabolic remodeling, that is a typical feature of the failing heart, with reduced activities of high energy phosphate generating pathways. In this study, we sought to identify metabolic abnormalities leading to HD-related cardiomyopathy in pre-clinical and clinical settings. We found that HD mouse models developed a profound deterioration in cardiac energy equilibrium, despite AMP-activated protein kinase hyperphosphorylation. This was accompanied by a reduced glucose usage and a significant deregulation of genes involved in de novo purine biosynthesis, in conversion of adenine nucleotides, and in adenosine metabolism. Consequently, we observed increased levels of nucleotide catabolites such as inosine, hypoxanthine, xanthine and uric acid, in murine and human HD serum. These effects may be caused locally by mutant HTT, via gain or loss of function effects, or distally by a lack of trophic signals from central nerve stimulation. Either may lead to energy equilibrium imbalances in cardiac cells, with activation of nucleotide catabolism plus an inhibition of re-synthesis. Our study suggests that future therapies should target cardiac mitochondrial dysfunction to ameliorate energetic dysfunction. Importantly, we describe the first set of biomarkers related to heart and skeletal muscle dysfunction in both pre-clinical and clinical HD settings. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrical detection and quantification of single and mixed DNA nucleotides in suspension

NASA Astrophysics Data System (ADS)

Ahmad, Mahmoud Al; Panicker, Neena G.; Rizvi, Tahir A.; Mustafa, Farah

2016-09-01

High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

NASA Astrophysics Data System (ADS)

Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

2017-06-01

We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

Some parameters relevant to affinity chromatography on immobilized nucleotides

PubMed Central

Lowe, C. R.; Harvey, M. J.; Craven, D. B.; Dean, P. D. G.

1973-01-01

1. The suitability of cellulose and Sepharose as supports for affinity chromatography of two groups of cofactor-linked enzymes, dehydrogenases and kinases, was examined. Sepharose was found to be superior. 2. The selective capacities of the columns were measured by frontal analysis and are discussed in relation to the nucleotide contents. 3. The effect of various concentrations of enzyme and of non-specific protein on the performance of the affinity columns, and the effects of equilibration time, flow rate, sample volume and dilution of the nucleotide were examined. 4. The effect of interposing polymethylene and polyglycine extension arms between the matrix backbone and the nucleotide was investigated for several cofactor-dependent enzymes. Maximum binding was observed with an extension arm 0.8–1nm long. PMID:4354739

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters*

PubMed Central

Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

2014-01-01

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081

Long-Range Charge Transport in Adenine-Stacked RNA:DNA Hybrids.

PubMed

Li, Yuanhui; Artés, Juan M; Hihath, Joshua

2016-01-27

An extremely important biological component, RNA:DNA can also be used to design nanoscale structures such as molecular wires. The conductance of single adenine-stacked RNA:DNA hybrids is rapidly and reproducibly measured using the break junction approach. The conductance decreases slightly over a large range of molecular lengths, suggesting that RNA:DNA can be used as an oligonucleotide wire. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lack of hepcidin ameliorates anemia and improves growth in an adenine-induced mouse model of chronic kidney disease

PubMed Central

Sureshbabu, Angara; Doty, Steve B.; Zhu, Yuan-Shan; Patino, Edwin; Cunningham-Rundles, Susanna; Choi, Mary E.; Boskey, Adele; Rivella, Stefano

2016-01-01

Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted. PMID:27440777

Energy level alignment at the interfaces between typical electrodes and nucleobases: Al/adenine/indium-tin-oxide and Al/thymine/indium-tin-oxide

NASA Astrophysics Data System (ADS)

Lee, Younjoo; Lee, Hyunbok; Park, Soohyung; Yi, Yeonjin

2012-12-01

We investigated the interfacial electronic structures of Al/adenine/indium-tin-oxide (ITO) and Al/thymine/ITO using in situ ultraviolet and x-ray photoemission spectroscopy and density functional theory calculations. Adenine shows both an interface dipole and level bending, whereas thymine shows only an interface dipole in contact with ITO. In addition, thymine possesses a larger ionization energy than adenine. These are understood with delocalized π states confirmed with theoretical calculations. For the interface between nucleobases and Al, both nucleobases show a prominent reduction of the electron injection barrier from Al to each base in accordance with a downward level shift.

The binding of glucose and nucleotides to hexokinase from Saccharomyces cerevisiae.

PubMed

Woolfitt, A R; Kellett, G L; Hoggett, J G

1988-01-29

The binding of glucose, ADP and AdoPP[NH]P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence. The data were analysed in terms of Qmax, the maximal quenching of fluorescence at saturating concentrations of ligand, and [L]0.5, the concentration of ligand at half-maximal quenching. No changes in fluorescence were observed with free enzyme and nucleotide alone. In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and [L]0.5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species. Qmax induced by glucose alone was between 22 and 25%, while [L]0.5 was approx. 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer. In the presence of 6 mM ADP or 2 mM AdoPP[NH]P, Qmax for glucose was increased by up to 4% and [L]0.5 was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer. The results are interpreted in terms of nucleotide-induced conformational change of hexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide.

High-Spin Ferric Ions in Saccharomyces cerevisiae Vacuoles Are Reduced to the Ferrous State during Adenine-Precursor Detoxification

PubMed Central

2015-01-01

The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) FeIII complexes with polyphosphate-related ligands. Some FeIII oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS FeIII. Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 μM FeIII citrate) accumulated two major NHHS FeII species as the vacuolar NHHS FeIII species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS FeII complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS FeIII with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS FeIII species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated. PMID:24919141

Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

DTIC Science & Technology

2009-03-01

mucin-like, hormone receptor- like 1 SLC25A5 -1.58342009 0.0202321 -1.20432 0.03115 solute carrier family 25 ( mitochondrial carrier ; adenine nucleotide...family 25 ( mitochondrial carrier ; adenine nucleotide translocator), member 5 CYP26B1 -1.45880527 0.019056 -1.20533 0.01287 cytochrome P450, family...FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 01-03-2009 2. REPORT TYPE Annual Summary 3. DATES COVERED 1 Mar 2006 – 28 Feb 2009 4 . TITLE AND SUBTITLE

Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors

PubMed Central

El-Gendy, Dalia M.; Ghany, Nabil A. Abdel; El Sherbini, E. E. Foad; Allam, Nageh K.

2017-01-01

A simple method is demonstrated to prepare spongy adenine-functionalized graphene (SFG) as interconnected, porous 3-dimensional (3D) network crinkly sheets. Such 3D network structure provides better contact at the electrode/electrolyte interface and facilitates the charge transfer kinetics. The fabricated SFG was characterized by X-ray diffraction (XRD), FTIR, scanning electron microscopy (FESEM), Raman spectroscopy, thermogravimetric analysis (TGA), UV−vis absorption spectroscopy, and transmission electron microscopy (TEM). The synthesized materials have been evaluated as supercapacitor materials in 0.5 M H2SO4 using cyclic voltammetry (CV) at different potential scan rates, and galvanostatic charge/discharge tests at different current densities. The SFG electrodes showed a maximum specific capacitance of 333 F/g at scan rate of 1 mV/s and exhibited excellent cycling retention of 102% after 1000 cycles at 200 mV/s. The energy density was 64.42 Wh/kg with a power density of 599.8 W/kg at 1.0 A/g. Those figures of merit are much higher than those reported for graphene-based materials tested under similar conditions. The observed high performance can be related to the synergistic effects of the spongy structure and the adenine functionalization. PMID:28216668

Clay catalysis of oligonucleotide formation: kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine

NASA Technical Reports Server (NTRS)

Kawamura, K.; Ferris, J. P.

1999-01-01

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine of substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.

Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.

1991-03-11

The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{supmore » 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.« less

Free amino acids and 5'-nucleotides in Finnish forest mushrooms.

PubMed

Manninen, Hanna; Rotola-Pukkila, Minna; Aisala, Heikki; Hopia, Anu; Laaksonen, Timo

2018-05-01

Edible mushrooms are valued because of their umami taste and good nutritional values. Free amino acids, 5'-nucleotides and nucleosides were analyzed from four Nordic forest mushroom species (Lactarius camphoratus, Boletus edulis, Cantharellus cibarius, Craterellus tubaeformis) using high precision liquid chromatography analysis. To our knowledge, these taste components were studied for the first time from Craterellus tubaeformis and Lactarius camphoratus. The focus was on the umami amino acids and 5'-nucleotides. The free amino acid and 5'-nucleotide/nucleoside contents of studied species differed from each other. In all studied samples, umami amino acids were among five major free amino acids. The highest concentration of umami amino acids was on L. camphoratus whereas B. edulis had the highest content of sweet amino acids and C. cibarius had the highest content of bitter amino acids. The content of umami enhancing 5'-nucleotides were low in all studied species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gum acacia mitigates genetic damage in adenine-induced chronic renal failure in rats.

PubMed

Ali, B H; Al Balushi, K; Al-Husseini, I; Mandel, P; Nemmar, A; Schupp, N; Ribeiro, D A

2015-12-01

Subjects with chronic renal failure (CRF) exhibit oxidative genome damage, which may predispose to carcinogenesis, and Gum acacia (GumA) ameliorates this condition in humans and animals. We evaluated here renal DNA damage and urinary excretion of four nucleic acid oxidation adducts namely 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxoguanosine (8-oxoGuo) and 8-hydroxy-2-deoxyguanisone (8-OHdg) in rats with adenine (ADE)-induced CRF with and without GumA treatment. Twenty-four rats were divided into four equal groups and treated for 4 weeks. The first group was given normal food and water (control). The second group was given normal food and GumA (15% w/v) in drinking water. The third group was fed powder diet containing adenine (ADE) (0·75% w/w in feed). The fourth group was fed like in the third group, plus GumA in drinking water (15%, w/v). ADE feeding induced CRF (as measured by several physiological, biochemical and histological indices) and also caused a significant genetic damage and significant decreases in urinary 8-oxo Gua and 8-oxoGuo, but not in the other nucleic acids. However, concomitant GumA treatment reduced the level of genetic damage in kidney cells as detected by Comet assay and significantly reversed the effect of adenine on urinary 8-oxoGuo. Treatment with GumA is able to mitigate genetic damage in renal tissues of rats with ADE-induced CRF. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

Probing adenine rings and backbone linkages using base specific isotope-edited Raman spectroscopy: application to group II intron ribozyme domain V.

PubMed

Chen, Yuanyuan; Eldho, Nadukkudy V; Dayie, T Kwaku; Carey, Paul R

2010-04-27

Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, "D5", which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all of its adenine residues labeled with (13)C and (15)N and utilizing Raman difference spectroscopy, we identify the conformationally sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg(2+) binding was explored by analyzing the Raman difference spectra for [D5 + Mg(2+)] minus [D5 no Mg(2+)], for D5 unlabeled, or D5 labeled with (13)C/(15)N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg(2+) binding at the N7 position. In the A-labeled spectra we attribute a Raman differential near 1450 cm(-1) and changes of intensity at 1296 cm(-1) to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg(2+) binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm(-1) upon magnesium binding is due to a "tightening up" (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine-labeled D5, small changes in the adenine backbone bond signatures in the 810-830 cm(-1) region suggest that small conformational changes occur in the tetraloop and bulge regions upon binding of Mg(2+). The PO(2)(-) stretching vibration, near 1100 cm(-1), from the nonbridging phosphate groups, probes the effect of Mg(2+)-hydrate inner-sphere interactions that cause an upshift. In turn, the upshift is modulated by the presence of monovalent cations since in the presence of Na(+) and Li(+) the upshift is 23 +/- 2 cm(-1) while in the presence of K(+) and Cs(+) it is 13 +/- 3 cm(-1), a finding that correlates

Intracellular ATP influences synaptic plasticity in area CA1 of rat hippocampus via metabolism to adenosine and activity-dependent activation of adenosine A1 receptors.

PubMed

zur Nedden, Stephanie; Hawley, Simon; Pentland, Naomi; Hardie, D Grahame; Doney, Alexander S; Frenguelli, Bruno G

2011-04-20

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to ≥ 0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with d-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A(1) receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

Calcium-dependent nonspecific permeability of the inner mitochondrial membrane is not induced in mitochondria of the yeast Endomyces magnusii.

PubMed

Deryabina, Y I; Isakova, E P; Shurubor, E I; Zvyagilskaya, R A

2004-09-01

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.

Raw coffee based dietary supplements contain carboxyatractyligenin derivatives inhibiting mitochondrial adenine-nucleotide-translocase.

PubMed

Lang, Roman; Fromme, Tobias; Beusch, Anja; Lang, Tatjana; Klingenspor, Martin; Hofmann, Thomas

2014-08-01

Capsules, powders and tablets containing raw coffee extract are advertised to the consumer as antioxidant rich dietary supplements as part of a healthy diet. We isolated carboxyatractyligenin (4), 2-O-β-d-glucopyranosyl carboxyatractyligenin (6) and 3'-O-β-d-glucopyranosyl-2'-O-isovaleryl-2β-(2-desoxy-carboxyatractyligenin)-β-d-glucopyranoside (8) from green coffee and found strong inhibitory effects on phosphorylating respiration in isolated mitochondria similar to the effects of the known phytotoxin carboxyatractyloside. LC-MS/MS analysis of commercial green coffee based dietary supplements revealed the occurrence of carboxyatractyligenin, 3'-O-β-d-glucopyranosyl-2'-O-isovaleryl-2β-(2-desoxy-carboxyatractyligenin)-β-d-glucopyranoside, and 2-O-β-d-glucopyranosyl carboxyatractyligenin in concentrations up to 4.0, 5.7, and 41.6μmol/g, respectively. These data might help to gain first insight into potential physiological side-effects of green coffee containing dietary supplement. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus.

PubMed

Jeon, Taeck J

2008-10-01

The expression of amoeba sams genes is switched from sams1 to sams2 when amoebae are infected with Legionella jeonii. To elucidate the mechanism for the inactivation of host sams1 gene by endosymbiotic bacteria, methylation states of the sams1 gene of D and xD amoebae was compared in this study. The sams1 gene of amoebae was methylated at an internal adenine residue of GATC site in symbiont-bearing xD amoebae but not in symbiont-free D amoebae, suggesting that the modification might have caused the inactivation of sams1 in xD amoebae. The sams1 gene of xD amoebae was inactivated at the transcriptional level. Analysis of DNA showed that adenine residues in L. jeonii sams were also methylated, implying that L. jeonii bacteria belong to a Dam methylase-positive strain. In addition, both SAM and Met appeared to act as negative regulators for the expression of sams1 whereas the expression of sams2 was not affected in amoebae.

DNA adenine methylation modulates pathogenicity of Klebsiella pneumoniae genotype K1.

PubMed

Fang, Chi-Tai; Yi, Wen-Ching; Shun, Chia-Tung; Tsai, Shih-Feng

2017-08-01

Klebsiella pneumoniae genotype K1 is a highly virulent pathogen that causes liver abscess and metastatic endophthalmitis/meningitis. Whether its pathogenicity is controlled by DNA adenine methylase (Dam), an epigenetic regulator of bacterial virulence gene expression, is yet unknown. We aimed to study the role of DNA adenine methylation in the pathogenicity of K. pneumoniae genotype K1. We identified the dam gene in the prototype tissue-invasive strain (NTUH-K2044) of K. pneumoniae genotype K1, using the strain's complete genome sequence in GenBank. We constructed a dam - mutant and compared it with the wild type, in terms of in vitro serum resistance and in vivo BALB/cByl mice inoculation. Loss of Dam activity in the mutant was verified by MboI restriction digestion of the genomic DNA and a 1000-fold increase in spontaneous mutation rate. The dam mutant lost at least 68% of serum resistance when compared with the wild type (survival ratio at 1 hour: 2.6 ± 0.4 vs. 8.2 ± 1.9; at 2 hours: 3.9 ± 1.6 vs. 17.4 ± 3.6; p values < 0.05). Likewise, virulence to mice decreased by 40-fold in an intraperitoneal injection model [lethal dose, 50% (LD 50 ): 2 × 10 3 colony-forming units (CFUs) vs. 5 × 10 1 CFUs] and by sixfold in a gastric ingestion model (LD 50 : 3 × 10 4 CFUs vs. 5 × 10 3 CFUs). Attenuation of the dam mutant was not attributable to its growth rate, which was similar to that of the wild type. Our results support the view that DNA adenine methylation plays an important role in modulating the pathogenicity of K. pneumoniae genotype K1. The incomplete attenuation indicates the existence of other regulatory factors. Copyright © 2015. Published by Elsevier B.V.

Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

PubMed

Tomaszewski, M; Buchowicz, J

1971-08-01

The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

Efficacy of Adenine in the Treatment of Leukopenia and Neutropenia Associated with an Overdose of Antipsychotics or Discontinuation of Lithium Carbonate Administration: Three Case Studies.

PubMed

Tomita, Takashi; Goto, Hidekazu; Sumiya, Kenji; Yoshida, Tadashi; Tanaka, Katsuya; Kohda, Yukinao

2016-11-30

Because adenine is effective for managing cases of radiation-induced and drug-induced leukopenia, it may be effective in cases of antipsychotic-induced leukopenia and neutropenia. Here, we report our experience with patients with leukopenia and neutropenia caused by an antipsychotic overdose or discontinuation of lithium carbonate, in whom adenine administration ameliorated the white blood cell and neutrophil counts. The progress of patients suggests that adenine is effective in cases of leukopenia and neutropenia associated with lithium carbonate discontinuation and an antipsychotic overdose.

Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo

NASA Astrophysics Data System (ADS)

Zhi, Xuan; Han, Baoqin; Sui, Xianxian; Hu, Rui; Liu, Wanshun

2015-02-01

The effects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigated in vivo and in vitro. Chitosan were hydrolyzed using chitosanase at pH 6-7 and 37° for 24 h to obtain LMWC. In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For the in vivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the control group, indicating that the rat chronic renal failure model worked successfully. The results after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100 mg kg-1 d-1.

Adenine radicals generated in alternating AT duplexes by direct absorption of low-energy UV radiation.

PubMed

Banyasz, Akos; Ketola, Tiia; Martínez-Fernández, Lara; Improta, Roberto; Markovitsi, Dimitra

2018-04-17

There is increasing evidence that the direct absorption of photons with energies that are lower than the ionization potential of nucleobases may result in oxidative damage to DNA. The present work, which combines nanosecond transient absorption spectroscopy and quantum mechanical calculations, studies this process in alternating adenine-thymine duplexes (AT)n. We show that the one-photon ionization quantum yield of (AT)10 at 266 nm (4.66 eV) is (1.5 ± 0.3) × 10-3. According to our PCM/TD-DFT calculations carried out on model duplexes composed of two base pairs, (AT)1 and (TA)1, simultaneous base pairing and stacking does not induce important changes in the absorption spectra of the adenine radical cation and deprotonated radical. The adenine radicals, thus identified in the time-resolved spectra, disappear with a lifetime of 2.5 ms, giving rise to a reaction product that absorbs at 350 nm. In parallel, the fingerprint of reaction intermediates other than radicals, formed directly from singlet excited states and assigned to AT/TA dimers, is detected at shorter wavelengths. PCM/TD-DFT calculations are carried out to map the pathways leading to such species and to characterize their absorption spectra; we find that, in addition to the path leading to the well-known TA* photoproduct, an AT photo-dimerization path may be operative in duplexes.

The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

PubMed

Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

2014-11-28

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Profiling of Sugar Nucleotides.

PubMed

Rejzek, Martin; Hill, Lionel; Hems, Edward S; Kuhaudomlarp, Sakonwan; Wagstaff, Ben A; Field, Robert A

2017-01-01

Sugar nucleotides are essential building blocks for the glycobiology of all living organisms. Detailed information on the types of sugar nucleotides present in a particular cell and how they change as a function of metabolic, developmental, or disease status is vital. The extraction, identification, and quantification of sugar nucleotides in a given sample present formidable challenges. In this chapter, currently used techniques for sugar nucleotide extraction from cells, separation from complex biological matrices, and detection by optical and mass spectrometry methods are discussed. © 2017 Elsevier Inc. All rights reserved.

Influence of nucleotide modifications at the C2' position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA.

PubMed

Copp, William; Denisov, Alexey Y; Xie, Jingwei; Noronha, Anne M; Liczner, Christopher; Safaee, Nozhat; Wilds, Christopher J; Gehring, Kalle

2017-09-29

Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Selective killing of tumors deficient in methylthioadenosine phosphorylase: a novel strategy.

PubMed

Lubin, Martin; Lubin, Adam

2009-05-29

The gene for methylthioadenosine phosphorylase (MTAP) lies on 9p21, close to the gene CDKN2A that encodes the tumor suppressor proteins p16 and p14ARF. MTAP and CDKN2A are homozygously co-deleted, with a frequency of 35 to 70%, in lung and pancreatic cancer, glioblastoma, osteosarcoma, soft-tissue sarcoma, mesothelioma, and T-cell acute lymphoblastic leukemia. In normal cells, but not in tumor cells lacking MTAP, MTAP cleaves the natural substrate, 5'-deoxy-5'-methylthioadenosine (MTA), to adenine and 5-methylthioribose-1-phosphate (MTR-1-P), which are then converted to adenine nucleotides and methionine. This distinct difference between normal MTAP-positive cells and tumor MTAP-negative cells led to several proposals for therapy. We offer a novel strategy in which both MTA and a toxic adenine analog, such as 2,6-diaminopurine (DAP), 6-methylpurine (MeP), or 2-fluoroadenine (F-Ade), are administered. In MTAP-positive cells, abundant adenine, generated from supplied MTA, competitively blocks the conversion of an analog, by adenine phosphoribosyltransferase (APRT), to its active nucleotide form. In MTAP-negative tumor cells, the supplied MTA cannot generate adenine; hence conversion of the analog is not blocked. We show that this combination treatment--adenine analog plus MTA--kills MTAP-negative A549 lung tumor cells, while MTAP-positive human fibroblasts (HF) are protected. In co-cultures of the breast tumor cell line, MCF-7, and HF cells, MCF-7 is inhibited or killed, while HF cells proliferate robustly. 5-Fluorouracil (5-FU) and 6-thioguanine (6-TG) may also be used with our strategy. Though neither analog is activated by APRT, in MTAP-positive cells, adenine produced from supplied MTA blocks conversion of 5-FU and 6-TG to their toxic nucleotide forms by competing for 5-phosphoribosyl-1-pyrophosphate (PRPP). The combination of MTA with 5-FU or 6-TG, in the treatment of MTAP-negative tumors, may produce a significantly improved therapeutic index. We describe a

A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota.

PubMed

Ketkar, Amit; Zafar, Maroof K; Banerjee, Surajit; Marquez, Victor E; Egli, Martin; Eoff, Robert L

2012-06-27

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

A nucleotide analogue induced gain of function corrects the error-prone nature of human DNA polymerase iota

PubMed Central

Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L

2012-01-01

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase. PMID:22632140

Lack of Association Between Toll-like Receptor 2 Polymorphisms (R753Q and A-16934T) and Atopic Dermatitis in Children from Thrace Region of Turkey

PubMed Central

Can, Ceren; Yazıcıoğlu, Mehtap; Gürkan, Hakan; Tozkır, Hilmi; Görgülü, Adnan; Süt, Necdet Hilmi

2017-01-01

Background: Atopic dermatitis is the most common chronic inflammatory skin disease. A complex interaction of both genetic and environmental factors is thought to contribute to the disease. Aims: To evaluate whether single nucleotide polymorphisms in the TLR2 gene c.2258C>T (R753Q) (rs5743708) and TLR2 c.-148+1614T>A (A-16934T) (rs4696480) (NM_0032643) are associated with atopic dermatitis in Turkish children. Study Design: Case-control study. Methods: The study was conducted on 70 Turkish children with atopic dermatitis aged 0.5-18 years. The clinical severity of atopic dermatitis was evaluated by the severity scoring of atopic dermatitis index. Serum total IgE levels, specific IgE antibodies to inhalant and food allergens were measured in both atopic dermatitis patients and controls, skin prick tests were done on 70 children with atopic dermatitis. Genotyping for TLR2 (R753Q and A-16934T) single nucleotide polymorphisms was performed in both atopic dermatitis patients and controls. Results: Cytosine-cytosine and cytosin-thymine genotype frequencies of the TLR2 R753Q single nucleotide polymorphism in the atopic dermatitis group were determined as being 98.6% and 1.4%, cytosine allele frequency for TLR2 R753Q single nucleotide polymorphism was determined as 99.29% and the thymine allele frequency was 0.71%, thymine-thymine, thymine-adenine, and adenine-adenine genotype frequencies of the TLR2 A-16934T single nucleotide polymorphism were 24.3%, 44.3%, and 31.4%. The thymine allele frequency for the TLR2 A-16934T single nucleotide polymorphism in the atopic dermatitis group was 46.43%, and the adenine allele frequency was 53.57%, respectively. There was not statistically significant difference between the groups for all investigated polymorphisms (p>0.05). For all single nucleotide polymorphisms studied, allelic distribution was analogous among atopic dermatitis patients and controls, and no significant statistical difference was observed. No homozygous carriers of

Lack of Association Between Toll-like Receptor 2 Polymorphisms (R753Q and A-16934T) and Atopic Dermatitis in Children from Thrace Region of Turkey.

PubMed

Can, Ceren; Yazıcıoğlu, Mehtap; Gürkan, Hakan; Tozkır, Hilmi; Görgülü, Adnan; Süt, Necdet Hilmi

2017-05-05

Atopic dermatitis is the most common chronic inflammatory skin disease. A complex interaction of both genetic and environmental factors is thought to contribute to the disease. To evaluate whether single nucleotide polymorphisms in the TLR2 gene c.2258C>T (R753Q) (rs5743708) and TLR2 c.-148+1614T>A (A-16934T) (rs4696480) (NM_0032643) are associated with atopic dermatitis in Turkish children. Case-control study. The study was conducted on 70 Turkish children with atopic dermatitis aged 0.5-18 years. The clinical severity of atopic dermatitis was evaluated by the severity scoring of atopic dermatitis index. Serum total IgE levels, specific IgE antibodies to inhalant and food allergens were measured in both atopic dermatitis patients and controls, skin prick tests were done on 70 children with atopic dermatitis. Genotyping for TLR2 (R753Q and A-16934T) single nucleotide polymorphisms was performed in both atopic dermatitis patients and controls. Cytosine-cytosine and cytosin-thymine genotype frequencies of the TLR2 R753Q single nucleotide polymorphism in the atopic dermatitis group were determined as being 98.6% and 1.4%, cytosine allele frequency for TLR2 R753Q single nucleotide polymorphism was determined as 99.29% and the thymine allele frequency was 0.71%, thymine-thymine, thymine-adenine, and adenine-adenine genotype frequencies of the TLR2 A-16934T single nucleotide polymorphism were 24.3%, 44.3%, and 31.4%. The thymine allele frequency for the TLR2 A-16934T single nucleotide polymorphism in the atopic dermatitis group was 46.43%, and the adenine allele frequency was 53.57%, respectively. There was not statistically significant difference between the groups for all investigated polymorphisms (p>0.05). For all single nucleotide polymorphisms studied, allelic distribution was analogous among atopic dermatitis patients and controls, and no significant statistical difference was observed. No homozygous carriers of the TLR2 R753Q single nucleotide polymorphism were

The effect of swimming exercise on adenine-induced kidney disease in rats, and the influence of curcumin or lisinopril thereon

PubMed Central

Karaca, Turan; Al Suleimani, Yousuf; Al Za'abi, Mohammed; Al Kalbani, Jamila; Ashique, Mohammed; Nemmar, Abderrahim

2017-01-01

Patients with chronic kidney disease (CKD) have been reported to benefit from different types of exercises. It has also been shown that the ACE inhibitor lisinopril, and the natural product curcumin are also beneficial in different models of CKD in rats. We assessed the influence of moderate swimming exercise (SE) on rats with adenine-induced CKD, and tested the possible effects of lisinopril and/or curcumin thereon using several physiological, biochemical, histopathological and immunohistochemical parameters. Rats (either sedentary or subjected to SE) were randomly divided into several groups, and given for five weeks either normal food or food mixed with adenine (0.25% w/w) to induce CKD. Some of these groups were also concomitantly treated orally with curcumin (75 mg/kg), or lisinopril (10 mg/kg) and were subjected to moderate SE (45 min/day three days each week). Rats fed adenine showed the typical biochemical, histopathological signs of CKD such as elevations in blood pressure, urinary albumin / creatinine ratio, and plasma urea, creatinine, indoxyl sulfate and phosphorus. SE, curcumin or lisinopril, given singly, significantly ameliorated all the adenine-induced actions. Administering curcumin or lisinopril with SE improved the histopathology of the kidneys, a salutary effect not seen with SE alone. Combining SE to the nephroprotective agents’ curcumin or lisinopril might offer additional nephroprotection. PMID:28445490

Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

NASA Astrophysics Data System (ADS)

Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

2015-07-01

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (ɛΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

Influence of nucleotide modifications at the C2’ position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA

PubMed Central

Copp, William; Denisov, Alexey Y.; Xie, Jingwei; Noronha, Anne M.; Liczner, Christopher; Safaee, Nozhat

2017-01-01

Abstract Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2′-deoxyribose, 2′-O-methyl-ribose, 2′-deoxy-2′-fluoro-ribose, arabinose and 2′-deoxy-2′-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2’ modifications gave a variety of effects. Arabinose and 2′-deoxy-2′-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2′-O-methyl and 2′-deoxy-2′-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability. PMID:28973475

CYTOLOGICAL STUDIES ON THE ANTIMETABOLITE ACTION OF 2,6-DIAMINOPURINE IN VICIA FABA ROOTS

PubMed Central

Setterfield, George; Duncan, Robert E.

1955-01-01

At a concentration of 9.6 x 10–5 M, 2,6-diaminopurine (DAP) completely inhibited cell enlargement, cell division, and DNA synthesis (determined by microphotometric measurement of Feulgen dye) in Vicia faba roots. Inhibition of cell enlargement was partially reversed by adenine, guanine, xanthine, adenosine, and desoxyadenosine. Guanine and the nucleosides gave the greatest reversal, suggesting that one point of DAP action upon cell enlargement is a disruption of nucleoside or nucleotide metabolism, possibly during pentosenucleic acid synthesis. DAP inhibited cell division by preventing onset of prophase. At the concentrations used it had no significant effect on the rate or appearance of mitoses in progress. Inhibition of entrance into prophase was not directly due to inhibition of DNA synthesis since approximately half of the inhibited nuclei had the doubled (4C) amount of DNA. Adenine competitively reversed DAP inhibition of cell division, giving an inhibition index of about 0.5. Guanine gave a slight reversal while xanthine, hypoxanthine, adenosine, and desoxyadenosine were inactive. A basic need for free adenine for the onset of mitosis was suggested by this reversal pattern. Meristems treated with DAP contained almost no nuclei with intermediate amounts of DNA, indicating that DAP prevented the onset of DNA synthesis while allowing that underway to reach completion. The inhibition of DNA synthesis was reversed by adenine, adenosine, and desoxyadenosine although synthesis appeared to proceed at a slower rate in reversals than in controls. Inhibition of DNA synthesis by DAP is probably through nucleoside or nucleotide metabolism. A small general depression of DNA content of nuclei in the reversal treatments was observed. This deviation from DNA "constancy" cannot be adequately explained at present although it may be a result of direct incorporation of DAP into DNA. The possible purine precursor, 4-amino-5-imidazolecarboxamide gave no reversal of DAP inhibition

Hydration properties of adenosine phosphate series as studied by microwave dielectric spectroscopy.

PubMed

Mogami, George; Wazawa, Tetsuichi; Morimoto, Nobuyuki; Kodama, Takao; Suzuki, Makoto

2011-02-01

Hydration properties of adenine nucleotides and orthophosphate (Pi) in aqueous solutions adjusted to pH=8 with NaOH were studied by high-resolution microwave dielectric relaxation (DR) spectroscopy at 20 °C. The dielectric spectra were analyzed using a mixture theory combined with a least-squares Debye decomposition method. Solutions of Pi and adenine nucleotides showed qualitatively similar dielectric properties described by two Debye components. One component was characterized by a relaxation frequency (f(c)=18.8-19.7 GHz) significantly higher than that of bulk water (17 GHz) and the other by a much lower f(c) (6.4-7.6 GHz), which are referred to here as hyper-mobile water and constrained water, respectively. By contrast, a hydration shell of only the latter type was found for adenosine (f(c)~6.7 GHz). The present results indicate that phosphoryl groups are mostly responsible for affecting the structure of the water surrounding the adenine nucleotides by forming one constrained water layer and an additional three or four layers of hyper-mobile water. Copyright © 2010 Elsevier B.V. All rights reserved.

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

PubMed Central

Worrell, V E; Nagle, D P

1990-01-01

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines. PMID:2345148

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Shanak, Siba; Helms, Volkhard

2014-12-01

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations.

PubMed

Shanak, Siba; Helms, Volkhard

2014-12-14

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Proton-impact ionization cross sections of adenine measured at 0.5 and 2.0 MeV by electron spectroscopy

NASA Astrophysics Data System (ADS)

Iriki, Y.; Kikuchi, Y.; Imai, M.; Itoh, A.

2011-11-01

Double-differential ionization cross sections (DDCSs) of vapor-phase adenine molecules (C5H5N5) by 0.5- and 2.0-MeV proton impact have been measured by the electron spectroscopy method. Electrons ejected from adenine were analyzed by a 45∘ parallel-plate electrostatic spectrometer over an energy range of 1.0-1000 eV at emission angles from 15∘ to 165∘. Single-differential cross sections (SDCSs) and total ionization cross sections (TICSs) were also deduced. It was found from the Platzman plot, defined as SDCSs divided by the classical Rutherford knock-on cross sections per target electron, that the SDCSs at higher electron energies are proportional to the total number of valence electrons (50) of adenine, while those at low-energy electrons are highly enhanced due to dipole and higher-order interactions. The present results of TICS are in fairly good agreement with recent classical trajectory Monte Carlo calculations, and moreover, a simple analytical formula gives nearly equivalent cross sections in magnitude at the incident proton energies investigated.

Kinetics and Thermodynamics of the Reaction between the (•)OH Radical and Adenine: A Theoretical Investigation.

PubMed

Milhøj, Birgitte O; Sauer, Stephan P A

2015-06-18

The accessibility of all possible reaction paths for the reaction between the nucleobase adenine and the (•)OH radical is investigated through quantum chemical calculations of barrier heights and rate constants at the ωB97X-D/6-311++G(2df,2pd) level with Eckart tunneling corrections. First the computational method is validated by considering the hydrogen abstraction from the heterocyclic N9 nitrogen in adenine as a test system. Geometries for all molecules in the reaction are optimized with four different DFT exchange-correlation functionals (B3LYP, BHandHLYP, M06-2X, and ωB97X-D), in combination with Pople and Dunning basis sets, all of which have been employed in similar investigations in the literature. Improved energies are obtained through single point calculations with CCSD(T) and the same basis sets, and reaction rate constants are calculated for all methods both without tunneling corrections and with the Wigner, Bell, and Eckart corrections. In comparison to CCSD(T)//BHandHLYP/aug-cc-pVTZ reference results, the ωB97X-D/6-311++G(2df,2pd) method combined with Eckart tunneling corrections provides a sensible compromise between accuracy and time. Using this method, all subreactions of the reaction between adenine and the (•)OH radical are investigated. The total rate constants for hydrogen abstraction and addition for adenine are predicted with this method to be 1.06 × 10(-12) and 1.10 × 10(-12) cm(3) molecules(-1) s(-1), respectively. Abstractions of H61 and H62 contribute the most, while only addition to the C8 carbon is found to be of any significance, in contrast to previous claims that addition is the dominant reaction pathway. The overall rate constant for the complete reaction is found to be 2.17 × 10(-12) cm(3) molecules(-1) s(-1), which agrees exceptionally well with experimental results.

Combined adrenal failure and testicular adrenal rest tumor in a patient with nicotinamide nucleotide transhydrogenase deficiency.

PubMed

Hershkovitz, Eli; Arafat, Maram; Loewenthal, Neta; Haim, Alon; Parvari, Ruti

2015-09-01

The nicotinamide nucleotide transhydrogenase (NNT) enzyme is the main generator of nicotinamide adenine dinucleotide phosphate-oxidase in the mitochondrion. Mutations of the NNT gene have been recently implicated in familial glucocorticoid deficiency. We describe the long-term clinical course of a NNT-deficient 20-year-old patient with combined adrenal failure who had developed a testicular adrenal rest tumor and precocious puberty. The patient's medical records were reviewed. Whole-exome sequencing was performed on DNA obtained from the patient and family members. The patient experienced Addisonian crisis at 10 months of age. Enlarged testicular volume and precocious puberty, accompanied by increased testosterone levels, were noted at 6 years. Testicular biopsy revealed a adrenal rest tumor, which regressed after intensification of glucocorticoid treatment. Genetic studies disclosed a c.1163A>C, p.Tyr388Ser substitution on the NNT gene. This mutation is predicted to be damaging to NNT function. We demonstrated for the first time that the clinical spectrum of NNT deficiency may consist of mineralocorticoid deficiency and testicular involvement as well.

Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

PubMed

Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun

2015-11-10

Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure. Published by Elsevier B.V.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation*♦

PubMed Central

Nebel, Merle; Schwoerer, Alexander P.; Warszta, Dominik; Siebrands, Cornelia C.; Limbrock, Ann-Christin; Swarbrick, Joanna M.; Fliegert, Ralf; Weber, Karin; Bruhn, Sören; Hohenegger, Martin; Geisler, Anne; Herich, Lena; Schlegel, Susan; Carrier, Lucie; Eschenhagen, Thomas; Potter, Barry V. L.; Ehmke, Heimo; Guse, Andreas H.

2013-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy. PMID:23564460

Theoretical Study of Tautomerization Reactions for the Ground and First Excited Electronic States of Adenine

NASA Technical Reports Server (NTRS)

Salter, Latasha M.; Chaban, Galina M.; Kwak, Dochan (Technical Monitor)

2002-01-01

Geometrical structures and energetic properties for different tautomers of adenine are calculated in this study, using multi-configurational wave functions. Both the ground and the lowest singlet excited state potential energy surfaces are studied. Four tautomeric forms are considered, and their energetic order is found to be different on the ground and the excited state potential energy surfaces. Minimum energy reaction paths are obtained for hydrogen atom transfer (tautomerization) reactions in the ground and the lowest excited electronic states. It is found that the barrier heights and the shapes of the reaction paths are different for the ground and the excited electronic states, suggesting that the probability of such tautomerization reaction is higher on the excited state potential energy surface. This tautomerization process should become possible in the presence of water or other polar solvent molecules and should play an important role in the photochemistry of adenine.

A new microplatform based on titanium dioxide nanofibers/graphene oxide nanosheets nanocomposite modified screen printed carbon electrode for electrochemical determination of adenine in the presence of guanine.

PubMed

Arvand, Majid; Ghodsi, Navid; Zanjanchi, Mohammad Ali

2016-03-15

The current techniques for determining adenine have several shortcomings such as high cost, high time consumption, tedious pretreatment steps and the requirements for highly skilled personnel often restrict their use in routine analytical practice. This paper describes the development and utilization of a new nanocomposite consisting of titanium dioxide nanofibers (TNFs) and graphene oxide nanosheets (GONs) for screen printed carbon electrode (SPCE) modification. The synthesized GONs and TNFs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). The modified electrode (TNFs/GONs/SPCE) was used for electrochemical characterization of adenine. The TNFs/GONs/SPCE exhibited an increase in peak current and the electron transfer kinetics and decrease in the overpotential for the oxidation reaction of adenine. Using differential pulse voltammetry (DPV), the prepared sensor showed good sensitivity for determining adenine in two ranges from 0.1-1 and 1-10 μM, with a detection limit (DL) of 1.71 nM. Electrochemical studies suggested that the TNFs/GONs/SPCE provided a synergistic augmentation on the voltammetric behavior of electrochemical oxidation of adenine, which was indicated by the improvement of anodic peak current and a decrease in anodic peak potential. The amount of adenine in pBudCE4.1 plasmid was determined via the proposed sensor and the result was in good compatibility with the sequence data of pBudCE4.1 plasmid. Copyright © 2015 Elsevier B.V. All rights reserved.

Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA

NASA Technical Reports Server (NTRS)

Prabahar, K. J.; Ferris, J. P.

1997-01-01

Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.

Dietary nucleotides and early growth in formula-fed infants: a randomized controlled trial.

PubMed

Singhal, Atul; Kennedy, Kathy; Lanigan, J; Clough, Helen; Jenkins, Wendy; Elias-Jones, Alun; Stephenson, Terrence; Dudek, Peter; Lucas, Alan

2010-10-01

Dietary nucleotides are nonprotein nitrogenous compounds that are found in high concentrations in breast milk and are thought to be conditionally essential nutrients in infancy. A high nucleotide intake has been suggested to explain some of the benefits of breastfeeding compared with formula feeding and to promote infant growth. However, relatively few large-scale randomized trials have tested this hypothesis in healthy infants. We tested the hypothesis that nucleotide supplementation of formula benefits early infant growth. Occipitofrontal head circumference, weight, and length were assessed in infants who were randomly assigned to groups fed nucleotide-supplemented (31 mg/L; n=100) or control formula without nucleotide supplementation (n=100) from birth to the age of 20 weeks, and in infants who were breastfed (reference group; n=101). Infants fed with nucleotide-supplemented formula had greater occipitofrontal head circumference at ages 8, 16, and 20 weeks than infants fed control formula (mean difference in z scores at 8 weeks: 0.4 [95% confidence interval: 0.1-0.7]; P=.006) even after adjustment for potential confounding factors (P=.002). Weight at 8 weeks and the increase in both occipitofrontal head circumference and weight from birth to 8 weeks were also greater in infants fed nucleotide-supplemented formula than in those fed control formula. Our data support the hypothesis that nucleotide supplementation leads to increased weight gain and head growth in formula-fed infants. Therefore, nucleotides could be conditionally essential for optimal infant growth in some formula-fed populations. Additional research is needed to test the hypothesis that the benefits of nucleotide supplementation for early head growth, a critical period for brain growth, have advantages for long-term cognitive development.

Labeled nucleotide phosphate (NP) probes

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2009-02-03

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Synthetic oligonucleotide separations by mixed-mode reversed-phase/weak anion-exchange liquid chromatography.

PubMed

Zimmermann, Aleksandra; Greco, Roberto; Walker, Isabel; Horak, Jeannie; Cavazzini, Alberto; Lämmerhofer, Michael

2014-08-08

Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes in biological sciences. If pharmaceutical-grade purities are required, chromatographic purification using ion-pair reversed-phase chromatography is commonly carried out. However, separation selectivity for structurally closely related impurities is often insufficient, especially at high sample loads. In this study, a "mixed-mode" reversed-phase/weak anion exchanger stationary phase has been investigated as an alternative tool for chromatographic separation of synthetic oligonucleotides with minor sequence variations. The employed mixed-mode phase shows great flexibility in method development. It has been run in various gradient elution modes, viz. one, two or three parameter (mixed) gradients (altering buffer pH, buffer concentration, and organic modifier) to find optimal elution conditions and gain further insight into retention mechanisms. Compared to ion-pair reversed-phase and mere anion-exchange separation, enhanced selectivities were observed with the mixed-mode phase for 20-23 nucleotide (nt) long oligonucleotides with similar sequences. Oligonucleotides differing by 1, 2 or 3 nucleotides in length could be readily resolved and separation factors for single nucleotide replacements declined in the order Cytosine (C)/Guanine (G)>Adenine (A)/Guanine∼Guanine/Thymine (T)>Adenine/Cytosine∼Cytosine/Thymine>Adenine/Thymine. Selectivities were larger when the modification was at the 3' terminal-end, declined when it was in the middle of the sequence and was smallest when it was located at the 5' terminus. Due to the lower surface area of the 200Å pore size mixed-mode stationary phase compared to the corresponding 100Å material, lower retention times with equal selectivities under milder elution conditions were achievable. Considering high sample loading capacities of the mixed-mode anion-exchanger phase, it should have great potential for chromatographic

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

PubMed

Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Fujimoto, Yuki; Furuie, Sumie; Yamamura, Ken-Ichi; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Moriyama, Mitsuaki; Sinasac, David S; Yamamoto, Takashi; Furukawa, Tatsuhiko; Kobayashi, Keiko

2017-04-01

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients. Copyright © 2017 Elsevier Inc. All rights reserved.

SiC nanoparticles-modified glassy carbon electrodes for simultaneous determination of purine and pyrimidine DNA bases.

PubMed

Ghavami, Raouf; Salimi, Abdollah; Navaee, Aso

2011-05-15

For the first time a novel and simple electrochemical method was used for simultaneous detection of DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment or separation process. Glassy carbon electrode modified with silicon carbide nanoparticles (SiCNP/GC), have been used for electrocatalytic oxidation of purine (guanine and adenine) and pyrimidine bases (thymine and cytosine) nucleotides. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) techniques were used to examine the structure of the SiCNP/GC modified electrode. The modified electrode shows excellent electrocatalytic activity toward guanine, adenine, thymine and cytosine. Differential pulse voltammetry (DPV) was proposed for simultaneous determination of four DNA bases. The effects of different parameters such as the thickness of SiC layer, pulse amplitude, scan rate, supporting electrolyte composition and pH were optimized to obtain the best peak potential separation and higher sensitivity. Detection limit, sensitivity and linear concentration range of the modified electrode toward proposed analytes were calculated for, guanine, adenine, thymine and cytosine, respectively. As shown this sensor can be used for nanomolar or micromolar detection of different DNA bases simultaneously or individually. This sensor also exhibits good stability, reproducibility and long lifetime. Copyright © 2011 Elsevier B.V. All rights reserved.

Nucleotide cleaving agents and method

DOEpatents

Que, Jr., Lawrence; Hanson, Richard S.; Schnaith, Leah M. T.

2000-01-01

The present invention provides a unique series of nucleotide cleaving agents and a method for cleaving a nucleotide sequence, whether single-stranded or double-stranded DNA or RNA, using and a cationic metal complex having at least one polydentate ligand to cleave the nucleotide sequence phosphate backbone to yield a hydroxyl end and a phosphate end.

De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

PubMed

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-04-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

Thermodynamics of Activation Gating in Olfactory-Type Cyclic Nucleotide-Gated (CNGA2) Channels

PubMed Central

Nache, Vasilica; Kusch, Jana; Biskup, Christoph; Schulz, Eckhard; Zimmer, Thomas; Hagen, Volker; Benndorf, Klaus

2008-01-01

Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21 kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order. PMID:18567637

Distinctive Spectral Features of Exciton and Excimer States in the Ultrafast Electronic Deactivation of the Adenine Dinucleotide

NASA Astrophysics Data System (ADS)

Stuhldreier, Mayra C.; Röttger, Katharina; Temps, Friedrich

We report the observation by transient absorption spectroscopy of distinctive spectro-temporal signatures of delocalized exciton versus relaxed, weakly bound excimer states in the ultrafast electronic deactivation after UV photoexcitation of the adenine dinucleotide.

Nucleosides and nucleotides. 192. Toward the total synthesis of cyclic ADP-carbocyclic-ribose. Formation of the intramolecular pyrophosphate linkage by a conformation-restriction strategy in a syn-form using a halogen substitution at the 8-position of the adenine ring.

PubMed

Sumita, Y; Shirato, M; Ueno, Y; Matsuda, A; Shuto, S

2000-01-01

The synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was investigated. Construction of the 18-membered backbone structure was successfully achieved by condensation of the two phosphate groups of 19, possibly due to restriction of the conformation of the substrate in a syn-form using an 8-chloro substituent at the adenine moiety. SN2 reactions between an optically active carbocyclic unit 8, which was constructed by a previously developed method, and 8-bromo-N6-trichloroacetyl-2',3'-O-isopropylideneadenosine 9c gave N-1-carbocyclic derivative, which was deprotected to give 5'-5"-diol derivatives 18. When 18 was treated with POCl3 in PO(OEt)3, the bromo group at the 8-position was replaced to give N-1-carbocyclic-8-chloroadenosine 5',5"-diphosphate derivative 19 in 43% yield. Treatment of 19 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride gave the desired intramolecular condensation product 20 in 10% yield. This is the first chemical construction of the 18-membered backbone structure containing an intramolecular pyrophosphate linkage of a cADPR-related compound with an adenine base.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Catalytic and regulatory roles of species involved in metal-nucleotide equilibriums in human pyridoxal kinase.

PubMed

Navarro, Freddy; Ramírez-Sarmiento, César A; Guixé, Victoria

2013-10-01

Pyridoxal 5'-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal-nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn(2+) the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP(4-) and HATP(3-) did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP(2-), although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn(2+); as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC50 of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal-nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.

Influence of gamma irradiation and benzyl adenine on keeping quality of custard apple fruits during storage.

PubMed

Chouksey, Swati; Singh, Alpana; Thakur, Rajendra Singh; Deshmukh, Reena

2013-10-01

The custard apple (Annona squamosa) fruits were procured from local market, irradiated with radiation doses 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 kGy and then treated with benzyl adenine (50 and 100 part per million) and stored at ambient temperature (25 ± 5 °C, Relative Humidity 90 ± 2%) for 12 days. The treated fruits were evaluated for sensory (viz; flavour, texture, internal and external colour) and chemical constituents (viz; Total Soluble Solids, titrable acidity, ascorbic acid, free soluble sugar, reducing sugar. non reducing sugar, carbohydrate) during storage. The study concluded that radiation dose of 1.5 kilo Gray along with 50 ppm benzyl adenine enhanced in shelf-life of custard apple fruits by 6 days at ambient temperature with good pulp texture, flavour, colour and nutritional quality as compared to control.

Studies with a reconstituted muscle glycolytic system. The anaerobic glycolytic response to simulated tetanic contraction

PubMed Central

Scopes, Robert K.

1974-01-01

By using a reconstituted glycolytic system and a highly active adenosine triphosphatase (ATPase), the metabolism during muscular tetanic contraction was simulated and observed. With an ATPase activity somewhat greater than can be maintained in muscle tissue, phosphocreatine was rapidly and completely utilized, lactate production commenced about 5s after the ATPase was added and after 15s adenine nucleotides were lost through deamination to IMP. By 40s, all metabolism ceased because of complete loss of adenine mononucleotides. With a lower ATPase activity, glycolytic regeneration of ATP was capable of maintaining the ATP concentration at its initial value and even by 80s, only one-half of the phosphocreatine had been utilized. No deamination occurred in this time. It is suggested that the metabolic events observed in the simulated system are basically the same as occur in muscle doing heavy work. PMID:4275706

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis[W

PubMed Central

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-01-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

Interaction between macrocyclic nickel complexes and the nucleotides GMP, AMP and ApG.

PubMed

Liu, Yangzhong; Sletten, Einar

2003-01-15

Reactions between the nucleotides GMP, AMP and ApG and the complexes Ni(tren), Ni(cyclam) and NiCR in aqueous solution have been monitored by (1)H, (15)N NMR and UV spectroscopy. The three nickel complexes display different properties in reactions with nucleotides. Ni(tren) which has a pseudo-octahedral coordination geometry was shown to bind to all three nucleotides. Ni(cyclam) and NiCR, both with four nitrogen atoms in a square planar arrangement are not able to bind to nucleotides efficiently because of steric hindrance. Oxidation of Ni(cyclam) by KHSO(5) to produce trivalent Ni(III)(cyclam) improves the coordination capacity, while oxidation of NiCR does not produce a similar effect. The nucleotides interact with trivalent nickel complexes to different extent. Ni(III)CR is seen to oxidize GMP gradually but does not affect AMP significantly. Ni(III)(cyclam), on the other hand, does not oxidize either GMP or AMP at the 1:1 concentration of oxidant used. This result is probably due to the lower redox potential of Ni(cyclam). ApG binds less efficiently to the Ni complexes but is easier oxidized than the mononucleotides.

Comparison of the Antiviral Effects of 5-Methoxymethyl-deoxyuridine with 5-Iododeoxyuridine, Cytosine Arabinoside, and Adenine Arabinoside

PubMed Central

Babiuk, Lorne A.; Meldrum, Blair; Gupta, V. Sagar; Rouse, Barry T.

1975-01-01

The antiviral activity of 5-methoxymethyl-2′-deoxyuridine (MMUdR) was compared with that of 5-iodo-2′-deoxyuridine (IUdR), cytosine arabinoside (Ara-C), and adenine arabinoside (Ara-A). At concentrations of 2 to 4 μg/ml, MMUdR was inhibitory to herpes simplex virus type 1, but concentrations as high as 128 μg/ml were not inhibitory to three other herpesviruses tested (equine rhinopneumonitis virus, murine cytomegalovirus, and feline rhinopneumonitis virus) or to vaccinia virus. The other nucleosides, in contrast, were inhibitory at similar concentrations (1 to 8 μg/ml) against all viruses tested. The inhibition of HSV-1 by MMUdR appeared to be the result of interference with virus replication rather than the result of drug toxicity to host cells. The drug was not toxic to host cells at 100 times the antiviral concentrations, and pretreatment of host cells with high concentrations of MMUdR had no effect on subsequent virus replication. Combination of MMUdR with either IUdR, Ara-A, or Ara-C gave an enhanced antiviral effect, suggesting that the mechanism of action of MMUdR is different from that of the other three drugs. Antiviral indexes were calculated for each compound and were found to be >250, 80, 40, and 8 for MMUdR, IUdR, Ara-A, and Ara-C, respectively. These were defined as the minimum dose at which toxicity was observed microscopically divided by the dose which reduced plaque numbers by 50%. PMID:1239978

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

PubMed

Michalakis, Stylianos; Becirovic, Elvir; Biel, Martin

2018-03-07

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca 2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

PubMed Central

Biel, Martin

2018-01-01

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application. PMID:29518895

Evidence for the role of hydrophobic forces on the interactions of nucleotide-monophosphates with cationic liposomes.

PubMed

Cuomo, Francesca; Mosca, Monica; Murgia, Sergio; Avino, Pasquale; Ceglie, Andrea; Lopez, Francesco

2013-11-15

In this work, the interaction of nucleotide-monophosphates (NMPs) with unilamellar liposomes made of 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) was investigated. Here, we demonstrate how adsorption is affected by the type of nucleotide-monophosphate. Dynamic light scattering (DLS) results revealed, for each NMP, that a distinguishable concentration exists at which a significant growth of the aggregates occurs. Adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) have shown a higher propensity to induce liposome aggregation process and in particular GMP appears to be the most effective. From ζ-potential experiments we found that liposomes loaded with purine based nucleotides (AMP and GMP) are able to decrease the ζ-potential values to a greater extent in comparison with the pyrimidine based nucleotides thimydine 5'-monophosphate (TMP) and uridine 5'-monophosphate (UMP). Moreover, a careful analysis of nucleotide-liposome interactions revealed that nucleotides have different capacity to induce the formation of nucleotide-liposome complexes, and purine based nucleotides have higher affinities with lipid membranes. On the whole, the data emphasize that the mechanisms driving the interactions between liposomes and NMPs are also influenced by the existence of hydrophobic forces. Copyright © 2013 Elsevier Inc. All rights reserved.

Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephansen, Anne B.; King, Sarah B.; Li, Wei-Li

2015-09-14

Dipole bound (DB) and valence bound (VB) anions of binary iodide-adenine complexes have been studied using one-color and time-resolved photoelectron imaging at excitation energies near the vertical detachment energy. The experiments are complemented by quantum chemical calculations. One-color spectra show evidence for two adenine tautomers, the canonical, biologically relevant A9 tautomer and the A3 tautomer. In the UV-pump/IR-probe time-resolved experiments, transient adenine anions can be formed by electron transfer from the iodide. These experiments show signals from both DB and VB states of adenine anions formed on femto- and picosecond time scales, respectively. Analysis of the spectra and comparison withmore » calculations suggest that while both the A9 and A3 tautomers contribute to the DB signal, only the DB state of the A3 tautomer undergoes a transition to the VB anion. The VB anion of A9 is higher in energy than both the DB anion and the neutral, and the VB anion is therefore not accessible through the DB state. Experimental evidence of the metastable A9 VB anion is instead observed as a shape resonance in the one-color photoelectron spectra, as a result of UV absorption by A9 and subsequent electron transfer from iodide into the empty π-orbital. In contrast, the iodide-A3 complex constitutes an excellent example of how DB states can act as doorway state for VB anion formation when the VB state is energetically available.« less

A hepcidin lowering agent mobilizes iron for incorporation into red blood cells in an adenine-induced kidney disease model of anemia in rats

PubMed Central

Sun, Chia Chi; Vaja, Valentina; Chen, Shanzhuo; Theurl, Igor; Stepanek, Aaron; Brown, Diane E.; Cappellini, Maria D.; Weiss, Guenter; Hong, Charles C.; Lin, Herbert Y.; Babitt, Jodie L.

2013-01-01

Background Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients. Methods We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model. Results Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes. Conclusions Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD. PMID:23345622

Adsorption of nucleotides onto ferromagnesian phyllosilicates: Significance for the origin of life

NASA Astrophysics Data System (ADS)

Pedreira-Segade, Ulysse; Feuillie, Cécile; Pelletier, Manuel; Michot, Laurent J.; Daniel, Isabelle

2016-03-01

The concentration of prebiotic organic building blocks may have promoted the formation of biopolymers in the environment of the early Earth. We therefore studied the adsorption of RNA monomers AMP, GMP, CMP, and UMP, and DNA monomers dGMP, dCMP, and TMP, on minerals that were abundant in the early Earth environment as the result of aqueous or hydrothermal alteration of the primitive oceanic crust. We focused our study on swelling clays, i.e. nontronite and montmorillonite, and non-swelling phyllosilicates, i.e. pyrophyllite, chlorite, lizardite and chrysotile suspended in an aqueous saline solution analog to seawater. In this reference study, adsorption experiments were carried out under standard conditions of pressure and temperature and controlled pH. Under such conditions, this work is also relevant to the preservation of nucleic acids in Fe-Mg-rich terrestrial and Martian soils. We compared the adsorption of the different monomers on individual minerals, as well as the adsorption of single monomers on the whole suite of minerals. We found that DNA monomers adsorb much more strongly than RNA monomers, and that any monomer containing the G nucleobase adsorbed more strongly than one containing the C nucleobase. At high surface loadings (greater than about 1 mM monomer in aqueous solution) we also found a dramatic increase in the slope of adsorption isotherm on the swelling clays, leading to large increases in the amounts adsorbed. Data were processed in order to understand the adsorption mechanism of nucleotides onto mineral surfaces. We infer that all nucleotides behave as homologous molecules in regard to their adsorption onto the studied mineral surfaces. At low to moderate surface loadings, their adsorption is best explained by a single mechanism common to the suite of minerals of the present study. At pH 7, adsorption certainly proceeds by ligand exchange between the phosphate group and the hydroxyls of the broken edges of phyllosilicates leading to the

Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC.

PubMed

Kochanowski, N; Blanchard, F; Cacan, R; Chirat, F; Guedon, E; Marc, A; Goergen, J-L

2006-01-15

Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.

Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

NASA Astrophysics Data System (ADS)

Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

1991-03-01

It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

PubMed

Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

2017-09-01

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

NASA Astrophysics Data System (ADS)

Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

2017-09-01

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

The GC-rich mitochondrial and plastid genomes of the green alga Coccomyxa give insight into the evolution of organelle DNA nucleotide landscape

DOE PAGES

Smith, David Roy; Burki, Fabien; Yamada, Takashi; ...

2011-08-26

Here, most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features ofmore » this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, David Roy; Burki, Fabien; Yamada, Takashi

2011-05-13

Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of thismore » species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

PubMed Central

James, Zachary M.; Borst, Andrew J.; Haitin, Yoni; Frenz, Brandon; DiMaio, Frank; Zagotta, William N.; Veesler, David

2017-01-01

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae—which shares sequence similarity to eukaryotic CNG and HCN channels—in the presence of a saturating concentration of cAMP. A short S4–S5 linker connects nearby voltage-sensing and pore domains to produce a non–domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies. PMID:28396445

Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate.

PubMed

Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís

2016-05-23

The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D-UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter-base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long-lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the (1) Lb , S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D-UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm(-1) in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D-UV spectroscopy to disentangle the photophysics of multichromophoric systems. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Apyrase (Nucleoside Triphosphate-Diphosphohydrolase) and Extracellular Nucleotides Regulate Cotton Fiber Elongation in Cultured Ovules1[W][OA

PubMed Central

Clark, Greg; Torres, Jonathan; Finlayson, Scott; Guan, Xueying; Handley, Craig; Lee, Jinsuk; Kays, Julia E.; Chen, Z. Jeffery; Roux, Stanley J.

2010-01-01

Ectoapyrase enzymes remove the terminal phosphate from extracellular nucleoside tri- and diphosphates. In Arabidopsis (Arabidopsis thaliana), two ectoapyrases, AtAPY1 and AtAPY2, have been implicated as key modulators of growth. In fibers of cotton (Gossypium hirsutum), transcript levels for GhAPY1 and GhAPY2, two closely related ectoapyrases that have high sequence similarity to AtAPY1 and AtAPY2, are up-regulated when fibers enter their rapid growth phase. In an ovule culture system, fibers release ATP as they grow, and when their ectoapyrase activity is blocked by the addition of polyclonal anti-apyrase antibodies or by two different small molecule inhibitors, the medium ATP level rises and fiber growth is suppressed. High concentrations of the poorly hydrolyzable nucleotides ATPγS and ADPβS applied to the medium inhibit fiber growth, and low concentrations of them stimulate growth, but treatment with adenosine 5′-O-thiomonophosphate causes no change in the growth rate. Both the inhibition and stimulation of growth by applied nucleotides can be blocked by an antagonist that blocks purinoceptors in animal cells, and by adenosine. Treatment of cotton ovule cultures with ATPγS induces increased levels of ethylene, and two ethylene antagonists, aminovinylglycine and silver nitrate, block both the growth stimulatory and growth inhibitory effects of applied nucleotides. In addition, the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, lowers the concentration of nucleotide needed to promote fiber growth. These data indicate that ectoapyrases and extracellular nucleotides play a significant role in regulating cotton fiber growth and that ethylene is a likely downstream component of the signaling pathway. PMID:20018604

[Effect of hypoxia on glycolysis in the adductor muscle and hepatopancreas of the marine mussel Mytilus galloprovincialis Lmk].

PubMed

Ibarguren, I; Villamarín, J A; Barcia, R; Ramos-Martínez, J I

1989-12-01

Concentrations of glycolytic intermediates and adenine nucleotides have been estimated in adductor muscle and hepatopancreas from the sea mussel Mytilus galloprovincialis Lmk. after various periods of valve closure. Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals hexokinase, phosphofructokinase, pyruvate kinase and fructose-1,6-bisphosphatase catalyze non-equilibrium reactions. The changes in the concentrations of the glycolytic intermediates and in the rate M.A.R./Keq during hypoxia suggest that the carbon flow after valve closure is first controlled by phophofructokinase, but later on the rate of transformation of phosphoenolyruvate regulates this flow.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.

PubMed

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar; Henning, Heiko

2016-11-15

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. © 2016. Published by The Company of Biologists Ltd.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

PubMed Central

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar

2016-01-01

ABSTRACT Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. PMID:27612509

Nucleotide-dependent conformational states of actin

PubMed Central

Pfaendtner, Jim; Branduardi, Davide; Parrinello, Michele; Pollard, Thomas D.; Voth, Gregory A.

2009-01-01

The influence of the state of the bound nucleotide (ATP, ADP-Pi, or ADP) on the conformational free-energy landscape of actin is investigated. Nucleotide-dependent folding of the DNase-I binding (DB) loop in monomeric actin and the actin trimer is carried out using all-atom molecular dynamics (MD) calculations accelerated with a multiscale implementation of the metadynamics algorithm. Additionally, an investigation of the opening and closing of the actin nucleotide binding cleft is performed. Nucleotide-dependent free-energy profiles for all of these conformational changes are calculated within the framework of metadynamics. We find that in ADP-bound monomer, the folded and unfolded states of the DB loop have similar relative free-energy. This result helps explain the experimental difficulty in obtaining an ordered crystal structure for this region of monomeric actin. However, we find that in the ADP-bound actin trimer, the folded DB loop is stable and in a free-energy minimum. It is also demonstrated that the nucleotide binding cleft favors a closed conformation for the bound nucleotide in the ATP and ADP-Pi states, whereas the ADP state favors an open confirmation, both in the monomer and trimer. These results suggest a mechanism of allosteric interactions between the nucleotide binding cleft and the DB loop. This behavior is confirmed by an additional simulation that shows the folding free-energy as a function of the nucleotide cleft width, which demonstrates that the barrier for folding changes significantly depending on the value of the cleft width. PMID:19620726

Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

PubMed

Abbas, Charles A; Sibirny, Andriy A

2011-06-01

Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B₂] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

PubMed Central

Abbas, Charles A.; Sibirny, Andriy A.

2011-01-01

Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

Single nucleotide polymorphism analysis using different colored dye dimer probes

NASA Astrophysics Data System (ADS)

Marmé, Nicole; Friedrich, Achim; Denapaite, Dalia; Hakenbeck, Regine; Knemeyer, Jens-Peter

2006-09-01

Fluorescence quenching by dye dimer formation has been utilized to develop hairpin-structured DNA probes for the detection of a single nucleotide polymorphism (SNP) in the penicillin target gene pbp2x, which is implicated in the penicillin resistance of Streptococcus pneumoniae. We designed two specific DNA probes for the identification of the pbp2x genes from a penicillin susceptible strain R6 and a resistant strain Streptococcus mitis 661 using green-fluorescent tetramethylrhodamine (TMR) and red-fluorescent DY-636, respectively. Hybridization of each of the probes to its respective target DNA sequence opened the DNA hairpin probes, consequently breaking the nonfluorescent dye dimers into fluorescent species. This hybridization of the target with the hairpin probe achieved single nucleotide specific detection at nanomolar concentrations via increased fluorescence.

Posttransfusional changes of 2,3-diphosphoglycerate and nucleotides in CPD-SAGM-preserved erythrocytes.

PubMed

Matthes, G; Strunk, S; Siems, W; Grune, T

1993-06-01

Posttransfusional changes of preserved red blood cells can influence the oxygen equilibrium curve which is mainly affected by the concentration of erythrocyte 2,3-diphosphoglycerate (DPG). The regeneration kinetics of DPG and nucleotides (ATP, ADP, AMP, GTP, GDP) was determined over a period of 0-48 h in surgically treated patients following transfusion of DPG-depleted packed red cells stored for 14 days in CPD-SAGM. 3 h after transfusion the DPG levels raised up to 40% of the patients' prior DPG concentrations. Complete regeneration of the DPG concentrations occurred 36-48 h after transfusion. Changes in the nucleotide pattern indicate, after a temporary decrease of ATP and GTP levels (after 10-30 min) and an activation phase (after 3-12 h), the full regeneration of these parameters 24-48 h after transfusion. The regeneration kinetics of DPG should be taken into consideration for transfusions with blood units stored for more than 14 days, especially in patients with reduced compensatory mechanisms (coronary and cerebral scleroses, pacemaker, etc.) and large transfusion volumes.

Studies related to primitive chemistry. A proton and nitrogen-14 nuclear magnetic resonance amino acid and nucleic acid constituents and a and their possible relation to prebiotic

NASA Technical Reports Server (NTRS)

Manatt, S. L.; Cohen, E. A.; Shiller, A. M.; Chan, S. I.

1973-01-01

Preliminary proton nuclear magnetic resonance (NMR) studies were made to determine the applicability of this technique for the study of interactions between monomeric and polymeric amino acids with monomeric nucleic acid bases and nucleotides. Proton NMR results for aqueous solutions (D2O) demonstrated interactions between the bases cytosine and adenine and acidic and aromatic amino acids. Solutions of 5'-AMP admixed with amino acids exhibited more complex behavior but stacking between aromatic rings and destacking at high amino acids concentration was evident. The multisite nature of 5'-AMP was pointed out. Chemical shift changes for adenine and 5'-AMP with three water soluble polypeptides demonstrated that significant interactions exist. It was found that the linewidth-pH profile of each amino acid is unique. It is concluded that NMR techniques can give significant and quantitative data on the association of amino acid and nucleic acid constituents.

The Effect of Nucleotides and Inhibitors on Respiration in Isolated Wheat Mitochondria 1

PubMed Central

Pomeroy, M. Keith

1975-01-01

The effect of mono-, di-, and trinucleoside phosphates and respiratory inhibitors on respiration in winter wheat (Triticum aestivum L. cv. Rideau) mitochondria has been examined. When added during state 4 respiration, subsequent to addition of ADP, all of the dinucleotides stimulated oxidation and induced respiratory control with all substrates examined. Similar results were obtained with AMP, but other mononucleotides and all trinucleotides did not affect the rate of oxidation. Nucleoside diphosphates did not stimulate respiration when added prior to the addition of ADP, but subsequent addition of AMP, ADP, or ATP re-established coupled respiration in the presence of the dinucleotides. The duration of 2, 4-dinitrophenol stimulated respiration during oxidation of α-ketoglutarate was found to be dependent on the amount of AMP, ADP, or ATP added, either prior, or subsequent to, addition of the uncoupler. The addition of oligomycin during 2, 4-dinitrophenol stimulated respiration reestablished coupled respiration with low ADP/O ratios, when added after addition of ATP or conditions which allow formation of ATP from added ADP. The nucleoside diphosphates, other than ADP, did not stimulate oxidation of α-ketoglutarate in the presence of 2, 4-dinitrophenol until a small amount of adenine nucleotide was added to the system. The results suggest that dinucleotides other than ADP, are able to participate in the energy conversion processs of the mitochondria, probably via transphosphorylation reactions. Images PMID:16659027

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, C.M.; Rohrmann, G.F.; Merrill, G.F., E-mail: merrillg@onid.orst.ed

2009-06-05

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involvedmore » in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.« less

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Long, C M; Rohrmann, G F; Merrill, G F

2009-06-05

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

Pressure-tuning infrared and Raman microscopy study of the DNA bases: adenine, guanine, cytosine, and thymine.

PubMed

Yang, Seung Yun; Butler, Ian S

2013-12-01

Diamond-anvil cell, pressure-tuning infrared (IR), and Raman microspectroscopic measurements have been undertaken to examine the effects of high pressures up to about 45 kbar on the vibrational spectra of the four DNA bases, adenine, cytosine, guanine, and thymine. Small structural changes were evident for all the four bases, viz., for adenine and cytosine at 28-31 kbar; for guanine at 16-19 kbar; and for thymine at 25-26 kbar. These changes are most likely associated with alterations in the intermolecular hydrogen-bonding interactions. The pressure dependences of the main peaks observed in the IR spectra of the two phases of guanine lie in the -0.07-0.66 (low-pressure phase) and 0.06-0.91 (high-pressure phase) cm⁻¹/kbar ranges. Also, in the Raman spectra of this nucleoside base, the dν/dP values range from -0.07-0.31 (low-pressure phase) to 0.08-0.50 (high-pressure phase) cm⁻¹/kbar. Similar ranges of dν/dP values were obtained for the other three nucleoside bases.

Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

PubMed Central

Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

2015-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

Effect of Electronic Excitation on Hydrogen Atom Transfer (Tautomerization) Reactions for the DNA Base Adenine

NASA Technical Reports Server (NTRS)

Chaban, Galina M.; Salter, Latasha M.; Kwak, Dochan (Technical Monitor)

2002-01-01

Geometrical structures and energetic properties for four different tautomers of adenine are calculated in this study, using multi-configurational wave functions. Both the ground and the lowest single excited state potential energy surface are studied. The energetic order of the tautomers on the ground state potential surface is 9H less than 7H less than 3H less than 1H, while on the excited state surface this order is found to be different: 3H less than 1H less than 9H less than 7H. Minimum energy reaction paths are obtained for hydrogen atom transfer (9 yields 3 tautomerization) reactions in the ground and the lowest excited electronic state. It is found that the barrier heights and the shapes of the reaction paths are different for the ground and the excited electronic state, suggesting that the probability of such tautomerization reaction is higher on the excited state potential energy surface. The barrier for this reaction in the excited state may become very low in the presence of water or other polar solvent molecules, and therefore such tautomerization reaction may play an important role in the solution phase photochemistry of adenine.

Sensitive colorimetric visualization of dihydronicotinamide adenine dinucleotide based on anti-aggregation of gold nanoparticles via boronic acid-diol binding.

PubMed

Liu, Shufeng; Du, Zongfeng; Li, Peng; Li, Feng

2012-05-15

A facile, highly sensitive colorimetric strategy for dihydronicotinamide adenine dinucleotide (NADH) detection is proposed based on anti-aggregation of gold nanoparticles (AuNPs) via boronic acid-diol binding chemistry. The aggregation agent, 4-mercaptophenylboronic acid (MPBA), has specific affinity for AuNPs through Au-S interaction, leading to the aggregation of AuNPs by self-dehydration condensation at a certain concentration, which is responsible for a visible color change of AuNPs from wine red to blue. With the addition of NADH, MPBA would prefer reacting with NADH to form stable borate ester via boronic acid-diol binding dependent on the pH and solvent, revealing an obvious color change from blue to red with increasing the concentration of NADH. The anti-aggregation effect of NADH on AuNPs was seen by the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for NADH is from 8.0 × 10(-9)M to 8.0 × 10(-6)M, with a low detection limit of 2.0 nM. The as-established colorimetric strategy opened a new avenue for NADH determination. Copyright © 2012 Elsevier B.V. All rights reserved.

Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

NASA Astrophysics Data System (ADS)

Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

1996-07-01

In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-β -S, pertussis toxin, and antibodies against the α -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

Salmonella DNA Adenine Methylase Mutants Confer Cross-Protective Immunity

PubMed Central

Heithoff, Douglas M.; Enioutina, Elena Y.; Daynes, Raymond A.; Sinsheimer, Robert L.; Low, David A.; Mahan, Michael J.

2001-01-01

Salmonella isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different Salmonella serovars. The protection afforded by the Salmonella enterica serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection. S. enterica serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also, S. enterica serovar Typhimurium Dam− vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts. PMID:11598044

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

PubMed Central

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

PubMed Central

Fasullo, Michael; Endres, Lauren

2015-01-01

Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases. PMID:25923076

Evolving nucleotide binding surfaces

NASA Technical Reports Server (NTRS)

Kieber-Emmons, T.; Rein, R.

1981-01-01

An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

Structural basis of AMPK regulation by adenine nucleotides and glycogen

DOE PAGES

Li, Xiaodan; Wang, Lili; Zhou, X. Edward; ...

2014-11-21

AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions.

PubMed

Yegutkin, Gennady G; Guerrero-Toro, Cindy; Kilinc, Erkan; Koroleva, Kseniya; Ishchenko, Yevheniia; Abushik, Polina; Giniatullina, Raisa; Fayuk, Dmitriy; Giniatullin, Rashid

2016-09-01

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca(2+) transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.

Erhuang Formula ameliorates renal damage in adenine-induced chronic renal failure rats via inhibiting inflammatory and fibrotic responses.

PubMed

Zhang, Chun-Yan; Zhu, Jian-Yong; Ye, Ying; Zhang, Miao; Zhang, Li-Jun; Wang, Su-Juan; Song, Ya-Nan; Zhang, Hong

2017-11-01

The present study aimed to evaluate the protective effects of Erhuang Formula (EHF) and explore its pharmacological mechanisms on adenine-induced chronic renal failure (CRF). The compounds in EHF were analyzed by HPLC/MS. Adenine-induced CRF rats were administrated by EHF. The effects were evaluated by renal function examination and histology staining. Immunostaining of some proteins related cell adhesion was performedin renal tissues, including E-cadherin, β-catenin, fibronectin and laminin. The qRT-PCR was carried out determination of gene expression related inflammation and fibrosis including NF-κB, TNF-α, TGF-β1, α-SMA and osteopontin (OPN). Ten compounds in EHF were identified including liquiritigenin, farnesene, vaccarin, pachymic acid, cycloastragenol, astilbin, 3,5,6,7,8,3',4'-heptemthoxyflavone, physcion, emodin and curzerene. Abnormal renal function and histology had significant improvements by EHF treatment. The protein expression of β-catenin, fibronectin and laminin were significantly increased and the protein expression of E-cadherin significantly decreased in CRF groups. However, these protein expressions were restored to normal levels in EHF group. Furthermore, low expression of PPARγ and high expression of NF-κB, TNF-α, TGF-β1, α-SMA and OPN were substantially restored by EHF treatment in a dose-dependent manner. EHF ameliorated renal damage in adenine-induced CRF rats, and the mechanisms might involve in the inhibition of inflammatory and fibrotic responses and the regulation of PPARγ, NF-κB and TGF-β signaling pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

PubMed Central

Thévenod, F; Gasser, K W; Hopfer, U

1990-01-01

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation

Cyclic Nucleotide Phosphodiesterases: important signaling modulators and therapeutic targets

PubMed Central

Ahmad, Faiyaz; Murata, Taku; Simizu, Kasumi; Degerman, Eva; Maurice, Donald; Manganiello, Vincent

2014-01-01

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases are critical regulators of their intracellular concentrations and their biological effects. Since these intracellular second messengers control many cellular homeostatic processes, dysregulation of their signals and signaling pathways initiate or modulate pathophysiological pathways related to various disease states, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication, chronic obstructive pulmonary disease, and psoriasis. Alterations in expression of PDEs and PDE-gene mutations (especially mutations in PDE6, PDE8B, PDE11A and PDE4) have been implicated in various diseases and cancer pathologies. PDEs also play important role in formation and function of multi-molecular signaling/regulatory complexes called signalosomes. At specific intracellular locations, individual PDEs, together with pathway-specific signaling molecules, regulators, and effectors, are incorporated into specific signalosomes, where they facilitate and regulate compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions. Currently, only a limited number of PDE inhibitors (PDE3, PDE4, PDE5 inhibitors) are used in clinical practice. Future paths to novel drug discovery include the crystal structure-based design approach, which has resulted in generation of more effective family-selective inhibitors, as well as burgeoning development of strategies to alter compartmentalized cyclic nucleotide signaling pathways by selectively targeting individual PDEs and their signalosome partners. PMID:25056711

The effect of pi-stacking, h-bonding, and electrostatic interactions on the ionization energies of nucleic acid bases: adenine-adenine, thymine-thymine and adenine-thymine dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bravaya, Ksenia B.; Kostko, Oleg; Ahmed, Musahid

A combined theoretical and experimental study of the ionized dimers of thymine and adenine, TT, AA, and AT, is presented. Adiabatic and vertical ionization energies(IEs) for monomers and dimers as well as thresholds for the appearance of the protonated species are reported and analyzed. Non-covalent interactions stronglyaffect the observed IEs. The magnitude and the nature of the effect is different for different isomers of the dimers. The computations reveal that for TT, the largestchanges in vertical IEs (0.4 eV) occur in asymmetric h-bonded and symmetric pi- stacked isomers, whereas in the lowest-energy symmetric h-bonded dimer the shiftin IEs is muchmore » smaller (0.1 eV). The origin of the shift and the character of the ionized states is different in asymmetric h-bonded and symmetric stacked isomers. Inthe former, the initial hole is localized on one of the fragments, and the shift is due to the electrostatic stabilization of the positive charge of the ionized fragment by thedipole moment of the neutral fragment. In the latter, the hole is delocalized, and the change in IE is proportional to the overlap of the fragments' MOs. The shifts in AAare much smaller due to a less effcient overlap and a smaller dipole moment. The ionization of the h-bonded dimers results in barrierless (or nearly barrierless) protontransfer, whereas the pi-stacked dimers relax to structures with the hole stabilized by the delocalization or electrostatic interactions.« less

Structural basis of reverse nucleotide polymerization

PubMed Central

Nakamura, Akiyoshi; Nemoto, Taiki; Heinemann, Ilka U.; Yamashita, Keitaro; Sonoda, Tomoyo; Komoda, Keisuke; Tanaka, Isao; Söll, Dieter; Yao, Min

2013-01-01

Nucleotide polymerization proceeds in the forward (5′-3′) direction. This tenet of the central dogma of molecular biology is found in diverse processes including transcription, reverse transcription, DNA replication, and even in lagging strand synthesis where reverse polymerization (3′-5′) would present a “simpler” solution. Interestingly, reverse (3′-5′) nucleotide addition is catalyzed by the tRNA maturation enzyme tRNAHis guanylyltransferase, a structural homolog of canonical forward polymerases. We present a Candida albicans tRNAHis guanylyltransferase-tRNAHis complex structure that reveals the structural basis of reverse polymerization. The directionality of nucleotide polymerization is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme’s active site from the opposite direction (180° flip) compared with similar nucleotide substrates of canonical 5′-3′ polymerases, and the finger domains are on opposing sides of the core palm domain. Structural, biochemical, and phylogenetic data indicate that reverse polymerization appeared early in evolution and resembles a mirror image of the forward process. PMID:24324136

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

PubMed

Danylovych, H V

2016-01-01

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Nucleotide Binding by Lhs1p Is Essential for Its Nucleotide Exchange Activity and for Function in Vivo*

PubMed Central

de Keyzer, Jeanine; Steel, Gregor J.; Hale, Sarah J.; Humphries, Daniel; Stirling, Colin J.

2009-01-01

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo. PMID:19759005

Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon

PubMed Central

2010-01-01

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5′-phosphosulfate or 3′,5′-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2′,3′-cylic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-l-arabinofuranose, guanosine 5'-diphosphate (GDP)-l-galactofuranose, UDP-l-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches. PMID:21043458

Control of box C/D snoRNP assembly by N6-methylation of adenine.

PubMed

Huang, Lin; Ashraf, Saira; Wang, Jia; Lilley, David Mj

2017-09-01

N 6 -methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N 6 -methyladenine at a key trans Hoogsteen-sugar A·G base pair, of which half are methylated in vivo The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5-kDa protein and the induced folding of the RNA Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N 6 -methylation of adenine prevents the formation of trans Hoogsteen-sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson-Crick base pairs) are more susceptible to disruption by N 6 mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

PubMed

Rösner, J; Liotta, A; Angamo, E A; Spies, C; Heinemann, U; Kovács, R

2016-11-01

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO 2 , field potentials and changes in extracellular potassium concentration ([K + ] o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K + ] o and pO 2 changes as compared to continuous illumination. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

PubMed

Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

2002-04-01

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

High-NaCl diet impairs dynamic renal blood flow autoregulation in rats with adenine-induced chronic renal failure.

PubMed

Saeed, Aso; DiBona, Gerald F; Grimberg, Elisabeth; Nguy, Lisa; Mikkelsen, Minne Line Nedergaard; Marcussen, Niels; Guron, Gregor

2014-03-15

This study examined the effects of 2 wk of high-NaCl diet on kidney function and dynamic renal blood flow autoregulation (RBFA) in rats with adenine-induced chronic renal failure (ACRF). Male Sprague-Dawley rats received either chow containing adenine or were pair-fed an identical diet without adenine (controls). After 10 wk, rats were randomized to either remain on the same diet (0.6% NaCl) or to be switched to high 4% NaCl chow. Two weeks after randomization, renal clearance experiments were performed under isoflurane anesthesia and dynamic RBFA, baroreflex sensitivity (BRS), systolic arterial pressure variability (SAPV), and heart rate variability were assessed by spectral analytical techniques. Rats with ACRF showed marked reductions in glomerular filtration rate and renal blood flow (RBF), whereas mean arterial pressure and SAPV were significantly elevated. In addition, spontaneous BRS was reduced by ∼50% in ACRF animals. High-NaCl diet significantly increased transfer function fractional gain values between arterial pressure and RBF in the frequency range of the myogenic response (0.06-0.09 Hz) only in ACRF animals (0.3 ± 4.0 vs. -4.4 ± 3.8 dB; P < 0.05). Similarly, a high-NaCl diet significantly increased SAPV in the low-frequency range only in ACRF animals. To conclude, a 2-wk period of a high-NaCl diet in ACRF rats significantly impaired dynamic RBFA in the frequency range of the myogenic response and increased SAPV in the low-frequency range. These abnormalities may increase the susceptibility to hypertensive end-organ injury and progressive renal failure by facilitating pressure transmission to the microvasculature.

Transcriptional regulation of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of wasabi (Eutrema wasabi Maxim).

PubMed

Hou, D X; Fukuda, M; Fujii, M; Fuke, Y

2000-12-20

Wasabi is a very popular pungent spice in Japan. This study examined the ability of 6-(methylsufinyl)hexyl isothiocyanate (6-MITC), an active principle of wasabi, to induce the cellular expression of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase (QR) in Hepa 1c1c7 cells. The cells were treated with various concentrations of 6-MITC, and were then assessed for cell growth, QR activity and QR mRNA expression. The induction of QR activity and QR mRNA expression was time- and dose-responsive over a narrow range of 0.1-5 microM, with declining induction at higher concentrations due to cell toxicity. Furthermore, transfection studies demonstrated that the induction of transcription of the QR gene by 6-MITC involved an antioxidant/electrophile-responsive element (ARE/EpRE) activation. Our results suggest a novel mechanism by which dietary wasabi 6-MITC may be implicated in cancer chemoprevention.

E-ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy.

PubMed

Bach, Barbara Charlotte; Leal, Daniela Bitencourt Rosa; Jaques, Jeandre Augusto dos Santos; Souza, Viviane do Carmo Gonçalves; Ruchel, Jader Betsch; Schlemmer, Karine Bizzi; Zanette, Régis Adriel; Hecktheuer, Pedro Abib; de Lima Pereira, Patrique; Casali, Emerson André; Alves, Sydney Hartz; Santurio, Janio Morais

2013-08-01

Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2-like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto-adenosine deaminase (E-ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E-ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E-ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E-ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy. Copyright © 2012 John Wiley & Sons, Ltd.

Role of Metal Oxides in Chemical Evolution: Interaction of Ribose Nucleotides with Alumina

NASA Astrophysics Data System (ADS)

Arora, Avnish Kumar; Kamaluddin

2009-03-01

Interaction of ribonucleotides—namely, 5‧-AMP, 5‧-GMP, 5‧-CMP, and 5‧-UMP—with acidic, neutral, and basic alumina has been studied. Purine nucleotides showed higher adsorption on alumina in comparison with pyrimidine nucleotides under acidic conditions. Adsorption data obtained followed Langmuir adsorption isotherm, and Xm and KL values were calculated. On the basis of infrared spectral studies of ribonucleotides, alumina, and ribonucleotide-alumina adducts, we propose that the nitrogen base and phosphate moiety of the ribonucleotides interact with the positive charge surface of alumina. Results of the present study may indicate the importance of alumina in concentrating organic molecules from dilute aqueous solutions in primeval seas in the course of chemical evolution on Earth.

Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine.

PubMed

Saito, Yoshio; Kugenuma, Kenji; Tanaka, Makiko; Suzuki, Azusa; Saito, Isao

2012-06-01

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids. Copyright © 2012 Elsevier Ltd. All rights reserved.

Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins.

PubMed

Talbot, Darren A; Duchamp, Claude; Rey, Benjamin; Hanuise, Nicolas; Rouanet, Jean Louis; Sibille, Brigitte; Brand, Martin D

2004-07-01

Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).

Relative reactivity of ribosyl 2'-OH vs. 3'-OH in concentrated aqueous solutions of phosphoimidazolide activated nucleotides

NASA Technical Reports Server (NTRS)

Kanavarioti, A.; Lee, L. F.; Gangopadhyay, S.

1999-01-01

Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN2' pN and pN3' pN) and the pyrophosphate-linked (N5' ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2'-OH vs. 3'-OH was determined from the ratio of the yields of pN2' pN vs. pN3' pN. Experiments were performed at 23 degrees C in the range 7.2 < or = pH < or = 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg2+ or Mn2+) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN2' pN: pN3' pN = 2'-5': 3'-5' was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2'-OH is about 2 to 3 times more reactive than the 3'-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN2' pN: pN3' pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN2' pN: pN3' pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5' = 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3'-OH of one molecule to the reactive

Polynucleotide: adenosine glycosidase activity of saporin-L1: effect on DNA, RNA and poly(A).

PubMed Central

Barbieri, L; Valbonesi, P; Gorini, P; Pession, A; Stirpe, F

1996-01-01

The ribosome-inactivating proteins (RIPs) are a family of plant enzymes for which a unique activity has been determined: rRNA N-glycosidase, which removes adenine at a specific universally conserved position (A4324 in the case of rat ribosomes). Here we report that saporin-L1, a RIP from the leaves of Saponaria officinalis, recognizes other substrates, including RNAs from different sources, DNA and poly(A). Saporin-L1 depurinated DNA extensively and released adenine from all adenine-containing polynucleotides tested. Adenine was the only base released from DNA or artificial polynucleotides. The characteristics of the reactions catalysed by saporin-L1 have been determined: optimal pH and temperature, ionic requirements, and the kinetic parameters Km and kcat. The reaction proceeded without cofactors, at low ionic strength, in the absence of Mg2+ and K+. Saporin-L1 had no activity towards various adenine-containing non-polynucleotide compounds (cytokinins, cofactors, nucleotides). This plant protein may now be classified as a polynucleotide: adenosine glycosidase. PMID:8912688

DNA-thioguanine nucleotide concentration and relapse-free survival during maintenance therapy of childhood acute lymphoblastic leukaemia (NOPHO ALL2008): a prospective substudy of a phase 3 trial.

PubMed

Nielsen, Stine Nygaard; Grell, Kathrine; Nersting, Jacob; Abrahamsson, Jonas; Lund, Bendik; Kanerva, Jukka; Jónsson, Ólafur Gísli; Vaitkeviciene, Goda; Pruunsild, Kaie; Hjalgrim, Lisa Lyngsie; Schmiegelow, Kjeld

2017-04-01

Adjustment of mercaptopurine and methotrexate maintenance therapy of acute lymphoblastic leukaemia by leucocyte count is confounded by natural variations. Cytotoxicity is primarily mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to establish whether DNA-TGN concentrations in blood leucocytes during maintenance therapy are associated with relapse-free survival. In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0-17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m 2 once per day and methotrexate 20 mg/m 2 once per week, targeted to a leucocyte count of 1·5-3·0 × 10 9 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2. Between Nov 26, 2008 and June 14, 2016, 1509 patients from the NOPHO ALL2008 study were assessed for eligibility in the DNA-TGN substudy, of which 918 (89%) of 1026 eligible patients had at least one DNA-TGN measurement and were included in the analyses. Median follow-up was 4·6 years (IQR 3·1-6·1). Relapse-free survival was

Constitutional self-organization of adenine-uracil-derived hybrid materials.

PubMed

Arnal-Hérault, Carole; Barboiu, Mihai; Pasc, Andreea; Michau, Mathieu; Perriat, Pascal; van der Lee, Arie

2007-01-01

The alkoxysilane nucleobase adenine (A) and uracil (U) precursors described in this paper generate in solution a complex library of hydrogen-bonded aggregates, which can be expressed in the solid state as discrete higher oligomers. The different interconverting outputs that nucleobases may form by oligomerization define a dynamic polyfunctional diversity that may be "extracted selectively" in solid state by sol-gel transcription, under the intrinsic stability of the system. After the sol-gel process, unique constitutional preference for specific geometries in hybrid materials is consistent with a preferential arrangement of nucleobase systems, favoring the self-assembly by the Hoogsteen geometry. FTIR and NMR spectroscopy and X-ray powder diffraction experiments demonstrate the formation of self-organized hybrid supramolecular materials. Electron microscopy reveals the micrometric platelike morphology of the hybrid materials. The M(A-U) hybrid material is nanostructured in ordered circular domains of 5 nm in diameter of alternative light and dark rows with an one-dimensional periodicity of 3.5 A.

A Molecular Vestige of the Origin of Life on Minerals: Phosphorybosyl-Disphosphate

NASA Astrophysics Data System (ADS)

Akouche, M.; Jaber, M.; Maurel, M.-C.; Lambert, J.-F.; Georgelin, T.

2017-07-01

Nucleotides were synthesized from Ribose, Adenine, and phosphates on silica surfaces in one pot process. A important molecular intermediate was enlightened, the phosphoribosyl pyrophosphate. This molecule is a molecular relic of early earth.

A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana

PubMed Central

Wang, Chenggang; Zhou, Mingqi; Zhang, Xudong; Yao, Jin; Zhang, Yanping; Mou, Zhonglin

2017-01-01

Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 ± 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants. DOI: http://dx.doi.org/10.7554/eLife.25474.001 PMID:28722654

pH profile of the adsorption of nucleotides onto montmorillonite. I - Selected homoionic clays

NASA Technical Reports Server (NTRS)

Lawless, J. G.; Church, F. M.; Mazzurco, J.; Banin, A.; Huff, R.; Kao, J.; Cook, A.; Lowe, T.; Orenberg, J. B.; Edelson, E.

1985-01-01

The effect of pH and adsorbed ions on the adsorption of purine and pyrimidine nucleotides on montmorillonite clay was studied experimentally. The specific nucleotides examined were: 5 prime-AMP; 3-prime AMP; and 5 prime-CMP. The pH of the clay samples was adjusted to various levels in the 2-12 pH range using microliter volumes of concentrated acid (1N HCl) and base (1NHNaOH). It was found that preferential adsorption among nulceotides was dependent on the pH level and on the characteristics of the substituted metal cation and anion exchange mechanisms. Below pH 4, adsorption was attributed to cation and anion exchange mechanisms. Above pH 4, however, adsorption was attributed to the complexation mechanisms occurring between the metal cations in the clay exchange site and in the biomolecule. The possible role of homoionic clays in the concentration mechanisms of biomonomers in the prebiotic environment is discussed.

Degradation of Adenine on the Martian Surface in the Presence of Perchlorates and Ionizing Radiation: A Reflectron Time-of-flight Mass Spectrometric Study

NASA Astrophysics Data System (ADS)

Góbi, Sándor; Bergantini, Alexandre; Kaiser, Ralf I.

2017-04-01

The aim of the present work is to unravel the radiolytic decomposition of adenine (C5H5N5) under conditions relevant to the Martian surface. Being the fundamental building block of (deoxy)ribonucleic acids, the possibility of survival of this biomolecule on the Martian surface is of primary importance to the astrobiology community. Here, neat adenine and adenine-magnesium perchlorate mixtures were prepared and irradiated with energetic electrons that simulate the secondary electrons originating from the interaction of the galactic cosmic rays with the Martian surface. Perchlorates were added to the samples since they are abundant—and therefore relevant oxidizers on the surface of Mars—and they have been previously shown to facilitate the radiolysis of organics such as glycine. The degradation of the samples were monitored in situ via Fourier transformation infrared spectroscopy and the electron ionization quadruple mass spectrometric method; temperature-programmed desorption profiles were then collected by means of the state-of-the-art single photon photoionization reflectron time-of-flight mass spectrometry (PI-ReTOF-MS), allowing for the detection of the species subliming from the sample. The results showed that perchlorates do increase the destruction rate of adenine by opening alternative reaction channels, including the concurrent radiolysis/oxidation of the sample. This new pathway provides a plethora of different radiolysis products that were identified for the first time. These are carbon dioxide (CO2), isocyanic acid (HNCO), isocyanate (OCN-), carbon monoxide (CO), and nitrogen monoxide (NO); an oxidation product containing carbonyl groups (R1R2-C=O) with a constrained five-membered cyclic structure could also be observed. Cyanamide (H2N-C≡N) was detected in both irradiated samples as well.

Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine and DNA uracil concentrations

USDA-ARS?s Scientific Manuscript database

Background: Folate is an essential nutrient which supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases including colorectal cancer. Folate status is also inversely related to plasma homocys...

Maintenance of cAMP in non-heart-beating donor lungs reduces ischemia-reperfusion injury.

PubMed

Hoffmann, S C; Bleiweis, M S; Jones, D R; Paik, H C; Ciriaco, P; Egan, T M

2001-06-01

Studies suggest that pulmonary vascular ischemia-reperfusion injury (IRI) can be attenuated by increasing intracellular cAMP concentrations. The purpose of this study was to determine the effect of IRI on capillary permeability, assessed by capillary filtration coeficient (Kfc), in lungs retrieved from non-heart-beating donors (NHBDs) and reperfused with the addition of the beta(2)-adrenergic receptor agonist isoproterenol (iso), and rolipram (roli), a phosphodiesterase (type IV) inhibitor. Using an in situ isolated perfused lung model, lungs were retrieved from NHBD rats at varying intervals after death and either ventilated with O(2) or not ventilated. The lungs were reperfused with Earle's solution with or without a combination of iso (10 microM) and roli (2 microM). Kfc, lung viability, and pulmonary hemodynamics were measured. Lung tissue levels of adenine nucleotides and cAMP were measured by HPLC. Combined iso and roli (iso/roli) reperfusion decreased Kfc significantly (p < 0.05) compared with non-iso/roli-reperfused groups after 2 h of postmortem ischemia. Total adenine nucleotide (TAN) levels correlated with Kfc in non-iso/roli-reperfused (r = 0.89) and iso/roli-reperfused (r = 0.97) lungs. cAMP levels correlated with Kfc (r = 0.93) in iso/roli-reperfused lungs. Pharmacologic augmentation of tissue TAN and cAMP levels might ameliorate the increased capillary permeability observed in lungs retrieved from NHBDs.

Ketose induced respiratory inhibition in isolated hepatocytes.

PubMed

Martínez, P; Carrascosa, J M; Núñez de Castro, I

1987-06-01

The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.

Phosphate-Modified Nucleotides for Monitoring Enzyme Activity.

PubMed

Ermert, Susanne; Marx, Andreas; Hacker, Stephan M

2017-04-01

Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

Nucleic acid analysis using terminal-phosphate-labeled nucleotides

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-04-22

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence.

PubMed

James, Allison E; Rogovskyy, Artem S; Crowley, Michael A; Bankhead, Troy

2016-01-01

DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, David B.; Lao, Guifang

1998-01-01

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

Patterns of purine nucleotides in fish erythrocytes.

PubMed

Leray, C

1979-01-01

1. The purine nucleotides were determined in the whole blood of 9 fresh water teleosts and 2 marine selachians. 2. GTP and ATP accounted for 88-99% of the total erythrocytes purines. 3. The ATP/ADP ratio ranged from 11 to 60 in the erythrocytes of the fish examined. 4. GTP is widely distributed in fish erythrocytes but its level ranged from 1 to 33 nmol/mg Hb (0.4 to 9 mumol/ml erythrocyte). 5. Lepomis and Esox exhibited a GTP/ATP ratio as elevated as in Anguilla; moreover the concentration of GTP per mol of Hb (physiologically most indicative) is higher in Lepomis, Esox, Ictalurus and Silurus than in Anguilla.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, D.B.; Lao, G.

1998-01-06

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

Structural Basis for Nucleotide Exchange in Heterotrimeric G Proteins

PubMed Central

Dror, Ron O.; Mildorf, Thomas J.; Hilger, Daniel; Manglik, Aashish; Borhani, David W.; Arlow, Daniel H.; Philippsen, Ansgar; Villanueva, Nicolas; Yang, Zhongyu; Lerch, Michael T.; Hubbell, Wayne L.; Kobilka, Brian K.; Sunahara, Roger K.; Shaw, David E.

2016-01-01

G protein–coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains—previously observed to separate widely upon receptor binding to expose the nucleotide-binding site—separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism. PMID:26089515

Prebiotic synthesis of adenine and amino acids under Europa-like conditions

NASA Technical Reports Server (NTRS)

Levy, M.; Miller, S. L.; Brinton, K.; Bada, J. L.

2000-01-01

In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites, we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 years at -20 and -78 degrees C. In addition, the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20 degrees C. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be wider than previously thought.

Prebiotic synthesis of adenine and amino acids under Europa-like conditions.

PubMed

Levy, M; Miller, S L; Brinton, K; Bada, J L

2000-06-01

In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites, we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 years at -20 and -78 degrees C. In addition, the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20 degrees C. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be wider than previously thought.

8-(2-Furyl)adenine derivatives as A₂A adenosine receptor ligands.

PubMed

Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Thomas, Ajiroghene; Klotz, Karl-Norbert; Federico, Stephanie; Cacciari, Barbara; Spalluto, Giampiero; Volpini, Rosaria

2013-01-01

Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2-furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A₂A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N(6)-position takes to different effects on the A₂A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A₂A receptor crystal structures provides an interpretation of the obtained biological results. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Prebiotic Synthesis of Adenine and Amino Acids Under Europa-like Conditions

NASA Technical Reports Server (NTRS)

Levy, Matthew; Miller, Stanley L.; Brinton, Karen; Bada, Jeffrey L.

2003-01-01

In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites. we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 year at -20 and -78 C. In addition the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20%. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be m der than previously thought.

Molecular recognition modes between adenine or adeniniun(1+) ion and binary M(II)(pdc) chelates (MCoZn; pdc=pyridine-2,6-dicarboxylate(2-) ion).

PubMed

Del Pilar Brandi-Blanco, María; Choquesillo-Lazarte, Duane; Domínguez-Martín, Alicia; Matilla-Hernández, Antonio; González-Pérez, Josefa María; Castiñeiras, Alfonso; Niclós-Gutiérrez, Juan

2013-10-01

Mixed ligand M(II)-complexes (MCoZn) with pyridine-2,6-dicarboxylate(2-) chelator (pdc) and adenine (Hade) have been synthesized and studied by X-ray diffraction and other spectral and thermal methods: [Cu(pdc)(H(N9)ade)(H2O)] (1), [Cu2(pdc)2(H2O)2(μ2-N3,N7-H(N9)ade)]·3H2O (2), trans-[M(pdc)(H(N9)ade)(H2O)2]·nH2O for MCo (3-L, 3-M, 3-H) or Zn (4-L, 4-H), where n is 0, 1 or 3 for the 'lowest' (L), 'medium' (M) and 'highest' (H) hydrated forms, and the salt trans-[Ni(pdc)(H2(N1,N9)ade)(H2O)2]Cl·2H2O (5). In all the nine compounds, both neutral and cationic adenine exist as their most stable tautomer and the molecular recognition pattern between the metal-pdc chelates and the adenine or adeninium(1+) ligands involves the MN7 bond in cooperation with an intra-molecular N6H⋯O(coordinated carboxylate) interligand interaction. In addition the dinuclear copper(II) compound (2) has the CuN3 bond and the N9H⋯O(coord. carboxylate) interaction. The structures of mononuclear ternary complexes proved that the molecular recognition pattern is the same irrespective of (a) the coordination geometry of the complex molecule, (b) the different hydrated forms of crystals with Co or Zn, and (c) the neutral of cationic form of the adenine ligand. These features are related to the mer-NO2 chelating ligand conformation (imposed by the planar rigidity of pdc) as a driving force for the observed metal binding mode. Copyright © 2013 Elsevier Inc. All rights reserved.

A novel adenine-based metal organic framework derived nitrogen-doped nanoporous carbon for flexible solid-state supercapacitor.

PubMed

Li, Haowen; Fu, Dongying; Zhang, Xian-Ming

2018-01-01

In this article, we have synthesized a series of nitrogen-doped nanoporous carbon (NPC) from metal organic framework of UiO-66 with different ratios of adenine and 1,4-benzendicarboxylate (H 2 BDC) coated on carbon nanotube film (CNTF) to obtain a flexible porous electrode (NPC/CNTF). It is worth noting that the introduction of adenine at different ratios did not change the structure of UiO-66. We also investigated the effect of carbonization temperature from 800 to 1000°C on the electrochemical properties of the NPC. The ratio (H 2 BDC:adenine) 9 : 1 and the NPC carbonized at 900°C (denoted as NPC-1-900) exhibits better electrochemical properties. The results show that NPC-1-900/CNTF electrode exhibits an exceptional areal capacitance of 121.5 mF cm -2 compared to that of PC-900/CNTF electrode (22.8 mF cm -2 ) at 5 mV s -1 in a three-electrode system, indicating that the incorporation of nitrogen is beneficial to the electrochemical properties of nanoporous carbon. A symmetric flexible solid-state supercapacitor of NPC-1-900/CNTF has also been assembled and tested. Electrochemical data show that the device exhibited superior areal capacitance (43.2 mF cm -2 ) at the scan rate of 5 mV s -1 ; the volumetric energy density is 57.3 µWh cm -3 and the volumetric power density is 2.4 mW cm -3 at the current density of 0.5 mA cm -2 based on poly(vinyl alcohol)/H 3 PO 4 gel electrolyte. For practical application, we have also studied the bending tests of the device, which show that the device exhibits outstanding mechanical stability under different bending angles. Furthermore, the flexible device shows excellent cyclic stability, which can retain 91.5% of the initial capacitance after 2000 cycles.

A novel adenine-based metal organic framework derived nitrogen-doped nanoporous carbon for flexible solid-state supercapacitor

PubMed Central

Li, Haowen; Zhang, Xian-Ming

2018-01-01

In this article, we have synthesized a series of nitrogen-doped nanoporous carbon (NPC) from metal organic framework of UiO-66 with different ratios of adenine and 1,4-benzendicarboxylate (H2BDC) coated on carbon nanotube film (CNTF) to obtain a flexible porous electrode (NPC/CNTF). It is worth noting that the introduction of adenine at different ratios did not change the structure of UiO-66. We also investigated the effect of carbonization temperature from 800 to 1000°C on the electrochemical properties of the NPC. The ratio (H2BDC:adenine) 9 : 1 and the NPC carbonized at 900°C (denoted as NPC-1-900) exhibits better electrochemical properties. The results show that NPC-1-900/CNTF electrode exhibits an exceptional areal capacitance of 121.5 mF cm−2 compared to that of PC-900/CNTF electrode (22.8 mF cm−2) at 5 mV s−1 in a three-electrode system, indicating that the incorporation of nitrogen is beneficial to the electrochemical properties of nanoporous carbon. A symmetric flexible solid-state supercapacitor of NPC-1-900/CNTF has also been assembled and tested. Electrochemical data show that the device exhibited superior areal capacitance (43.2 mF cm−2) at the scan rate of 5 mV s−1; the volumetric energy density is 57.3 µWh cm−3 and the volumetric power density is 2.4 mW cm−3 at the current density of 0.5 mA cm−2 based on poly(vinyl alcohol)/H3PO4 gel electrolyte. For practical application, we have also studied the bending tests of the device, which show that the device exhibits outstanding mechanical stability under different bending angles. Furthermore, the flexible device shows excellent cyclic stability, which can retain 91.5% of the initial capacitance after 2000 cycles. PMID:29410815

Removal of interfering nucleotides from brain extracts containing substance p. Effect of drugs on brain concentrations of substance p

PubMed Central

Laszlo, I.

1963-01-01

Several methods for removing interfering nucleotides, adenosine-5'-monophosphate and adenosine 5'-triphosphate from brain extracts have been studied. An enzymic method, using adenylic acid deaminase, has been found suitable. This deaminates adenosine monophosphate to 5'-inosinic acid, an inactive compound which does not influence the estimations of substance P. Owing to the adenosine triphosphatase content of the enzyme extract, adenosine triphosphate was also inactivated. For the estimation of adenosine monophosphate-deaminase activity, a simple colorimetric method is described which measures the ammonia liberated from adenosine monophosphate. Substance P in mouse brain extracts was estimated after treatment of the animals with various drugs, and after the enzymic removal of interfering nucleotides from the brain extracts. The drugs had no effect on the substance P content of mouse brain. The effect of drugs on the contractions of the guinea-pig ileum induced by substance P was also investigated, and the effect of drugs on the estimations of substance P in brain extracts is discussed. PMID:14066136

Use of supernatant refractive index and supernatant hemoglobin concentration to assess residual glycerol concentration in cryopreserved red blood cells.

PubMed

Wong, Kenneth A; Nsier, Nada; Acker, Jason P

2009-10-01

Red blood cells (RBCs) cryopreserved in glycerol must be deglycerolized prior to transfusion. The adequacy of glycerol removal is commonly assessed by measurement of the refractive index (RI) of the supernatant fluid. However, the presence of free hemoglobin in the supernatant falsely increases the RI and may lead to discard of units that have an acceptable residual glycerol concentration. We performed an analysis of the diagnostic accuracy of 3 methods for residual glycerol measurement - refractometry, osmometry, and a glycerol assay kit. Residual glycerol measurement using these methods was performed on 12 deglycerolized, citrate-phosphate-dextrose (CPD)/saline-adenine-glucose-mannitol (SAGM) leukoreduced RBCs. A calculation that estimates the glycerol concentration based on the refractive index and supernatant hemoglobin concentration was developed and ensures that units with an elevated RI due to the presence of hemoglobin are not discarded if their residual glycerol concentration was <1.0% (w/v). Osmometry was an accurate method for estimating residual glycerol concentration. Refractometry overestimated the residual glycerol concentration due to the interference from hemoglobin. However, when supernatant hemoglobin values were measured and used in the calculation for glycerol concentration, refractometry accurately estimated the residual glycerol concentration. The residual glycerol concentration of cryopreserved, deglycerolized CPD/SAGM RBCs can be accurately estimated using the supernatant refractive index and an equation that accounts for the supernatant hemoglobin concentration.

New perspectives in cyclic nucleotide-mediated functions in the CNS: the emerging role of cyclic nucleotide-gated (CNG) channels.

PubMed

Podda, Maria Vittoria; Grassi, Claudio

2014-07-01

Cyclic nucleotides play fundamental roles in the central nervous system (CNS) under both physiological and pathological conditions. The impact of cAMP and cGMP signaling on neuronal and glial cell functions has been thoroughly characterized. Most of their effects have been related to cyclic nucleotide-dependent protein kinase activity. However, cyclic nucleotide-gated (CNG) channels, first described as key mediators of sensory transduction in retinal and olfactory receptors, have been receiving increasing attention as possible targets of cyclic nucleotides in the CNS. In the last 15 years, consistent evidence has emerged for their expression in neurons and astrocytes of the rodent brain. Far less is known, however, about the functional role of CNG channels in these cells, although several of their features, such as Ca(2+) permeability and prolonged activation in the presence of cyclic nucleotides, make them ideal candidates for mediators of physiological functions in the CNS. Here, we review literature suggesting the involvement of CNG channels in a number of CNS cellular functions (e.g., regulation of membrane potential, neuronal excitability, and neurotransmitter release) as well as in more complex phenomena, like brain plasticity, adult neurogenesis, and pain sensitivity. The emerging picture is that functional and dysfunctional cyclic nucleotide signaling in the CNS has to be reconsidered including CNG channels among possible targets. However, concerted efforts and multidisciplinary approaches are still needed to get more in-depth knowledge in this field.

Detection of a single nucleotide polymorphism in the human alpha-lactalbumin gene: implications for human milk proteins.

PubMed

Chowanadisai, Winyoo; Kelleher, Shannon L; Nemeth, Jennifer F; Yachetti, Stephen; Kuhlman, Charles F; Jackson, Joan G; Davis, Anne M; Lien, Eric L; Lönnerdal, Bo

2005-05-01

Variability in the protein composition of breast milk has been observed in many women and is believed to be due to natural variation of the human population. Single nucleotide polymorphisms (SNPs) are present throughout the entire human genome, but the impact of this variation on human milk composition and biological activity and infant nutrition and health is unclear. The goals of this study were to characterize a variant of human alpha-lactalbumin observed in milk from a Filipino population by determining the location of the polymorphism in the amino acid and genomic sequences of alpha-lactalbumin. Milk and blood samples were collected from 20 Filipino women, and milk samples were collected from an additional 450 women from nine different countries. alpha-Lactalbumin concentration was measured by high-performance liquid chromatography (HPLC), and milk samples containing the variant form of the protein were identified with both HPLC and mass spectrometry (MS). The molecular weight of the variant form was measured by MS, and the location of the polymorphism was narrowed down by protein reduction, alkylation and trypsin digestion. Genomic DNA was isolated from whole blood, and the polymorphism location and subject genotype were determined by amplifying the entire coding sequence of human alpha-lactalbumin by PCR, followed by DNA sequencing. A variant form of alpha-lactalbumin was observed in HPLC chromatograms, and the difference in molecular weight was determined by MS (wild type=14,070 Da, variant=14,056 Da). Protein reduction and digestion narrowed the polymorphism between the 33rd and 77th amino acid of the protein. The genetic polymorphism was identified as adenine to guanine, which translates to a substitution from isoleucine to valine at amino acid 46. The frequency of variation was higher in milk from China, Japan and Philippines, which suggests that this polymorphism is most prevalent in Asia. There are SNPs in the genome for human milk proteins and their

Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins

PubMed Central

Talbot, Darren A; Duchamp, Claude; Rey, Benjamin; Hanuise, Nicolas; Rouanet, Jean Louis; Sibille, Brigitte; Brand, Martin D

2004-01-01

Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate). PMID:15146050

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria.

PubMed

Kristián, Tibor; Weatherby, Tina M; Bates, Timothy E; Fiskum, Gary

2002-12-01

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.

Proteomic Analysis of Prostate Cancer Field Effect

DTIC Science & Technology

2011-02-01

Homo sapiens] profilin 1 [Homo sapiens] enolase 1 [Homo sapiens] sorbitol dehydrogenase [Homo sapiens] calmodulin 1 [Homo sapiens] apolipoprotein... sorbitol dehydrogenase [Homo sapiens] solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 [Homo sapiens] DJ-1

Structure of a eukaryotic cyclic nucleotide-gated channel

PubMed Central

Li, Minghui; Zhou, Xiaoyuan; Wang, Shu; Michailidis, Ioannis; Gong, Ye; Su, Deyuan; Li, Huan; Li, Xueming; Yang, Jian

2018-01-01

Summary Cyclic nucleotide-gated (CNG) channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5 Å-resolution single-particle electron cryomicroscopy structure of a CNG channel from C. elegans in the cGMP-bound open state. The channel has an unusual voltage-sensor-like domain (VSLD), accounting for its deficient voltage dependence. A C-terminal linker connecting S6 and the cyclic nucleotide-binding domain interacts directly with both the VSLD and pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of CNG channels and cyclic nucleotide modulation of related channels. PMID:28099415

Energy efficiency trade-offs drive nucleotide usage in transcribed regions

PubMed Central

Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J.

2016-01-01

Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of ‘A' versus ‘T' and ‘G' versus ‘C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides ‘U' and ‘C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides ‘A' and ‘G' at non-synonymous coding sites. PMID:27098217

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells.

PubMed

Woollhead, A M; Sivagnanasundaram, J; Kalsi, K K; Pucovsky, V; Pellatt, L J; Scott, J W; Mustard, K J; Hardie, D G; Baines, D L

2007-08-01

AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells

PubMed Central

Woollhead, A M; Sivagnanasundaram, J; Kalsi, K K; Pucovsky, V; Pellatt, L J; Scott, J W; Mustard, K J; Hardie, D G; Baines, D L

2007-01-01

Background and purpose: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (Iamiloride) and ouabain-sensitive basolateral (Iouabain) short circuit current in H441 lung epithelial cells. Experimental approach: H441 cells were grown on permeable filters at air interface. Iamiloride, Iouabain and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Key results: Phenformin, AICAR and metformin increased AMPK (α1) activity and decreased Iamiloride. The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased Iouabain across H441 monolayers and decreased monolayer resistance. The decrease in Iamiloride was closely related to Iouabain with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Conclusions and implications: Activation of α1-AMPK is associated with inhibition of apical amiloride-sensitive Na+ channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on Iouabain, with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component. PMID:17603555

Functional analysis of regulatory single-nucleotide polymorphisms.

PubMed

Pampín, Sandra; Rodríguez-Rey, José C

2007-04-01

The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed 'haploChIP' allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.

Probing phenylalanine/adenine pi-stacking interactions in protein complexes with explicitly correlated and CCSD(T) computations.

PubMed

Copeland, Kari L; Anderson, Julie A; Farley, Adam R; Cox, James R; Tschumper, Gregory S

2008-11-13

To examine the effects of pi-stacking interactions between aromatic amino acid side chains and adenine bearing ligands in crystalline protein structures, 26 toluene/(N9-methyl)adenine model configurations have been constructed from protein/ligand crystal structures. Full geometry optimizations with the MP2 method cause the 26 crystal structures to collapse to six unique structures. The complete basis set (CBS) limit of the CCSD(T) interaction energies has been determined for all 32 structures by combining explicitly correlated MP2-R12 computations with a correction for higher-order correlation effects from CCSD(T) calculations. The CCSD(T) CBS limit interaction energies of the 26 crystal structures range from -3.19 to -6.77 kcal mol (-1) and average -5.01 kcal mol (-1). The CCSD(T) CBS limit interaction energies of the optimized complexes increase by roughly 1.5 kcal mol (-1) on average to -6.54 kcal mol (-1) (ranging from -5.93 to -7.05 kcal mol (-1)). Corrections for higher-order correlation effects are extremely important for both sets of structures and are responsible for the modest increase in the interaction energy after optimization. The MP2 method overbinds the crystal structures by 2.31 kcal mol (-1) on average compared to 4.50 kcal mol (-1) for the optimized structures.

Effects of four additive solutions on canine leukoreduced red cell concentrate quality during storage.

PubMed

Lacerda, Luciana A; Hlavac, Nicole R C; Terra, Silvia R; Back, Franciele P; Jane Wardrop, K; González, Félix H D

2014-09-01

Additive solutions (AS) and prestorage leukoreduction (LR) are important tools used to maintain erythrocyte viability during storage and avoid transfusion reactions in recipients, respectively. The purpose of the study was to determine the efficacy of a WBC filter (Immugard IIIRC) and compare the effect of 4 AS (phosphate-adenine-glucose-guanosine-gluconate-mannitol [PAGGGM], saline-adenine-glucose-mannitol [SAGM], Adsol, Optisol) on the in vitro quality of canine leukoreduced packed RBC units (pRBC) stored for 41 days. Five hundred milliliters of blood were collected from 8 healthy dogs each into 70 mL of citrate-phosphate-dextrose (CPD) solution, and were leukoreduced by a polyurethane filter. pRBC of each dog were divided equally into 4 bags containing a different AS. Bags were stored for 41 days at 4°C and evaluated every 10 days. Variables analyzed included pH, PCV, and% hemolysis, and lactate, glucose, potassium, sodium, ATP, and 2,3-diphosphoglycerate (2,3-DPG) concentrations. The LR resulted in residual WBC counts comparable to human standards. During storage, pH, and glucose, 2,3-DPG, and ATP concentrations decreased, and hemolysis, and lactate, sodium, and potassium concentrations increased (P < .05). Significant differences between AS were seen in the glucose and sodium concentrations, due to the composition of AS. Also, the pH maintained by PAGGGM at day 21 was significantly higher than that seen with SAGM or Adsol. All AS used gave satisfactory results during the first 21 days of storage based on the degree of hemolysis, and on ATP and 2,3-DPG concentrations. When compared with day 1 values, significant changes were seen in these variables by day 31 with all AS. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

Regulation of Ion Channels by Pyridine Nucleotides

PubMed Central

Kilfoil, Peter J.; Tipparaju, Srinivas M.; Barski, Oleg A.; Bhatnagar, Aruni

2014-01-01

Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion–transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide–binding β-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP+ to Kvβ removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K+ transporters. These nucleotides also have been shown to modify the activity of the plasma membrane KATP channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit—the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP+ metabolite, NAADP+, regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias. PMID:23410881

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance.

PubMed

Elliott, Michael R; Chekeni, Faraaz B; Trampont, Paul C; Lazarowski, Eduardo R; Kadl, Alexandra; Walk, Scott F; Park, Daeho; Woodson, Robin I; Ostankovich, Marina; Sharma, Poonam; Lysiak, Jeffrey J; Harden, T Kendall; Leitinger, Norbert; Ravichandran, Kodi S

2009-09-10

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y(2)(-/-) (also known as P2ry2(-/-)) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to

Effects of nucleotide supplementation in milk replacer on small intestinal absorptive capacity in dairy calves.

PubMed

Kehoe, S I; Heinrichs, A J; Baumrucker, C R; Greger, D L

2008-07-01

Milk replacer was supplemented with nucleotides and fed to dairy calves from birth through weaning to examine the potential for enhancing recovery of small intestinal function after enteric infection. Three treatments of 23 calves each were fed milk replacer (10% body weight/d) supplemented with no nucleotides (C), purified nucleotides (N), or nucleotides from an extract of Saccharomyces cerevisiae (S). Average daily gain, health scores, fecal dry matter, and fecal bacteria were monitored, and blood was analyzed for packed cell volume, glucose, blood urea nitrogen (BUN), and creatinine. Calves were monitored twice daily for fecal score, and 48 h after increased fecal fluidity was recorded, intestinal function was evaluated by measuring absorption of orally administered xylose (0.5 g/kg of body weight). Packed cell volume of blood was greater for treatment N for wk 2 and 5 compared with other treatment groups. Four calves per treatment were killed, and intestinal tissue was evaluated for morphology, enzyme activities, and nucleoside transporter mRNA expression. Treatment S calves had increased abundance of nucleoside transporter mRNA, numerically longer villi, and lower alkaline phosphatase than other groups. Growth measurements and plasma concentrations of glucose, BUN, creatinine, and IgG were not different between treatments; however, BUN-to-creatinine ratio was higher for treatment N, possibly indicating decreased kidney function. There were also no treatment effects on fecal dry matter and fecal bacteria population. However, N-treated calves had the highest detrimental and lowest beneficial bacteria overall, indicating an unfavorable intestinal environment. Supplementation of purified nucleotides did not improve intestinal morphology or function and resulted in higher fecal water loss and calf dehydration. Supplementation of nucleotides derived from yeast tended to increase calf intestinal function, provide a more beneficial intestinal environment, and improve

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zoghbi, M. E.; Altenberg, G. A.

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we usedmore » luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.« less

Adenylate and Nicotinamide Nucleotides in Developing Soybean Seeds During Seed-Fill 1

PubMed Central

Quebedeaux, Bruno

1981-01-01

Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity. Of the oxidized (NAD, NADP) and reduced (NADH, NADPH) nicotinamide nucleotide forms, NAD was the most abundant. Levels as high as 17.5 micrograms per seed were observed during the mid-seed-filling stages. NADP was found almost exclusively in the reduced form with a NADP: NADPH ratio of less than 0.35, whereas the reverse was noted for NAD which was found mainly in the oxidized form with a NAD:NADH ratio in the range of 5 to 25. NADP was detected in low concentrations compared to the other adenylate and nicotinamide nucleotides. The nicotinamide redox charge defined as (NADH + NADPH)/(NAD + NADH) + (NADP + NADPH) was calculated to express the state of the energy balance between the oxidized and reduced nicotinamide nucleotide forms. The nicotinamide redox charge varied between 0.15 and 0.30 during seed development and was significantly lower than that found for the adenylate energy charge. PMID:16661875

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A*

PubMed Central

Yang, Yidai; Ye, Qilu; Jia, Zongchao; Côté, Graham P.

2015-01-01

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min−1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PMID:26260792

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-05-11

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed Central

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-01-01

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process. Images PMID:1645868

Development of an ion-pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro-2a cell line.

PubMed

Chen, Yunyun; Xing, Dongming; Wang, Wei; Ding, Yi; Du, Lijun

2007-06-01

The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post-ischemic recovery. An IP-HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro-2a cell line. The chromatographic conditions were: a Zorbax SB-C18 reversed-phase column; mobile phase 100 mM KH(2)PO(4), 1 mM tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 M), filtered through a 0.45 microm Millipore filter and degassed prior to use. The flow-rate was 1.0 mL/min. The injection volume was 20 microL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 +/- 1 degrees C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4 degrees C) in 1 mL of an ice-cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform. Copyright 2007 John Wiley & Sons, Ltd.

A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

PubMed

Kopra, Kari; Ligabue, Alessio; Wang, Qi; Syrjänpää, Markku; Blaževitš, Olga; Veltel, Stefan; van Adrichem, Arjan J; Hänninen, Pekka; Abankwa, Daniel; Härmä, Harri

2014-07-01

A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

Cyclic nucleotide content of tobacco BY-2 cells.

PubMed

Richards, Helen; Das, Swadipa; Smith, Christopher J; Pereira, Louisa; Geisbrecht, Alan; Devitt, Nicola J; Games, David E; van Geyschem, Jan; Gareth Brenton, A; Newton, Russell P

2002-11-01

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

PubMed

Florea, Mara; Nau, Werner M

2010-03-07

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Nucleotide binding properties of bovine brain uncoating ATPase.

PubMed

Gao, B; Emoto, Y; Greene, L; Eisenberg, E

1993-04-25

Many functions of the 70-kDa heat-shock proteins (hsp70s) appear to be regulated by bound nucleotide. In this study we examined the nucleotide binding properties of purified bovine brain uncoating ATPase, one of the constitutively expressed members of the hsp70 family. We found that uncoating ATPase purified by ATP-agarose column chromatography retained one ADP molecule bound per enzyme molecule which could not be removed by extensive dialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociation but rather to strong binding of the nucleotide to the uncoating ATPase. In confirmation of this view, equilibrium dialysis experiments suggested that the dissociation constants for both ADP and ATP were less than 0.1 microM. Schmid et al. (Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem 260, 10057-10062) suggested that the uncoating ATPase had two sites for bound nucleotide, one specific for ATP and one binding both ATP and ATP analogues but not ADP. In contrast, we found that enzyme with bound ADP did not bind further adenosine 5'-(beta,gamma-imino)triphosphate or dATP, nor did more than one ATP molecule bind per enzyme even in 200 microM free ATP. These results strongly suggest that the enzyme has only one binding site for nucleotide. During steady-state ATP hydrolysis, 85% of the bound nucleotide at this site was determined to be ATP and 15% ADP; this is consistent with the rate of ADP release determined in the exchange experiments noted above, where ADP release was found to be six times faster than the overall rate of ATP hydrolysis.

Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions.

PubMed

Bean, Greg J; Amann, Kurt J

2008-01-15

MreB is a bacterial orthologue of actin that affects cell shape, polarity, and chromosome segregation. Although a significant body of work has explored its cellular functions, we know very little about the biochemical behavior of MreB. We have cloned, overexpressed in Escherichia coli, and purified untagged MreB1 from Thermotoga maritima. We have characterized the conditions that regulate its monomer-to-polymer assembly reaction, the critical concentrations of that reaction, the manner in which MreB uses nucleotides, its stability, and the structure of the assembled polymer. MreB requires a bound purine nucleotide for polymerization and rapidly hydrolyzes it following assembly. MreB assembly contains two distinct components, one that does not require divalent cations and one that does, which may comprise the nucleation and elongation phases of assembly, respectively. MreB assembly is strongly favored by increasing temperature or protein concentration but inhibited differentially by high concentrations of monovalent salts. The polymerization rate increases and the bulk critical concentration decreases with increasing temperature, but in contrast to previous reports, MreB is capable of polymerizing across a broad range of temperatures. MreB polymers are shorter and stiffer and scatter more light than eukaryotic actin filaments. Due to rapid ATP hydrolysis and phosphate release, we suggest that most assembled MreB in cells is in the ADP-bound state. Because of only moderate differences between the ATP and ADP critical concentrations, treadmilling may occur, but we do not predict dynamic instability in cells. Because of the relatively low cellular concentration of MreB and the observed structural properties of the polymer, a single MreB assembly may exist in cells.

GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

PubMed

Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

2010-11-19

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

The EMBL nucleotide sequence database

PubMed Central

Stoesser, Guenter; Baker, Wendy; van den Broek, Alexandra; Camon, Evelyn; Garcia-Pastor, Maria; Kanz, Carola; Kulikova, Tamara; Lombard, Vincent; Lopez, Rodrigo; Parkinson, Helen; Redaschi, Nicole; Sterk, Peter; Stoehr, Peter; Tuli, Mary Ann

2001-01-01

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) is maintained at the European Bioinformatics Institute (EBI) in an international collaboration with the DNA Data Bank of Japan (DDBJ) and GenBank at the NCBI (USA). Data is exchanged amongst the collaborating databases on a daily basis. The major contributors to the EMBL database are individual authors and genome project groups. Webin is the preferred web-based submission system for individual submitters, whilst automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via ftp, email and World Wide Web interfaces. EBI’s Sequence Retrieval System (SRS), a network browser for databanks in molecular biology, integrates and links the main nucleotide and protein databases plus many specialized databases. For sequence similarity searching a variety of tools (e.g. Blitz, Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. PMID:11125039

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling.

PubMed

Abbott, Jason R; Hodges, Timothy R; Daniels, R Nathan; Patel, Pratiq A; Kennedy, Jack Phillip; Howes, Jennifer E; Akan, Denis T; Burns, Michael C; Sai, Jiqing; Sobolik, Tammy; Beesetty, Yugandhar; Lee, Taekyu; Rossanese, Olivia W; Phan, Jason; Waterson, Alex G; Fesik, Stephen W

2018-06-01

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at sub-micromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway, resulting in a decrease in pERK1/2 T202/Y204 protein levels at higher compound concentrations.

Obstructive uropathy and severe acute kidney injury from renal calculi due to adenine phosphoribosyltransferase deficiency.

PubMed

Chong, Siew Le; Ng, Yong Hong

2016-05-01

Adenine phosphoribosyltransferase (APRT) deficiency is an uncommon genetic cause of chronic kidney disease due to crystalline nephropathy. A case of a Chinese boy with APRT deficiency presenting with severe acute kidney injury secondary to obstructive uropathy from multiple renal calculi was reviewed. The patient underwent staged removal of the calculi. Infrared spectrometry of the renal calculi showed 2,8-dihydroxyadenine. APRT deficiency was confirmed with abolished APRT enzyme activity in red blood cells. He was started on allopurinol and low purine diet with complete resolution of the residual calculi. APRT deficiency should be considered in patients with multiple radiolucent renal calculi.

Modified formulation of CPDA for storage of whole blood, and of SAGM for storage of red blood cells, to maintain the concentration of 2,3-diphosphoglycerate.

PubMed

Kurup, P A; Arun, P; Gayathri, N S; Dhanya, C R; Indu, A R

2003-11-01

A dramatic decrease in the level of 2,3-diphosphoglycerate (2,3-DPG) takes place during the storage of whole blood (WB) in CPDA (citrate-phosphate-dextrose-adenine) and a similar decrease occurs during the storage of red blood cells (RBCs) in SAGM (saline-adenine-glucose-mannitol). The aim of the present study was to prevent this decrease by modifying CPDA and SAGM. The pH of WB anticoagulant or RBC preservative solution was maintained at 7.6 by autoclaving the dextrose solution separately, by incorporating ascorbic acid and nicotinic acid into both CPDA and SAGM (to produce modified CPDA and SAGM solutions), and by reducing the concentration of adenine and adding citrate to the modified SAGM solution. The concentration of 2,3-DPG in WB after 28 days of storage in modified CPDA, and in RBCs stored in modified SAGM, was compared with that in WB or RBCs stored in unmodified solutions. The initial 2,3-DPG levels were maintained after 28 days in the modified formulations [10.63 +/- 2.58 microM/g of haemoglobin (Hb) in the case of modified CPDA and 12.07 +/- 1.47 microM/g of Hb in the case of modified SAGM], whereas in standard CPDA and SAGM solutions, the concentration of 2,3-DPG decreased to very low levels (0.86 +/- 0.97 microM/g Hb for CPDA and 0.12 +/- 0.008 for SAGM). Our modification in the formulation of CPDA or SAGM is effective in arresting the dramatic decrease in the level of 2,3-DPG that occurs during storage of WB and RBCs in unmodified solutions.

Bacterial nucleotide-based second messengers.

PubMed

Pesavento, Christina; Hengge, Regine

2009-04-01

In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

Polymerization of amino acids containing nucleotide bases

NASA Technical Reports Server (NTRS)

Ben Cheikh, Azzouz; Orgel, Leslie E.

1990-01-01

The nucleoamino acids 1-(3'-amino,3'-carboxypropyl)uracil (3) and 9-(3'-amino,3'-carboxypropyl)adenine (4) have been prepared as (L)-en-antiomers and as racemic mixtures. When 3 or 4 is suspended in water and treated with N,N'-carbon-yldiimidazole, peptides are formed in good yield. The products formed from the (L)-enantiomers are hydrolyzed to the monomeric amino acids by pronase. Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (L)-enantiomer of 3 did not act as templates to facilitate the oligomerization of 4.

Supra-molecular architecture in a co-crystal of the N(7)-H tautomeric form of N (6)-benzoyl-adenine with adipic acid (1/0.5).

PubMed

Swinton Darious, Robert; Thomas Muthiah, Packianathan; Perdih, Franc

2016-06-01

The asymmetric unit of the title co-crystal, C12H9N5O·0.5C6H10O4, consists of one mol-ecule of N (6)-benzoyl-adenine (BA) and one half-mol-ecule of adipic acid (AA), the other half being generated by inversion symmetry. The dihedral angle between the adenine and phenyl ring planes is 26.71 (7)°. The N (6)-benzoyl-adenine mol-ecule crystallizes in the N(7)-H tautomeric form with three non-protonated N atoms. This tautomeric form is stabilized by intra-molecular N-H⋯O hydrogen bonding between the carbonyl (C=O) group and the N(7)-H hydrogen atom on the Hoogsteen face of the purine ring, forming an S(7) ring motif. The two carboxyl groups of adipic acid inter-act with the Watson-Crick face of the BA mol-ecules through O-H⋯N and N-H⋯O hydrogen bonds, generating an R 2 (2)(8) ring motif. The latter units are linked by N-H⋯N hydrogen bonds, forming layers parallel to (10-5). A weak C-H⋯O hydrogen bond is also present, linking adipic acid mol-ecules in neighbouring layers, enclosing R (2) 2(10) ring motifs and forming a three-dimensional structure. C=O⋯π and C-H⋯π inter-actions are also present in the structure.

Resonant two-photon ionization and laser induced fluorescence spectroscopy of jet-cooled adenine

NASA Astrophysics Data System (ADS)

Kim, Nam Joon; Jeong, Gawoon; Kim, Yung Sam; Sung, Jiha; Keun Kim, Seong; Park, Young Dong

2000-12-01

Electronic spectra of the jet-cooled DNA base adenine were obtained by the resonant two-photon ionization (R2PI) and the laser induced fluorescence (LIF) techniques. The 0-0 band to the lowest electronically excited state was found to be located at 35 503 cm-1. Well-resolved vibronic structures were observed up to 1100 cm-1 above the 0-0 level, followed by a slow rise of broad structureless absorption. The lowest electronic state was proposed to be of nπ* character, which lies ˜600 cm-1 below the onset of the ππ* state. The broad absorption was attributed to the extensive vibronic mixing between the nπ* state and the high-lying ππ* state.

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein. Characterization and regulation by uridine and guanosine nucleotides

PubMed Central

Jørgensen, Casper Møller; Fields, Christopher J.; Chander, Preethi; Watt, Desmond; Burgner, John W.; Smith, Janet L.; Switzer, Robert L.

2011-01-01

Summary The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5’ leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. Apparent dissociation constants at 0° C ranged from 0.13 to 0.87 nM in the absence of effectors; dissociation constants were decreased by 3 to 12 fold by uridine nucleotides and increased by 40 to 200 fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. PMID:18190533

Identification of protein-interacting nucleotides in a RNA sequence using composition profile of tri-nucleotides.

PubMed

Panwar, Bharat; Raghava, Gajendra P S

2015-04-01

The RNA-protein interactions play a diverse role in the cells, thus identification of RNA-protein interface is essential for the biologist to understand their function. In the past, several methods have been developed for predicting RNA interacting residues in proteins, but limited efforts have been made for the identification of protein-interacting nucleotides in RNAs. In order to discriminate protein-interacting and non-interacting nucleotides, we used various classifiers (NaiveBayes, NaiveBayesMultinomial, BayesNet, ComplementNaiveBayes, MultilayerPerceptron, J48, SMO, RandomForest, SMO and SVM(light)) for prediction model development using various features and achieved highest 83.92% sensitivity, 84.82 specificity, 84.62% accuracy and 0.62 Matthew's correlation coefficient by SVM(light) based models. We observed that certain tri-nucleotides like ACA, ACC, AGA, CAC, CCA, GAG, UGA, and UUU preferred in protein-interaction. All the models have been developed using a non-redundant dataset and are evaluated using five-fold cross validation technique. A web-server called RNApin has been developed for the scientific community (http://crdd.osdd.net/raghava/rnapin/). Copyright © 2015 Elsevier Inc. All rights reserved.

Surface-Enhanced Hyper-Raman Spectra of Adenine, Guanine, Cytosine, Thymine, and Uracil

PubMed Central

2016-01-01

Using picosecond excitation at 1064 nm, surface-enhanced hyper-Raman scattering (SEHRS) spectra of the nucleobases adenine, guanine, cytosine, thymine, and uracil with two different types of silver nanoparticles were obtained. Comparing the SEHRS spectra with SERS data from the identical samples excited at 532 nm and with known infrared spectra, the major bands in the spectra are assigned. Due to the different selection rules for the one- and two-photon excited Raman scattering, we observe strong variation in relative signal strengths of many molecular vibrations obtained in SEHRS and SERS spectra. The two-photon excited spectra of the nucleobases are found to be very sensitive with respect to molecule–nanoparticle interactions. Using both the SEHRS and SERS data, a comprehensive vibrational characterization of the interaction of nucleobases with silver nanostructures can be achieved. PMID:28077982

DNA bases thymine and adenine in bio-organic light emitting diodes.

PubMed

Gomez, Eliot F; Venkatraman, Vishak; Grote, James G; Steckl, Andrew J

2014-11-24

We report on the use of nucleic acid bases (NBs) in organic light emitting diodes (OLEDs). NBs are small molecules that are the basic building blocks of the larger DNA polymer. NBs readily thermally evaporate and integrate well into the vacuum deposited OLED fabrication. Adenine (A) and thymine (T) were deposited as electron-blocking/hole-transport layers (EBL/HTL) that resulted in increases in performance over the reference OLED containing the standard EBL material NPB. A-based OLEDs reached a peak current efficiency and luminance performance of 48 cd/A and 93,000 cd/m(2), respectively, while T-based OLEDs had a maximum of 76 cd/A and 132,000 cd/m(2). By comparison, the reference OLED yielded 37 cd/A and 113,000 cd/m(2). The enhanced performance of T-based devices is attributed to a combination of energy levels and structured surface morphology that causes more efficient and controlled hole current transport to the emitting layer.

Nuclear Overhauser effect studies on the conformation of magnesium adenosine 5'-triphosphate bound to rabbit muscle creatine kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosevear, P.R.; Powers, V.M.; Dowhan, D.

1987-08-25

Nuclear Overhauser effects were used to determine interproton distances on MgATP bound to rabbit muscle creatine kinase. The internuclear distances were used in a distance geometry program that objectively determines both the conformation of the bound MgATP and its uniqueness. Two classes of structures were found that satisfied the measured interproton distances. Both classes had the same anti glycosidic torsional angle (X = 78 +/- 10/sup 0/) but differed in their ribose ring puckers (O1'-endo or C4'-exo). The uniqueness of the glycosidic torsional angle is consistent with the preference of creatine kinase for adenine nucleotides. One of these conformations ofmore » MgATP bound to creatine kinase is indistinguishable from the conformation found for Co(NH/sub 3/)/sub 4/ ATP bound to the catalytic subunit of protein kinase, which also has a high specificity for adenine nucleotides. Distance geometry calculations also suggest that upper limit distances, when low enough (less than or equal to 3.4 A), can be used instead of measured distances to define, within experimental error, the glycosidic torsional angle of bound nucleotides. However, this approach does not permit an evaluation of the ribose ring pucker.« less

Blood constituents as phagostimulants for the bed bug Cimex lectularius L.

PubMed

Romero, Alvaro; Schal, Coby

2014-02-15

Many hematophagous arthropods are stimulated by blood constituents to initiate feeding. We used a membrane-based feeding system to identify chemicals that stimulate acceptance and engorgement responses in various life stages of bed bugs. Water was fortified with a variety of compounds (e.g. salts, amino acids, vitamins, nucleotides, cholesterol and fatty acids) in these bioassays. ATP was the most effective phagostimulant in adults and nymphs, resulting in >70% of bed bugs fully engorging. Addition of NaCl to low ATP solutions that alone elicited <50% engorgement significantly enhanced feeding responses of bed bugs. A comparison of feeding responses with solutions of various adenine nucleotides showed that ATP was more stimulatory than ADP, which was more effective than AMP. Feeding assays with physiological levels of other blood constituents such as d-glucose, albumin, globulin, cholesterol and mixtures of vitamins and amino acids did not stimulate engorgement, suggesting that adenine nucleotides are the most important feeding stimulants in bed bugs. Identification of phagostimulants for bed bugs will contribute towards the development of artificial diets for rearing purposes, as well as for the development of alternative methods to eliminate bed bug infestations.

Quantum-chemical studies on the favored and rare tautomers of neutral and redox adenine.

PubMed

Raczyńska, Ewa D; Makowski, Mariusz; Zientara-Rytter, Katarzyna; Kolczyńska, Katarzyna; Stępniewski, Tomasz M; Hallmann, Małgorzata

2013-02-21

All possible twenty-three prototropic tautomers of neutral and redox adenine (nine amine and fourteen imine forms, including geometric isomerism of the exo ═NH group) were examined in vacuo {DFT(B3LYP)/6-311+G(d,p)}. The NH → NH conversions as well as those usually omitted, NH → CH and CH → CH, were considered. An interesting change of the tautomeric preference occurs when proceeding from neutral to reduced adenine. One-electron reduction favors the nonaromatic amine C8H-N10H tautomer. This tautomeric preference is similar to that (C2H) for reduced imidazole. Water molecules (PCM model) seem to not change this trend. They influence solely the relative energies. The DFT vertical detachment energy in the gas phase is positive for each tautomer, e.g., 0.03 eV for N9H-N10H and 1.84 eV for C8H-N10H. The DFT adiabatic electron affinity for the favored process, neutral N9H-N10H → reduced C8H-N10H (ground states), is equal to 0.18 eV at 0 K (ZPE included). One-electron oxidation does not change the tautomeric preference in the gas phase. The aromatic amine N9H-N10H tautomer is favored for the oxidized molecule similarly as for the neutral one. The DFT adiabatic ionization potential for the favored process, neutral N9H-N10H → oxidized N9H-N10H (ground states), is equal to 8.12 eV at 0 K (ZPE included). Water molecules (PCM model) seem to influence solely the composition of the tautomeric mixture and the relative energies. They change the energies of the oxidation and reduction processes by ca. 2 eV.

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

PubMed

Fujita, Akiko; Sato, Chihiro; Münster-Kühnel, Anja-K; Gerardy-Schahn, Rita; Kitajima, Ken

2005-02-01

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay

Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.

PubMed

Nakajima, Kazuki; Kitazume, Shinobu; Angata, Takashi; Fujinawa, Reiko; Ohtsubo, Kazuaki; Miyoshi, Eiji; Taniguchi, Naoyuki

2010-07-01

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.

Cyclic nucleotide binding proteins in the Arabidopsis thaliana and Oryza sativa genomes

PubMed Central

Bridges, Dave; Fraser, Marie E; Moorhead, Greg BG

2005-01-01

Background Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules. Results Our analysis found that several ion channels and a class of thioesterases constitute the possible cyclic nucleotide binding proteins in plants. Contrary to some reports, we found no biochemical or bioinformatic evidence for a plant cyclic nucleotide regulated protein kinase, suggesting that cyclic nucleotide functions in plants have evolved differently than in mammals. Conclusion This paper provides a molecular framework for the discussion of cyclic nucleotide function in plants, and resolves a longstanding debate about the presence of a cyclic nucleotide dependent kinase in plants. PMID:15644130

Enzymatic Incorporation of Modified Purine Nucleotides in DNA.

PubMed

Abu El Asrar, Rania; Margamuljana, Lia; Abramov, Mikhail; Bande, Omprakash; Agnello, Stefano; Jang, Miyeon; Herdewijn, Piet

2017-12-14

A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with 5-methylisocytosine were tested as potential pairing partners of N 8 -glycosylated nucleotides with an 8-azaguanine or 8-aza-9-deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5-methylisocytosine nucleotide followed by the 1-methyl-8-aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8-aza-9-deazaguanine represents a self-complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

PubMed

Kandeel, Mahmoud; Kitade, Yukio

2018-02-01

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

Adenine phosphoribosyltransferase deficiency in the United Kingdom: two novel mutations and a cross-sectional survey

PubMed Central

Arenas-Hernandez, Monica; Escuredo, Emilia; Fairbanks, Lynette; Marinaki, Tony; Mapplebeck, Sarah; Sheaff, Michael; Almond, Michael K.

2016-01-01

Background Adenine phosphoribosyltransferase deficiency is an inborn error of metabolism that can cause kidney disease from crystalline nephropathy or kidney stones. Methods We present three cases from a single centre with varied presentations to illustrate how increasing awareness led to better patient identification. We then undertook a cross-sectional survey of all the patients identified from the Purine Research Laboratory in the UK since 1974. Results Our index case presented with recurrent nephrolithiasis and was diagnosed on stone analysis, the second case presented with acute kidney injury and the third case was identified from a biopsy undertaken for acute on chronic kidney injury. Genetic studies identified two novel mutations. Twenty patients were retrospectively identified. The mean age at diagnosis was 25 years (range 2–70); eight were <20 years, seven were 20–40 years and five were >40 years. Five of the 20 patients were deceased, 3 after end-stage renal disease (ESRD). Twelve have normal renal function, one had CKD stage 3, one had severe kidney disease and one was on dialysis. Conclusions Adenine phosphoribosyltransferase deficiency presents in a wide spectrum in all age groups. Patients can be completely asymptomatic and kidney disease may be incorrectly attributed to other conditions. Outcome is poor in late diagnosis and there is a high prevalence of ESRD. Patients with unexplained renal stone disease or deterioration in kidney function should be considered for screening. Identification and surveillance of patients in the UK can improve. There is now a rare disease registry with meetings organized that include patients, families and health care providers to improve awareness. PMID:27994857

Diadenosine polyphosphate-stimulated gluconeogenesis in isolated rat proximal tubules.

PubMed Central

Edgecombe, M; Craddock, H S; Smith, D C; McLennan, A G; Fisher, M J

1997-01-01

Diadenosine polyphosphates released into the extracellular environment influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the impact of these novel nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis was stimulated following exposure of isolated proximal tubules to a range of adenine-containing nucleotides including ADP, ATP, Ap3A, Ap4A, Ap5A and Ap6A. The concentration-dependence of ATP-, Ap3A- and Ap4A-mediated stimulation of gluconeogenesis was similar and was consistent with a role for these agents in the physiological control of renal metabolism. Nucleotide-stimulated gluconeogenesis was diminished in the presence of agents that interfere with phospholipase C activation or intracellular Ca2+ metabolism, indicative of a role for polyphosphoinositide-mediated Ca2+ mobilization in the mechanism of action of ATP, Ap3A and Ap4A. The characteristics of binding of [2-3H]Ap4A to renal plasma-membrane preparations suggest that Ap4A mediates its effects on proximal tubule gluconeogenesis via interaction with P2y-like purinoceptor(s) also recognized by extracellular ATP. PMID:9163337

A novel MALDI–TOF based methodology for genotyping single nucleotide polymorphisms

PubMed Central

Blondal, Thorarinn; Waage, Benedikt G.; Smarason, Sigurdur V.; Jonsson, Frosti; Fjalldal, Sigridur B.; Stefansson, Kari; Gulcher, Jeffery; Smith, Albert V.

2003-01-01

A new MALDI–TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3′-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis. PMID:14654708

Effect of Microcystin-LR on Cultured Rat Endothelial Cells

DTIC Science & Technology

1989-01-10

changes, as indicated bv the release of (14C]adenine nucleotides, and a small reduction of cell devsity in entlothelial cell monolayers. Silymarin at...oxidants, dithioerythritol (DTE) (CLELAND, 1964), or silymarin (SM) (FRAGA et al., 1987), in order to determine if these agents could prevent changes

Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

PubMed

Ido, Yasuo

2016-07-01

Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. © 2016 The Author. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Acceleration of adventitious shoots by interaction between exogenous hormone and adenine sulphate in Althaea officinalis L.

PubMed

Naz, Ruphi; Anis, M

2012-11-01

In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog's (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2 ± 0.58 buds in central vein explants. Addition of different growth regulators (cytokinins-6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins-indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4 ± 0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.

Manganese(II) Chloride Alters Nucleotide and Nucleoside Catabolism in Zebrafish (Danio rerio) Adult Brain.

PubMed

Altenhofen, Stefani; Nabinger, Débora Dreher; Pereira, Talita Carneiro Brandão; Leite, Carlos Eduardo; Bogo, Maurício Reis; Bonan, Carla Denise

2018-05-01

ATP and adenosine, the main signaling molecules of purinergic system, are involved in toxicological effects induced by metals. The manganese (Mn) exposure induces several cellular changes, which could interfere with signaling pathways, such as the purinergic system. In this study, we evaluated the effects of exposure to manganese(II) chloride (MnCl 2 ) during 96 h on nucleoside triphosphate diphosphohydrolase (NTPDase), ecto-5'-nucleotidase, and adenosine deaminase (ADA) activities, followed by analyzing the gene expression patterns of NTPDases (entpd1, entpd2a.1, entpd2a.2, entpd2-like, entpd3) and ADA (ADA 1 , ADA 2.1 , ADA 2.2 , ADAasi, ADAL) families in zebrafish brain. In addition, the brain metabolism of nucleotides and nucleosides was evaluated after MnCl 2 exposure. The results showed that MnCl 2 exposure during 96 h inhibited the NTPDase (1.0 and 1.5 mM) and ecto-ADA (0.5, 1.0, and 1.5 mM) activities, further decreasing ADA2.1 expression at all MnCl 2 concentrations analyzed. Purine metabolism was also altered by the action of MnCl 2 . An increased amount of ADP appeared at all MnCl 2 concentrations analyzed; however, AMP and adenosine levels are decreased at the concentrations of 1.0 and 1.5 mM MnCl 2 , whereas decreased inosine (INO) levels were observed at all concentrations tested. The findings of this study demonstrated that MnCl 2 may inhibit NTPDase and ecto-ADA activities, consequently modulating nucleotide and nucleoside levels, which may contribute for the toxicological effects induced by this metal.

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

PubMed

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

2015-04-14

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase*

PubMed Central

Jin, Zhinan; Johnson, Kenneth A.

2011-01-01

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches. PMID:20978284

Acid-soluble nucleotides of pinto bean leaves at different stages of development.

PubMed

Weinstein, L H; McCune, D C; Mancini, J F; van Leuken, P

1969-11-01

Acid-soluble nucleotides of unifoliate leaves of Pinto bean plants (Phaseolus vulgaris L.) were determined at young, mature, and senescent stages of development. At least 25 components could be distinguished on the basis of inorganic phosphorus determinations and 37 or more fractions on the basis of (32)P labeling, with adenosine di- and triphosphates accounting for 60% of the total moles of nucleotide. The total nucleotide P and inorganic P, on a fresh weight basis, decreased about 44% between each stage of leaf development, but decrements in the levels of individual nucleotides varied from this over-all pattern.Minor changes in the relative abundance of the individual nucleotides accompanied aging although the percentage of purine-containing nucleotides decreased with age. Total (32)P activity per leaf in the nucleotide pool increased about 3-fold between the young and mature leaves and decreased slightly as leaves became senescent. In general, the specific activities of the nucleotides increased with increased age and adenosine-, guanosine-, uridine-, and cytidine triphosphates and adenosine diphosphate accounted for approximately 90% of the total activity. The changes in the relative sizes and energy status of the nucleotide pools were not so obvious as the changes in other metabolites that have been reported to accompany aging in leaf tissue.

Blood vitamin concentrations in privately owned dogs fed non-standardized commercial diets and after intake of diets with specified vitamin concentrations.

PubMed

Tran, J L; Horvath, C; Krammer, S; Höller, U; Zentek, J

2007-02-01

The objective was to investigate in a survey study the blood vitamin concentrations in healthy dogs fed non-specified commercial complete diets and in an intervention study to determine the effects of defined dietary vitamin intakes on blood vitamin levels and hair and skin condition. Sixty-four privately owned dogs, aged from 1 to 8 years, without history of skin or coat problems were included. All animals were fed commercial complete diets with uncertain vitamin concentrations before enrolment. The animals were assigned, according to weight and gender, to four groups with graded vitamin intakes. The blood vitamin levels and skin and coat quality of the dogs were investigated at days 0 and day 122. Coat and hair condition was not influenced by the experimental diets. The retinol concentrations were reduced at the end of the experiment compared with the baseline levels, retinyl esters were not influenced. 25-Hydroxycholecalciferol decreased in all groups, alpha-tocopherol was constant or tended to decrease. Ascorbic acid, thiamine pyrophosphate and riboflavin concentrations were not affected by treatment, flavin adenine dinucleotide and pyridoxal-5'-phosphate were partially reduced on day 122. Cobalamin, pantothenate and biotin concentrations increased with higher dietary intakes, folate levels in tendency. In conclusion, this study gives a survey of blood vitamin concentrations in healthy dogs and provides a data base for the evaluation of the vitamin status in health and disease.

Analysis of cytokinin nucleotides in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry after solid-phase extraction.

PubMed

Ge, Liya; Yong, Jean Wan Hong; Tan, Swee Ngin; Yang, Xin Hao; Ong, Eng Shi

2006-11-10

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 microM for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5'-monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.

Kidney Disease in Adenine Phosphoribosyltransferase Deficiency.

PubMed

Runolfsdottir, Hrafnhildur Linnet; Palsson, Runolfur; Agustsdottir, Inger M; Indridason, Olafur S; Edvardsson, Vidar O

2016-03-01

Adenine phosphoribosyltransferase (APRT) deficiency is a purine metabolism disorder causing kidney stones and chronic kidney disease (CKD). The course of nephrolithiasis and CKD has not been well characterized. The objective of this study was to examine long-term kidney outcomes in patients with APRT deficiency. An observational cohort study. All patients enrolled in the APRT Deficiency Registry of the Rare Kidney Stone Consortium. Kidney stones, acute kidney injury (AKI), stage of CKD, end-stage renal disease, estimated glomerular filtration rate (eGFR), and changes in eGFR. Serum creatinine and eGFR calculated using creatinine-based equations. Of 53 patients, 30 (57%) were females and median age at diagnosis was 37.0 (range, 0.6-67.9) years. Median duration of follow-up was 10.3 (range, 0.0-31.5) years. At diagnosis, kidney stones had developed in 29 (55%) patients and 20 (38%) had CKD stages 3 to 5, including 11 (21%) patients with stage 5. At latest follow-up, 33 (62%) patients had experienced kidney stones; 18 (34%), AKI; and 22 (42%), CKD stages 3 to 5. Of 14 (26%) patients with stage 5 CKD, 12 had initiated renal replacement therapy. Kidney stones recurred in 18 of 33 (55%) patients. The median eGFR slope was -0.38 (range, -21.99 to 1.42) mL/min/1.73m(2) per year in patients receiving treatment with an xanthine dehydrogenase inhibitor and -5.74 (range, -75.8 to -0.10) mL/min/1.73m(2) per year in those not treated prior to the development of stage 5 CKD (P=0.001). Use of observational registry data. Progressive CKD and AKI episodes are major features of APRT deficiency, whereas nephrolithiasis is the most common presentation. Advanced CKD without a history of kidney stones is more prevalent than previously reported. Our data suggest that timely therapy may retard CKD progression. Copyright © 2016 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Modification by protons of frog skeletal muscle KATP channels: effects on ion conduction and nucleotide inhibition.

PubMed Central

Vivaudou, M; Forestier, C

1995-01-01

1. The molecular mechanisms underlying pH regulation of skeletal muscle ATP-sensitive K+ (KATP) channels were studied using the patch clamp technique in the inside-out configuration. Two effects of intracellular protons were studied in detail: the decrease in magnitude of single-channel currents and the increase in open probability (Po) of nucleotide-inhibited channels. 2. The pH dependence of inward unit currents under different ionic conditions was in poor agreement with either a direct block of the pore by protons or an indirect proton-induced conformational change, but was compatible with the protonation of surface charges located near the cytoplasmic entrance of the pore. This latter electrostatic mechanism was modelled using Gouy-Chapman-Stern theory, which predicted the data accurately with a surface charge density of about 0.1 negative elementary charges per square nanometre and a pK (pH value for 50% effect) value for protonation of these charges of 6.25. The same mechanism, i.e. neutralization of negative surface charges by cation binding, could also account for the previously reported reduction of inward unit currents by Mg2+. 3. Intracellular alkalization did not affect Po of the KATP channels. Acidification increased Po. In the presence of 0.1 mM ATP (no Mg2+), the channel activation vs. pH relationship could be fitted with a sigmoid curve with a Hill coefficient slightly above 2 and a pK value of 6. This latter value was dependent on the ATP concentration, decreasing from 6.3 in 30 microM ATP to 5.3 in 1 microM ATP. 4. Conversely, the channel inhibition vs. ATP concentration curve was shifted to the right when the pH was lowered. At pH 7.1, the ATP concentration causing half-maximal inhibition was about 10 microM. At pH 5.4, it was about 400 microM. The Hill coefficient values remained slightly below 2. Similar effects were observed when ADP was used as the inhibitory nucleotide. 5. These results confirm that a reciprocal competitive link exists

In female rat heart mitochondria, oophorectomy results in loss of oxidative phosphorylation.

PubMed

Pavón, Natalia; Cabrera-Orefice, Alfredo; Gallardo-Pérez, Juan Carlos; Uribe-Alvarez, Cristina; Rivero-Segura, Nadia A; Vazquez-Martínez, Edgar Ricardo; Cerbón, Marco; Martínez-Abundis, Eduardo; Torres-Narvaez, Juan Carlos; Martínez-Memije, Raúl; Roldán-Gómez, Francisco-Javier; Uribe-Carvajal, Salvador

2017-02-01

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca 2+ buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn 2+ superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction. © 2017 Society for Endocrinology.

Reduced ischemia-reperfusion injury with isoproterenol in non-heart-beating donor lungs.

PubMed

Jones, D R; Hoffmann, S C; Sellars, M; Egan, T M

1997-05-01

Transplantation of lungs retrieved from non-heart-beating donors could expand the donor pool. Recent studies suggest that the ischemia-reperfusion injury (IRI) to the lung can be attenuated by increasing intracellular cAMP concentrations. The purpose of this study was to determine the effect of IRI on capillary permeability, as measured by Kfc, in lungs retrieved from non-heart-beating donors and reperfused with or without isoproterenol (iso). Using an in situ isolated perfused lung model, lungs were retrieved from non-heart-beating donor rats ventilated with O2 or not at varying intervals after death. The lungs were reperfused with or without iso (10 microM). Kfc, lung viability, and pulmonary hemodynamics were measured, and tissue levels of adenine nucleotides and cAMP were measured by HPLC. Iso-reperfusion decreased Kfc significantly (P < 0.05) compared to non-iso-reperfused groups at all postmortem ischemic times, irrespective of preharvest ventilation status. Pulmonary arterial pressures and resistances increased and venous resistances decreased with iso-reperfusion. Total adenine nucleotide (TAN) levels correlated with Kfc in non-iso-reperfused (r = 0.65) and iso-perfused (r = 0.84) lungs. cAMP levels increased significantly with iso-reperfusion. cAMP levels correlated with Kfc (r = 0.87) in iso-reperfused lungs. Iso-reperfusion of lungs retrieved from non-heart-beating donor rats results in decreased capillary permeability and increased lung tissue cAMP levels. Pharmacologic augmentation of tissue TAN and cAMP levels may further ameliorate the increased capillary permeability seen in lungs retrieved from non-heart-beating donors.

High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling

PubMed Central

Till, Bradley J.; Zerr, Troy; Bowers, Elisabeth; Greene, Elizabeth A.; Comai, Luca; Henikoff, Steven

2006-01-01

Human individuals differ from one another at only ∼0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease. PMID:16893952

Formation of amino acids and nucleotide bases in a Titan atmosphere simulation experiment.

PubMed

Hörst, S M; Yelle, R V; Buch, A; Carrasco, N; Cernogora, G; Dutuit, O; Quirico, E; Sciamma-O'Brien, E; Smith, M A; Somogyi, A; Szopa, C; Thissen, R; Vuitton, V

2012-09-01

The discovery of large (>100 u) molecules in Titan's upper atmosphere has heightened astrobiological interest in this unique satellite. In particular, complex organic aerosols produced in atmospheres containing C, N, O, and H, like that of Titan, could be a source of prebiotic molecules. In this work, aerosols produced in a Titan atmosphere simulation experiment with enhanced CO (N(2)/CH(4)/CO gas mixtures of 96.2%/2.0%/1.8% and 93.2%/5.0%/1.8%) were found to contain 18 molecules with molecular formulae that correspond to biological amino acids and nucleotide bases. Very high-resolution mass spectrometry of isotopically labeled samples confirmed that C(4)H(5)N(3)O, C(4)H(4)N(2)O(2), C(5)H(6)N(2)O(2), C(5)H(5)N(5), and C(6)H(9)N(3)O(2) are produced by chemistry in the simulation chamber. Gas chromatography-mass spectrometry (GC-MS) analyses of the non-isotopic samples confirmed the presence of cytosine (C(4)H(5)N(3)O), uracil (C(5)H(4)N(2)O(2)), thymine (C(5)H(6)N(2)O(2)), guanine (C(5)H(5)N(5)O), glycine (C(2)H(5)NO(2)), and alanine (C(3)H(7)NO(2)). Adenine (C(5)H(5)N(5)) was detected by GC-MS in isotopically labeled samples. The remaining prebiotic molecules were detected in unlabeled samples only and may have been affected by contamination in the chamber. These results demonstrate that prebiotic molecules can be formed by the high-energy chemistry similar to that which occurs in planetary upper atmospheres and therefore identifies a new source of prebiotic material, potentially increasing the range of planets where life could begin.

Formation Of Amino Acids And Nucleotide Bases In A Titan Atmosphere Simulation Experiment

NASA Astrophysics Data System (ADS)

Horst, Sarah; Yelle, R. V.; Buch, A.; Carrasco, N.; Cernogora, G.; Dutuit, O.; Quirico, E.; Sciamma-O'Brien, E.; Smith, M. A.; Somogyi, A.; Szopa, C.; Thissen, R.; Vuitton, V.

2010-10-01

Titan has been a subject of astrobiological interest since the Voyager spacecraft first revealed the diversity of the organic chemistry occurring in the atmosphere. However, it was not until the arrival of Cassini-Huygens that the chemical complexity of Titan's atmosphere was fully appreciated. The Cassini Plasma Spectrometer (CAPS) observed negative ions with m/z values up to 10,000 u/q at 950 km [1] and positive ions with m/z up to 400 u/q [2]. CAPS has also observed O+ flowing into Titan's atmosphere [3]. While Titan's atmosphere is relatively oxygen poor compared to terrestrial planets, CO is the fourth most abundant molecule in the atmosphere (˜50 ppm). The fact that the observed O+ flux is deposited in the region now known to contain large organic molecules leads to the exciting possibility that oxygen can be incorporated into these molecules resulting in the production of prebiotic molecules. In this work, Titan aerosol analogues (or "tholins") produced in PAMPRE, a Titan atmosphere simulation experiment, have been analyzed in a very high resolution LTQ Orbitrap mass spectrometer. These PAMPRE tholins were produced by capacitively coupled RF discharge in a mixture of N2, CH4 and CO. The tholins were found to contain 18 molecules with molecular formulae corresponding to biological amino acids and nucleotide bases. GC-MS measurements have confirmed the structure of seven: adenine, cytosine, uracil, thymine, guanine, glycine and alanine. The production of prebiotic molecules under atmospheric conditions presents a new source of prebiotic material and may increase the range of planets where life could begin. [1] Coates AJ, et al. (2007). Geophys. Res. Lett. 34:22103- +. [2] Crary FJ, et al. (2009). Planet. Space Sci. 57:1847- 1856. [3] Hartle RE, et al. (2006). Geophys. Res. Lett. 33:8201-+.

Reducing nontemplated 3' nucleotide addition to polynucleotide transcripts

DOEpatents

Kao, C. Cheng

2000-01-01

Non-template 3' nucleotide addition to a transcript is reduced by transcribing a transcript from a template comprising an ultimate and/or penultimate 5' ribose having a C'2 substituent such as methoxy, which reduces non-template 3' nucleotide addition to the transcript. The methods are shown to be applicable to a wide variety of polymerases, including Taq, T7 RNA polymerase, etc.

Profile of nucleosides and nucleotides in donkey's milk.

PubMed

Vincenzetti, Silvia; Pucciarelli, Stefania; Nucci, Chiara; Polzonetti, Valeria; Cammertoni, Natalina; Polidori, Paolo

2014-01-01

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkey's milk may be considered a good replacer for cow's milk in feeding children with severe Ig-E mediated cow's milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.

Compositions and methods for detecting single nucleotide polymorphisms

DOEpatents

Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

2016-11-22

Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

Information Entropy of Influenza A Segment 7

NASA Astrophysics Data System (ADS)

Thompson, William A.; Fan, Shaohua; Weltman, Joel K.

2008-12-01

Information entropy (H) is a measure of uncertainty at each position within in a sequence of nucleotides.H was used to characterize a set of influenza A segment 7 nucleotide sequences. Nucleotide locations of high entropy were identified near the 5’ start of all of the sequences and the sequences were assigned to subsets according to synonymous nucleotide variants at those positions: either uracil at position six (U6), cytosine at position six (C6), adenine (A12) at position 12, guanine at position 12 (G12), adenine at position 15 (A15) or cytosine (C15) at position 15. H values were found to be correlated/corresponding (Kendall tau) along the lengths of the nucleotide segments of the subset pairs at each position. However, the H values of each subset of sequences were statistically distinguishable from those of the other member of the pair (Kolmogorov-Smirnov test). The joint probability of uncorrelated distributions of U6 and C6 sequences to viral subtypes and to viral host species was 34 times greater than for the A12:G12 subset pair and 214 times greater than for the A15:C15 pair. This result indicates that the high entropy position six of segment 7 is either a reporter or a sentinel location. The fact that not one of the H5N1 sequences in the dataset was a member of the C6 subset, but all 125 H5N1 sequences are members of the U6 subset suggests a non-random sentinel function.

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals.

PubMed

El Houmami, Nawal; Seligmann, Hervé

2017-01-01

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

Relationships between laser powers and photoacoustic signal intensities of flavin adenine dinucleotide and beta-carotene dissolved in solutions

NASA Astrophysics Data System (ADS)

Imakubo, Keiichi

1994-10-01

Ar ion laser-induced photoacoustic spectroscopy has been performed on 0.01 mu M flavin adenine dinucleotide in H2O and 0.01 mu M beta-carotene in n-hexane where the optical absorption spectroscopy is not applicable. On the basis of the linear relationships between laser powers and photoacoustic signal intensities up to 500 mW, it may be concluded that laser power ranging from 10 to 50 mW is required for the successful observation of photoacoustic signals without any photochemical or photobiological effects.

Cytokinin nucleotides contents in sexual buds of Douglas-fir

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imbault, N.; Doumas, P.; Bonnet-Masimbert, N.

1989-04-01

Cytokinin nucleotides were extracted from male and female buds of Pseudotsuga menxiesii by 10 % perchloric acid. They were prepurified on cation exchanger columns (CBA, Amersham) and then separated by two HPLC systems. The first one (Partisil 10 SAX, 10{mu}m, Wathman) separates the mono-, di- and tri-phosphates groups which were collected. The second one (Ultraspher, 5 {mu}m, Beckman) separates the cytokinin nucleotides inside each group. After separation, cytokinin nucleotides were assayed by radioimmunoassay with anti ribosyl zeatin (RZ) and anti isopentenyladenosine (iPA) antibodies. The analysis showed in the monophosphate (mono-P) group one immunoreactant peak in RZ fraction which co-chromatographied withmore » RZ-5{prime}-mono-P and two peaks in the iPA fraction. One of them co-chromatographied with iPA-5{prime}-mono-P. In the diphosphate group, there were three peaks which reacted with anti RZ antibodies and one with anti iPA antibodies. The nucleotides obtained after the first HPLC system, were hydrolysed by a 5{prime}-nucleotidase showed compounds co-chromatographing with RZ and iPA. We did not observe any qualitative differences between the male and female buds. This is the first evidence of cytokinin nucleotides in tissue from woody plants.« less

Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

PubMed Central

Cohen, G. N.; Stanier, R. Y.; Bras, Gisele Le

1969-01-01

The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed. PMID:4391829

The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

PubMed Central

Root, D. D.; Reisler, E.

1992-01-01

Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304380

Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

PubMed

Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

2007-12-27

A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells.

Nucleotide excision repair by dual incisions in plants.

PubMed

Canturk, Fazile; Karaman, Muhammet; Selby, Christopher P; Kemp, Michael G; Kulaksiz-Erkmen, Gulnihal; Hu, Jinchuan; Li, Wentao; Lindsey-Boltz, Laura A; Sancar, Aziz

2016-04-26

Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct.

VarDetect: a nucleotide sequence variation exploratory tool

PubMed Central

Ngamphiw, Chumpol; Kulawonganunchai, Supasak; Assawamakin, Anunchai; Jenwitheesuk, Ekachai; Tongsima, Sissades

2008-01-01

Background Single nucleotide polymorphisms (SNPs) are the most commonly studied units of genetic variation. The discovery of such variation may help to identify causative gene mutations in monogenic diseases and SNPs associated with predisposing genes in complex diseases. Accurate detection of SNPs requires software that can correctly interpret chromatogram signals to nucleotides. Results We present VarDetect, a stand-alone nucleotide variation exploratory tool that automatically detects nucleotide variation from fluorescence based chromatogram traces. Accurate SNP base-calling is achieved using pre-calculated peak content ratios, and is enhanced by rules which account for common sequence reading artifacts. The proposed software tool is benchmarked against four other well-known SNP discovery software tools (PolyPhred, novoSNP, Genalys and Mutation Surveyor) using fluorescence based chromatograms from 15 human genes. These chromatograms were obtained from sequencing 16 two-pooled DNA samples; a total of 32 individual DNA samples. In this comparison of automatic SNP detection tools, VarDetect achieved the highest detection efficiency. Availability VarDetect is compatible with most major operating systems such as Microsoft Windows, Linux, and Mac OSX. The current version of VarDetect is freely available at . PMID:19091032

DNA Nucleotides Detection via capacitance properties of Graphene

NASA Astrophysics Data System (ADS)

Khadempar, Nahid; Berahman, Masoud; Yazdanpanah, Arash

2016-05-01

In the present paper a new method is suggested to detect the DNA nucleotides on a first-principles calculation of the electronic features of DNA bases which chemisorbed to a graphene sheet placed between two gold electrodes in a contact-channel-contact system. The capacitance properties of graphene in the channel are surveyed using non-equilibrium Green's function coupled with the Density Functional Theory. Thus, the capacitance properties of graphene are theoretically investigated in a biological environment, and, using a novel method, the effect of the chemisorbed DNA nucleotides on electrical charges on the surface of graphene is deciphered. Several parameters in this method are also extracted including Electrostatic energy, Induced density, induced electrostatic potential, Electron difference potential and Electron difference density. The qualitative and quantitative differences among these parameters can be used to identify DNA nucleotides. Some of the advantages of this approach include its ease and high accuracy. What distinguishes the current research is that it is the first experiment to investigate the capacitance properties of gaphene changes in the biological environment and the effect of chemisorbed DNA nucleotides on the surface of graphene on the charge.

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

PubMed Central

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-01-01

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes. PMID:28341698

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats.

PubMed

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-05-05

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade + reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2 FEN1 Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe , but contributes to DNA repeat stability in MMR-independent processes. Copyright © 2017 Villahermosa et al.

Strong coupling between adenine nucleobases in DNA single strands revealed by circular dichroism using synchrotron radiation

NASA Astrophysics Data System (ADS)

Kadhane, Umesh; Holm, Anne I. S.; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

2008-02-01

Circular dichroism (CD) experiments on DNA single strands (dAn) at the ASTRID synchrotron radiation facility reveal that eight adenine (A) bases electronically couple upon 190nm excitation. After n=8 , the CD signal increases linearly with n with a slope equal to the sum of the coupling terms. Nearest neighbor interactions account for only 24% of the CD signal whereas electronic communication is limited to nearest neighbors for two other exciton bands observed at 218 and 251nm (i.e., dimer excited states). Electronic coupling between bases in DNA is important for nonradiative deexcitation of electronically excited states since the hazardous energy is spread over a larger spatial region.

Degradation of Adenine on the Martian Surface in the Presence of Perchlorates and Ionizing Radiation: A Reflectron Time-of-flight Mass Spectrometric Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Góbi, Sándor; Bergantini, Alexandre; Kaiser, Ralf I., E-mail: ralfk@hawaii.edu

The aim of the present work is to unravel the radiolytic decomposition of adenine (C{sub 5}H{sub 5}N{sub 5}) under conditions relevant to the Martian surface. Being the fundamental building block of (deoxy)ribonucleic acids, the possibility of survival of this biomolecule on the Martian surface is of primary importance to the astrobiology community. Here, neat adenine and adenine–magnesium perchlorate mixtures were prepared and irradiated with energetic electrons that simulate the secondary electrons originating from the interaction of the galactic cosmic rays with the Martian surface. Perchlorates were added to the samples since they are abundant—and therefore relevant oxidizers on the surfacemore » of Mars—and they have been previously shown to facilitate the radiolysis of organics such as glycine. The degradation of the samples were monitored in situ via Fourier transformation infrared spectroscopy and the electron ionization quadruple mass spectrometric method; temperature-programmed desorption profiles were then collected by means of the state-of-the-art single photon photoionization reflectron time-of-flight mass spectrometry (PI-ReTOF-MS), allowing for the detection of the species subliming from the sample. The results showed that perchlorates do increase the destruction rate of adenine by opening alternative reaction channels, including the concurrent radiolysis/oxidation of the sample. This new pathway provides a plethora of different radiolysis products that were identified for the first time. These are carbon dioxide (CO{sub 2}), isocyanic acid (HNCO), isocyanate (OCN{sup −}), carbon monoxide (CO), and nitrogen monoxide (NO); an oxidation product containing carbonyl groups (R{sub 1}R{sub 2}–C=O) with a constrained five-membered cyclic structure could also be observed. Cyanamide (H{sub 2}N–C≡N) was detected in both irradiated samples as well.« less

Streptozotocin Diabetes CORRELATION WITH EXTENT OF DEPRESSION OF PANCREATIC ISLET NICOTINAMIDE ADENINE DINUCLEOTIDE

PubMed Central

Anderson, Tom; Schein, Philip S.; McMenamin, Mary G.; Cooney, David A.

1974-01-01

The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 μmol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 μmol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 μmol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 μmol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-14C]streptozotocin by islets was 3.8 times that of [methyl-14C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets. PMID

DNA Nucleotide Sequence Restricted by the RI Endonuclease

PubMed Central

Hedgpeth, Joe; Goodman, Howard M.; Boyer, Herbert W.

1972-01-01

The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage λ has been determined. The 5′-terminal nucleotide labeled with 32P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in λ DNA was deduced from these data and from the 3′-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virus [Formula: see text] The RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5′-phosphoryls and short cohesive termini of four nucleotides, pApApTpT. The most striking feature of the sequence is its symmetry. PMID:4343974

A step into the RNA world: Conditional analysis of hydrogel formation of adenosine 5'-monophosphate induced by cyanuric acid.

PubMed

Yokosawa, Takumi; Enomoto, Ryota; Uchino, Sho; Hirasawa, Ito; Umehara, Takuya; Tamura, Koji

2017-12-01

Nucleotide polymerization occurs by the nucleophilic attack of 3'-oxygen of the 3'-terminal nucleotide on the α-phosphorus of the incoming nucleotide 5'-triphosphate. The π-stacking of mononucleotides is an important factor for prebiotic RNA polymerization in terms of attaining the proximity of two reacting moieties. Adenosine and adenosine 5'-monophosphate (AMP) are known to form hydrogel in the presence of cyanuric acid at neutral pH. However, we observed that other canonical ribonucleotides did not gel under the same condition. The π-stacking-induced hydrogel formation of AMP was destroyed at pH 2.0, suggesting that the protonation of N at position 1 of adenine abolished hydrogen bonding with the NH of cyanuric acid and resulted in the deformation of the hexad of adenine and cyanuric acid. A liquid-like gel was formed in the case of adenosine with cyanuric acid and boric acid, whereas AMP caused the formation of a solid gel, implying that the negative charge inherent to AMP prevented the formation of esters of boric acid with the cis-diols of ribose. Cyanuric acid-driven oligomerizations of AMP might have been the first crucial event in the foundation of the RNA world. Copyright © 2017 Elsevier B.V. All rights reserved.

Rationalizing the structural variability of the exocyclic amino groups in nucleobases and their metal complexes: cytosine and adenine.

PubMed

Fonseca Guerra, Célia; Sanz Miguel, Pablo J; Cebollada, Andrea; Bickelhaupt, F Matthias; Lippert, Bernhard

2014-07-28

The exocyclic amino groups of cytosine and adenine nucleobases are normally almost flat, with the N atoms essentially sp(2) hybridized and the lone pair largely delocalized into the heterocyclic rings. However, a change to marked pyramidality of the amino group (N then sp(3) hybridized, lone pair essentially localized at N) occurs during i) involvement of an amino proton in strong hydrogen bonding donor conditions or ii) with monofunctional metal coordination following removal of one of the two protons. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains.

PubMed

He, Daniel; Lorenz, Robin; Kim, Choel; Herberg, Friedrich W; Lim, Chinten James

2017-12-15

The cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases (PKA and PKG) are key effectors of cyclic nucleotide signaling. Both share structural features that include tandem cyclic nucleotide-binding (CNB) domains, CNB-A and CNB-B, yet their functions are separated through preferential activation by either cAMP or cGMP. Based on structural studies and modeling, key CNB contact residues have been identified for both kinases. In this study, we explored the requirements for conversion of PKA activation from cAMP-dependent to cGMP-dependent. The consequences of the residue substitutions T192R/A212T within CNB-A or G316R/A336T within CNB-B of PKA-RIα on cyclic nucleotide binding and holoenzyme activation were assessed in vitro using purified recombinant proteins, and ex vivo using RIα-deficient mouse embryonic fibroblasts genetically reconstituted with wild-type or mutant PKA-RIα. In vitro, a loss of binding and activation selectivity was observed when residues in either one of the CNB domains were mutated, while mutations in both CNB domains resulted in a complete switch of selectivity from cAMP to cGMP. The switch in selectivity was also recapitulated ex vivo, confirming their functional roles in cells. Our results highlight the importance of key cyclic nucleotide contacts within each CNB domain and suggest that these domains may have evolved from an ancestral gene product to yield two distinct cyclic nucleotide-dependent protein kinases.

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells.

PubMed

Kurosaki, F; Kaburaki, H; Nishi, A

1994-03-07

Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 microM of free Ca2+, while Ca2+ lower than 0.1 microM did not affect the Ca(2+)-release. The Ca2+ flux across plasma membrane was restored from this Ca(2+)-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca2+ channel.

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca2+-induced cysteine relative mobility/mitochondrial permeability transition pore.

PubMed

Pestana, Cezar R; Silva, Carlos H T P; Uyemura, Sérgio A; Santos, Antonio C; Curti, Carlos

2010-08-01

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca(2+)-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys(56) relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca(2+)-induced ANT conformational change and mitochondrial swelling. ANT-Pro(61) isomerization increased ANT-Cys(56) relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca(2+) induced ANT "c" conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca(2+)-induced ANT "c" conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca(2+) induces the ANT "c" conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.

Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

ERIC Educational Resources Information Center

Strumilo, Slawomir

2005-01-01

Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

Fixed-Gap Tunnel Junction for Reading DNA Nucleotides

PubMed Central

2015-01-01

Previous measurements of the electronic conductance of DNA nucleotides or amino acids have used tunnel junctions in which the gap is mechanically adjusted, such as scanning tunneling microscopes or mechanically controllable break junctions. Fixed-junction devices have, at best, detected the passage of whole DNA molecules without yielding chemical information. Here, we report on a layered tunnel junction in which the tunnel gap is defined by a dielectric layer, deposited by atomic layer deposition. Reactive ion etching is used to drill a hole through the layers so that the tunnel junction can be exposed to molecules in solution. When the metal electrodes are functionalized with recognition molecules that capture DNA nucleotides via hydrogen bonds, the identities of the individual nucleotides are revealed by characteristic features of the fluctuating tunnel current associated with single-molecule binding events. PMID:25380505

Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis.

PubMed

Kalia, Dimpy; Merey, Gökçe; Nakayama, Shizuka; Zheng, Yue; Zhou, Jie; Luo, Yiling; Guo, Min; Roembke, Benjamin T; Sintim, Herman O

2013-01-07

For an organism to survive, it must be able to sense its environment and regulate physiological processes accordingly. Understanding how bacteria integrate signals from various environmental factors and quorum sensing autoinducers to regulate the metabolism of various nucleotide second messengers c-di-GMP, c-di-AMP, cGMP, cAMP and ppGpp, which control several key processes required for adaptation is key for efforts to develop agents to curb bacterial infections. In this review, we provide an update of nucleotide signaling in bacteria and show how these signals intersect or integrate to regulate the bacterial phenotype. The intracellular concentrations of nucleotide second messengers in bacteria are regulated by synthases and phosphodiesterases and a significant number of these metabolism enzymes had been biochemically characterized but it is only in the last few years that the effector proteins and RNA riboswitches, which regulate bacterial physiology upon binding to nucleotides, have been identified and characterized by biochemical and structural methods. C-di-GMP, in particular, has attracted immense interest because it is found in many bacteria and regulate both biofilm formation and virulence factors production. In this review, we discuss how the activities of various c-di-GMP effector proteins and riboswitches are modulated upon c-di-GMP binding. Using V. cholerae, E. coli and B. subtilis as models, we discuss how both environmental factors and quorum sensing autoinducers regulate the metabolism and/or processing of nucleotide second messengers. The chemical syntheses of the various nucleotide second messengers and the use of analogs thereof as antibiofilm or immune modulators are also discussed.

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

PubMed

Speck, Janina; Stebel, Sabine C; Arndt, Katja M; Müller, Kristian M

2011-01-01

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.

Nucleotide Intermediates in the Biosynthesis of Heteropolymeric Polysaccharides

PubMed Central

Strominger, Jack L.

1964-01-01

The role of nucleotides as “carriers” of small molecules for biosynthetic reactions is discussed. Following this introduction, the particular problem of nucleotide intermediates in chondroitin sulfate synthesis is presented. The egg shell of the hen contains a form of chondroitin sulfate and particular emphasis is placed on the biosynthesis of sulfated polysaccharides in the hen oviduct, which has been studied in the author's laboratory. ImagesFigure 16Figure 19 PMID:14104074

Nucleotide sequences specific to Brucella and methods for the detection of Brucella

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCready, Paula M; Radnedge, Lyndsay; Andersen, Gary L

Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

A movie of the RNA polymerase nucleotide addition cycle.

PubMed

Brueckner, Florian; Ortiz, Julio; Cramer, Patrick

2009-06-01

During gene transcription, RNA polymerase (Pol) passes through repetitive cycles of adding a nucleotide to the growing mRNA chain. Here we obtained a movie of the nucleotide addition cycle by combining structural information on different functional states of the Pol II elongation complex (EC). The movie illustrates the two-step loading of the nucleoside triphosphate (NTP) substrate, closure of the active site for catalytic nucleotide incorporation, and the presumed two-step translocation of DNA and RNA, which is accompanied by coordinated conformational changes in the polymerase bridge helix and trigger loop. The movie facilitates teaching and a mechanistic analysis of transcription and can be downloaded from http://www.lmb.uni-muenchen.de/cramer/pr-materials.

Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

NASA Technical Reports Server (NTRS)

Mullins, D. W., Jr.; Lacey, J. C., Jr.

1981-01-01

The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

Stimulation of inorganic pyrophosphate elaboration by cultured cartilage and chondrocytes.

PubMed

Ryan, L M; Kurup, I; Rosenthal, A K; McCarty, D J

1989-08-01

Inorganic pyrophosphate elaboration by articular cartilage may favor calcium pyrophosphate dihydrate crystal deposition. Frequently crystal deposits form in persons affected with metabolic diseases. The cartilage organ culture system was used to model these metabolic conditions while measuring the influence on extracellular pyrophosphate elaboration. Alterations of ambient pH, thyroid stimulating hormone levels, and parathyroid hormone levels did not change pyrophosphate accumulation in the media. However, subphysiologic ambient calcium concentrations (25, 100, 500 microM) increased pyrophosphate accumulation about chondrocytes 3- to 10-fold. Low calcium also induced release of [14C]adenine-labeled nucleotides from chondrocytes, potential substrates for generation of extracellular pyrophosphate by ectoenzymes. Exposing cartilage to 10% fetal bovine serum also enhanced by 50% the egress of inorganic pyrophosphate from the tissue.

37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide and...

37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide and...

Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA gamma.

PubMed

Gustafson, G; Hunter, B; Hanau, R; Armour, S L; Jackson, A O

1987-06-01

The complete nucleotide sequences of RNA gamma from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5'-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3'-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNA gamma ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNA gamma ORF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNA gamma ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3'-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984, Virology 139, 375-383). In the genomic RNA gamma, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.

Exchange transfusion with red blood cells preserved in adenine clears a child of severe falciparum malaria.

PubMed

Boctor, F N; Ali, N M; Choi, Y J; Morse, E E

1997-01-01

Falciparum malaria may be associated with significant morbidity and mortality. The degree of mortality and morbidity usually corresponds to the degree of parasitemia. Quinine and other antimalarial drugs are relatively slow acting and not always effective owing to the presence of drug resistance falciparum. Rapid reduction of the number of circulating parasites may be required. Exchange transfusion has been used as a safe and quick approach to decreasing the parasitemia and antimalaria drugs used to eradicate the rest of the Plasmodium. In the present report, a case is described of a child with severe falciparum malaria who was successfully treated with exchange transfusion using the new adenine and mannitol enriched preservative media, Adsol.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

PubMed Central

Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Houlden, Henry

2017-01-01

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. PMID:28360103

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

PubMed

Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

2017-05-26

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of Gum Arabic on Oxidative Stress and Inflammation in Adenine–Induced Chronic Renal Failure in Rats

PubMed Central

Ali, Badreldin H.; Al-Husseni, Isehaq; Beegam, Sumyia; Al-Shukaili, Ahmed; Nemmar, Abderrahim; Schierling, Simone; Queisser, Nina; Schupp, Nicole

2013-01-01

Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in chronic renal failure (CRF) in rats. The aim of this work was to study the role of inflammation and oxidative stress in adenine-induced CRF and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in feed (0.75%, w/w), GA in drinking water (15%, w/v) and adenine+GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration). In addition, the concentrations of the pro-inflammatory cytokine TNF-α and the oxidative stress markers glutathione and superoxide dismutase, renal apoptosis, superoxide formation and DNA double strand break frequency, detected by immunohistochemistry for γ-H2AX, were measured. Adenine significantly increased the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance and induced significant increases in the concentration of the measured inflammatory mediators. Further, it caused oxidative stress and DNA damage. Treatment with GA significantly ameliorated these actions. The mechanism of the reported salutary effect of GA in adenine-induced CRF is associated with mitigation of the adenine-induced inflammation and generation of free radicals. PMID:23383316

The Coding of Biological Information: From Nucleotide Sequence to Protein Recognition

NASA Astrophysics Data System (ADS)

Štambuk, Nikola

The paper reviews the classic results of Swanson, Dayhoff, Grantham, Blalock and Root-Bernstein, which link genetic code nucleotide patterns to the protein structure, evolution and molecular recognition. Symbolic representation of the binary addresses defining particular nucleotide and amino acid properties is discussed, with consideration of: structure and metric of the code, direct correspondence between amino acid and nucleotide information, and molecular recognition of the interacting protein motifs coded by the complementary DNA and RNA strands.

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

PubMed Central

Hoffmeyer, J.; Neuhard, J.

1971-01-01

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate. PMID:4928005

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA

PubMed Central

Naorem, Santa S.; Han, Jin; Wang, Shufang; Lee, William R.; Heng, Xiao; Miller, Jeff F.

2017-01-01

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation. PMID:29109248

Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5'-monophosphorothioate.

PubMed Central

Fedan, J. S.

1987-01-01

The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter. PMID:3038248

Molecular action of sulphonylureas on KATP channels: a real partnership between drugs and nucleotides.

PubMed

de Wet, Heidi; Proks, Peter

2015-10-01

Sulphonylureas stimulate insulin secretion from pancreatic β-cells primarily by closing ATP-sensitive K(+) channels in the β-cell plasma membrane. The mechanism of channel inhibition by these drugs is unusually complex. As direct inhibitors of channel activity, sulphonylureas act only as partial antagonists at therapeutic concentrations. However, they also exert an additional indirect inhibitory effect via modulation of nucleotide-dependent channel gating. In this review, we summarize current knowledge and recent advances in our understanding of the molecular mechanism of action of these drugs. © 2015 Authors; published by Portland Press Limited.

Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes.

PubMed

Braidy, Nady; Berg, Jade; Clement, James; Khorshidi, Fatemeh; Poljak, Anne; Jayasena, Tharusha; Grant, Ross; Sachdev, Perminder

2018-05-11

Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD + depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD + , and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD + levels. Recent studies have shown that enhancing NAD + levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD + anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD + synthesis pathway in mammalian cells. NAD + can also be produced by the NAD + salvage pathway. Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD + precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD + decline in degenerative disease states and physiological aging. Results obtained in recent years have shown that NAD + precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD + synthesis via their location in the NAD + anabolic pathway. Increased synthesis of NAD + promotes protective cell responses, further demonstrating that NAD + is a regulatory molecule associated with several

Toxicity associated with high dosage 9-[(2R,5R-2,5-dihydro-5-phosphonomethoxy)-2-furanyl]adenine therapy off attempts to abort early FIV infection.

PubMed

Hartmann, K; Ferk, G; North, T W; Pedersen, N C

1997-09-01

9-[(2R,5R-2,5-dihydro-5-phosphonomethoxy)-2-furanyl]adenine, or D4API, was tested in the feline immunodeficiency virus (FIV) infection model and found to be significantly more inhibitory in vitro than its parent compound 9-phosphonylmethoxethyl adenine (PMEA). Cytotoxicity was less than for PMEA or azidothymidine (AZT) for culture periods of 7 days, but more toxic after 10 days. D4API was rapidly absorbed by cats following subcutaneous inoculation, with a plasma half-life of less than 1 h after intravenous inoculation and between 2 and 3 h after subcutaneous injection. Peripheral blood mononuclear cells collected from cats given a single dose of D4API were refractory, however, to FIV infection in vitro for up to 24 h. Given its prolonged intracellular phase and high selectivity index, high dose D4API therapy was tested for its ability to abort an acute (i.e. 2 week) FIV infection. A divided daily dose of D4API, which was one-fourth the toxic dose and 125 times the concentration that would totally inhibit virus replication in vitro, completely abrogated the anticipated viremia and antibody responses. Unfortunately, a majority of treated/uninfected and treated/infected test cats died acutely of drug toxicity after 47 days of treatment. Toxicity in vivo mirrored what was observed in vitro, being precipitous and cumulative in nature. Toxic signs included widespread hepatic and lymphoid necrosis. A surviving treated/FIV infected cat remained healthy to day 175 when the study was terminated; antibodies appeared 2 months later than in untreated/infected cats and virus was only detectable at low levels on day 175. In contrast, untreated/infected cats were viremic and antibody positive from 3 to 4 weeks post-infection onwards. Therefore, it was possible to alter, but not abort, an early FIV infection with prolonged, high-dose D4API treatment.

Antiviral Nucleotide Incorporation by Recombinant Human Mitochondrial RNA Polymerase Is Predictive of Increased In Vivo Mitochondrial Toxicity Risk

PubMed Central

Lin, Xiaodong; Yokokawa, Fumiaki; Sweeney, Zachary; Saunders, Oliver; Xie, Lili; Lim, Siew Pheng; Uteng, Marianne; Uehara, Kyoko; Warne, Robert; Gang, Wang; Jones, Christopher; Yendluri, Satya; Gu, Helen; Mansfield, Keith; Boisclair, Julie; Heimbach, Tycho; Catoire, Alexandre; Bracken, Kathryn; Weaver, Margaret; Moser, Heinz; Zhong, Weidong

2016-01-01

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2′-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 μM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 μM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, β, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated. PMID:27645237

Role of conserved nucleotides in building the 16S rRNA binding site of E. coli ribosomal protein S8.

PubMed Central

Allmang, C; Mougel, M; Westhof, E; Ehresmann, B; Ehresmann, C

1994-01-01

Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a question of debate. Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region. These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site. The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already proposed non canonical U598-U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone. Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove. The third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598-U641 pair should not be directly in contact with the protein. Images PMID:7937081

Effects of luminal flow and nucleotides on [Ca(2+)](i) in rabbit cortical collecting duct.

PubMed

Woda, Craig B; Leite, Maurilo; Rohatgi, Rajeev; Satlin, Lisa M

2002-09-01

Nucleotide binding to purinergic P2 receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Whereas P2 receptors have been functionally identified at the basolateral membrane of the cortical collecting duct (CCD), a final regulatory site of urinary Na(+), K(+), and acid-base excretion, controversy exists as to whether apical purinoceptors exist in this segment. Nor has the distribution of receptor subtypes present on the unique cell populations that constitute Ca(2+) the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [Ca(2+)](i) did not differ between principal and intercalated cells, averaging approximately 120 nM. An acute increase in tubular fluid flow rate, associated with a 20% increase in tubular diameter, led to increases in [Ca(2+)](i) in both cell types. Luminal perfusion of 100 microM UTP or ATP-gamma-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [Ca(2+)](i) in both cell types (P < 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [Ca(2+)](i) transients. Luminal perfusion with a P2X (alpha,beta-methylene-ATP), P2X(7) (benzoyl-benzoyl-ATP), P2Y(1) (2-methylthio-ATP), or P2Y(4)/P2Y(6) (UDP) receptor agonist had no effect on [Ca(2+)](i). The nucleotide-induced [Ca(2+)](i) transients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal Ca(2+) stores, luminal perfusion with a Ca(2+)-free perfusate, or the L-type Ca(2+) channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2Y(2) receptors in the CCD via pathways that require both internal Ca(2+) mobilization and extracellular Ca(2+) entry. The flow-induced rise in [Ca(2+)](i) is

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

PubMed Central

Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

2017-01-01

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains. PMID:24107129

Organization of Nucleotides in Different Environments and the Formation of Pre-Polymers

NASA Astrophysics Data System (ADS)

Himbert, Sebastian; Chapman, Mindy; Deamer, David W.; Rheinstädter, Maikel C.

2016-08-01

RNA is a linear polymer of nucleotides linked by a ribose-phosphate backbone. Polymerization of nucleotides occurs in a condensation reaction in which phosphodiester bonds are formed. However, in the absence of enzymes and metabolism there has been no obvious way for RNA-like molecules to be produced and then encapsulated in cellular compartments. We investigated 5‧-adenosine monophosphate (AMP) and 5‧-uridine monophosphate (UMP) molecules confined in multi-lamellar phospholipid bilayers, nanoscopic films, ammonium chloride salt crystals and Montmorillonite clay, previously proposed to promote polymerization. X-ray diffraction was used to determine whether such conditions imposed a degree of order on the nucleotides. Two nucleotide signals were observed in all matrices, one corresponding to a nearest neighbour distance of 4.6 Å attributed to nucleotides that form a disordered, glassy structure. A second, smaller distance of 3.4 Å agrees well with the distance between stacked base pairs in the RNA backbone, and was assigned to the formation of pre-polymers, i.e., the organization of nucleotides into stacks of about 10 monomers. Such ordering can provide conditions that promote the nonenzymatic polymerization of RNA strands under prebiotic conditions. Experiments were modeled by Monte-Carlo simulations, which provide details of the molecular structure of these pre-polymers.

Pyrophosphorolytic dismutation of oligodeoxy-nucleotides by terminal deoxynucleotidyltransferase.

PubMed Central

Anderson, R S; Bollum, F J; Beattie, K L

1999-01-01

Terminal transferase (TdT), when incubated with a purified(32)P-5"-end-labeled oligonucleotide of defined length in the presence of Co(2+), Mn(2+)or Mg(2+)and 2-mercaptoethanol in cacodylate or HEPES buffer, pH 7.2, exhibits the ability to remove a 3"-nucleotide from one oligonucleotide and add it to the 3"-end of another. When analyzed by urea-PAGE, this activity is observed as a disproportionation of the starting oligonucleotide into a ladder of shorter and longer oligonucleotides distributed around the starting material. Optimal metal ion concentration is 1-2 mM. All three metal ions support this activity with Co(2+)> Mn(2+) congruent with Mg(2+). Oligonucleotides p(dT) and p(dA) are more efficient substrates than p(dG) and p(dC) because the latter may form secondary structures. The dismutase activity is significant even in the presence of dNTP concentrations comparable to those that exist in the nucleus during the G(1)phase of the cell cycle. Using BetaScope image analysis the rate of pyrophosphorolytic dismutase activity was found to be only moderately slower than the poly-merization activity. These results may help explain the GC-richness of immunoglobulin gene segment joins (N regions) and the loss of bases that occur during gene rearrangements in pre-B and pre-T cells. PMID:10454617

Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations.

PubMed

Chugun, Akihito; Sato, Osamu; Takeshima, Hiroshi; Ogawa, Yasuo

2007-01-01

To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca(2+)-dependent [(3)H]ryanodine binding, a biochemical measure of Ca(2+)-induced Ca(2+) release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca(2+) dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [(3)H]ryanodine binding initially increased as [Ca(2+)] increased, with a plateau in the range of 10-100 microM Ca(2+), and thereafter further increased to an apparent peak around 1 mM Ca(2+), followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3-1 mM Mg(2+) unexpectedly increased the binding two- to threefold and enhanced the affinity for [(3)H]ryanodine at 10-100 microM Ca(2+), resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg(2+). Ca(2+) could be a substitute for Mg(2+). Mg(2+) also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg(2+) on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg(2+) concentration.

Synthesis and characterization of zinc adeninate metal-organic frameworks (bioMOF1) as potential anti-inflammatory drug delivery material

NASA Astrophysics Data System (ADS)

Usman, Ken Aldren S.; Buenviaje, Salvador C.; Razal, Joselito M.; Conato, Marlon T.; Payawan, Leon M.

2018-05-01

Zn8(ad)4(BPDC)6O•2Me2NH2 (bioMOF1), a porous metal-organic framework with zinc-adeninate secondary building units (SBUs), interconnected via biphenyldicarboxylate linkers, shows great potential for drug delivery applications due to its non-toxic and biocompatible components (zinc and adenine). In this study, bioMOF1 crystals synthesized solvothermally at 130°C for 24 hours, were characterized thoroughly and loaded with a known anti-inflammatory drug, nimesulide (NIM). The crystalline nature of the material was confirmed using powder x-ray diffraction crystallography (PXRD) along with morphology assessment using focused-ion beam/field emission scanning electron microscopy (FIB/FESEM). NIM was introduced to the crystals via solvent exchange accompanied with vigorous stirring and quantified using thermogravimetric analysis (TGA) with loading saturation of ˜30% attained during the 2nd to 3rd day of drug immersion. Drug release in phosphate buffer saline and in deionized water was done to monitor the kinetic of drug release in vitro. The drug release showed a controlled discharge profile which slowed down at the 24th and 48th hour of release. Drug release in buffer showed a faster release of drug from the material, which means that the presence of cations in the solution could further trigger the release of drug. Slow drug release was observed for all of the set-ups with maximum % drug release of 24.47%, and 16.14% for the bioMOF1 in buffer and bioMOF1 in water respectively for the span of 48 hours.

n-Nucleotide circular codes in graph theory.

PubMed

Fimmel, Elena; Michel, Christian J; Strüngmann, Lutz

2016-03-13

The circular code theory proposes that genes are constituted of two trinucleotide codes: the classical genetic code with 61 trinucleotides for coding the 20 amino acids (except the three stop codons {TAA,TAG,TGA}) and a circular code based on 20 trinucleotides for retrieving, maintaining and synchronizing the reading frame. It relies on two main results: the identification of a maximal C(3) self-complementary trinucleotide circular code X in genes of bacteria, eukaryotes, plasmids and viruses (Michel 2015 J. Theor. Biol. 380, 156-177. (doi:10.1016/j.jtbi.2015.04.009); Arquès & Michel 1996 J. Theor. Biol. 182, 45-58. (doi:10.1006/jtbi.1996.0142)) and the finding of X circular code motifs in tRNAs and rRNAs, in particular in the ribosome decoding centre (Michel 2012 Comput. Biol. Chem. 37, 24-37. (doi:10.1016/j.compbiolchem.2011.10.002); El Soufi & Michel 2014 Comput. Biol. Chem. 52, 9-17. (doi:10.1016/j.compbiolchem.2014.08.001)). The univerally conserved nucleotides A1492 and A1493 and the conserved nucleotide G530 are included in X circular code motifs. Recently, dinucleotide circular codes were also investigated (Michel & Pirillo 2013 ISRN Biomath. 2013, 538631. (doi:10.1155/2013/538631); Fimmel et al. 2015 J. Theor. Biol. 386, 159-165. (doi:10.1016/j.jtbi.2015.08.034)). As the genetic motifs of different lengths are ubiquitous in genes and genomes, we introduce a new approach based on graph theory to study in full generality n-nucleotide circular codes X, i.e. of length 2 (dinucleotide), 3 (trinucleotide), 4 (tetranucleotide), etc. Indeed, we prove that an n-nucleotide code X is circular if and only if the corresponding graph [Formula: see text] is acyclic. Moreover, the maximal length of a path in [Formula: see text] corresponds to the window of nucleotides in a sequence for detecting the correct reading frame. Finally, the graph theory of tournaments is applied to the study of dinucleotide circular codes. It has full equivalence between the combinatorics

The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an “Ajar” Intermediate Conformation in the Nucleotide Selection Mechanism*

PubMed Central

Wu, Eugene Y.; Beese, Lorena S.

2011-01-01

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established “open” and “closed” states. In this “ajar” conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation. PMID:21454515

Febuxostat for the Prevention of Recurrent 2,8-dihydroxyadenine Nephropathy due to Adenine Phosphoribosyltransferase Deficiency Following Kidney Transplantation.

PubMed

Nanmoku, Koji; Kurosawa, Akira; Shinzato, Takahiro; Shimizu, Toshihiro; Kimura, Takaaki; Yagisawa, Takashi

2017-01-01

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder that results in irreversible renal damage due to 2,8-dihydroxyadenine (DHA) nephropathy. A 28-year-old man underwent living-related kidney transplantation for chronic kidney disease of unknown etiology. Numerous spherical brownish crystals observed in his urinary sediment on postoperative day 3 and were observed within the tubular lumen of renal allograft biopsy specimens on postoperative day 7. After a genetic diagnosis, febuxostat treatment was started on postoperative day 7, with the dosage gradually increased to 80 mg/day until complete the disappearance of 2,8-DHA crystals. Febuxostat prevented secondary 2,8-DHA nephropathy after kidney transplantation.

Febuxostat for the Prevention of Recurrent 2,8-dihydroxyadenine Nephropathy due to Adenine Phosphoribosyltransferase Deficiency Following Kidney Transplantation

PubMed Central

Nanmoku, Koji; Kurosawa, Akira; Shinzato, Takahiro; Shimizu, Toshihiro; Kimura, Takaaki; Yagisawa, Takashi

2017-01-01

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder that results in irreversible renal damage due to 2,8-dihydroxyadenine (DHA) nephropathy. A 28-year-old man underwent living-related kidney transplantation for chronic kidney disease of unknown etiology. Numerous spherical brownish crystals observed in his urinary sediment on postoperative day 3 and were observed within the tubular lumen of renal allograft biopsy specimens on postoperative day 7. After a genetic diagnosis, febuxostat treatment was started on postoperative day 7, with the dosage gradually increased to 80 mg/day until complete the disappearance of 2,8-DHA crystals. Febuxostat prevented secondary 2,8-DHA nephropathy after kidney transplantation. PMID:28566603

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Savabi, F.; Geiger, P.J.; Bessman, S.P.

1984-03-01

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphorus-labeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P/sub i/) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P/sub i/ is described. 20 references.

Formation of Amino Acids and Nucleotide Bases in a Titan Atmosphere Simulation Experiment

PubMed Central

Yelle, R.V.; Buch, A.; Carrasco, N.; Cernogora, G.; Dutuit, O.; Quirico, E.; Sciamma-O'Brien, E.; Smith, M.A.; Somogyi, Á.; Szopa, C.; Thissen, R.; Vuitton, V.

2012-01-01

Abstract The discovery of large (>100 u) molecules in Titan's upper atmosphere has heightened astrobiological interest in this unique satellite. In particular, complex organic aerosols produced in atmospheres containing C, N, O, and H, like that of Titan, could be a source of prebiotic molecules. In this work, aerosols produced in a Titan atmosphere simulation experiment with enhanced CO (N2/CH4/CO gas mixtures of 96.2%/2.0%/1.8% and 93.2%/5.0%/1.8%) were found to contain 18 molecules with molecular formulae that correspond to biological amino acids and nucleotide bases. Very high-resolution mass spectrometry of isotopically labeled samples confirmed that C4H5N3O, C4H4N2O2, C5H6N2O2, C5H5N5, and C6H9N3O2 are produced by chemistry in the simulation chamber. Gas chromatography–mass spectrometry (GC-MS) analyses of the non-isotopic samples confirmed the presence of cytosine (C4H5N3O), uracil (C5H4N2O2), thymine (C5H6N2O2), guanine (C5H5N5O), glycine (C2H5NO2), and alanine (C3H7NO2). Adenine (C5H5N5) was detected by GC-MS in isotopically labeled samples. The remaining prebiotic molecules were detected in unlabeled samples only and may have been affected by contamination in the chamber. These results demonstrate that prebiotic molecules can be formed by the high-energy chemistry similar to that which occurs in planetary upper atmospheres and therefore identifies a new source of prebiotic material, potentially increasing the range of planets where life could begin. Key Words: Astrochemistry—Planetary atmospheres—Titan—Astrobiology. Astrobiology 12, 809–817. PMID:22917035

Database of amino acid-nucleotide contacts in contacts in DNA-homeodomain protein

NASA Astrophysics Data System (ADS)

Grokhlina, T. I.; Zrelov, P. V.; Ivanov, V. V.; Polozov, R. V.; Chirgadze, Yu. N.; Sivozhelezov, V. S.

2013-09-01

The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

Prokaryotic Nucleotide Composition Is Shaped by Both Phylogeny and the Environment

DOE PAGES

Reichenberger, Erin R.; Rosen, Gail; Hershberg, Uri; ...

2015-04-09

Here, the causes of the great variation in nucleotide composition of prokaryotic genomes have long been disputed. Here, we use extensive metagenomic and whole-genome data to demonstrate that both phylogeny and the environment shape prokaryotic nucleotide content. We show that across environments, various phyla are characterized by different mean guanine and cytosine (GC) values as well as by the extent of variation on that mean value. At the same time, we show that GC-content varies greatly as a function of environment, in a manner that cannot be entirely explained by disparities in phylogenetic composition. We find environmentally driven differences inmore » nucleotide content not only between highly diverged environments (e.g., soil, vs. aquatic vs. human gut) but also within a single type of environment. More specifically, we demonstrate that some human guts are associated with a microbiome that is consistently more GC-rich across phyla, whereas others are associated with a more AT-rich microbiome. These differences appear to be driven both by variations in phylogenetic composition and by environmental differences—which are independent of these phylogenetic composition differences. Combined, our results demonstrate that both phylogeny and the environment significantly affect nucleotide composition and that the environmental differences affecting nucleotide composition are far subtler than previously appreciated.« less

Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels.

PubMed Central

Brown, R L; Snow, S D; Haley, T L

1998-01-01

In the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels convert stimulus-induced changes in the internal concentrations of cGMP and cAMP into changes in membrane potential. Although it is known that significant activation of these channels requires the binding of three or more molecules of ligand, the detailed molecular mechanism remains obscure. We have probed the structural changes that occur during channel activation by using sulfhydryl-reactive methanethiosulfonate (MTS) reagents and N-ethylmaleimide (NEM). When expressed in Xenopus oocytes, the alpha-subunit of the bovine retinal channel forms homomultimeric channels that are activated by cGMP with a K1/2 of approximately 100 microM. Cyclic AMP, on the other hand, is a very poor activator; a saturating concentration elicits only 1% of the maximum current produced by cGMP. Treatment of excised patches with MTS-ethyltrimethylamine (MTSET) or NEM dramatically potentiated the channel's response to both cyclic nucleotides. After MTSET treatment, the dose-response relation for cGMP was shifted by over two orders of magnitude to lower concentrations. The effect on channel activation by cAMP was even more striking. After modification, the channels were fully activated by cAMP with a K1/2 of approximately 60 microM. This potentiation was abolished by conversion of Cys481 to a nonreactive alanine residue. Potentiation occurred more rapidly in the presence of saturating cGMP, indicating that this region of the channel is more accessible when the channel is open. Cys481 is located in a linker region between the transmembrane and cGMP-binding domains of the channel. These results suggest that this region of the channel undergoes significant movement during the activation process and is critical for coupling ligand binding to pore opening. Potentiation, however, is not mediated by the recently reported interaction between the amino- and carboxy-terminal regions of the alpha-subunit. Deletion of the

Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

PubMed

Nilforoushan, Arman; Furrer, Antonia; Wyss, Laura A; van Loon, Barbara; Sturla, Shana J

2015-04-15

Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.

Absorption and luminescence spectroscopy of mass-selected flavin adenine dinucleotide mono-anions

NASA Astrophysics Data System (ADS)

Giacomozzi, L.; Kjær, C.; Langeland Knudsen, J.; Andersen, L. H.; Brøndsted Nielsen, S.; Stockett, M. H.

2018-06-01

We report the absorption profile of isolated Flavin Adenine Dinucleotide (FAD) mono-anions recorded using photo-induced dissociation action spectroscopy. In this charge state, one of the phosphoric acid groups is deprotonated and the chromophore itself is in its neutral oxidized state. These measurements cover the first four optical transitions of FAD with excitation energies from 2.3 to 6.0 eV (210-550 nm). The S0 → S2 transition is strongly blue shifted relative to aqueous solution, supporting the view that this transition has a significant charge-transfer character. The remaining bands are close to their solution-phase positions. This confirms that the large discrepancy between quantum chemical calculations of vertical transition energies and solution-phase band maxima cannot be explained by solvent effects. We also report the luminescence spectrum of FAD mono-anions in vacuo. The gas-phase Stokes shift for S1 is 3000 cm-1, which is considerably larger than any previously reported for other molecular ions and consistent with a significant displacement of the ground and excited state potential energy surfaces. Consideration of the vibronic structure is thus essential for simulating the absorption and luminescence spectra of flavins.

Getting it Right: How DNA Polymerases Select the Right Nucleotide.

PubMed

Ludmann, Samra; Marx, Andreas

2016-01-01

All living organisms are defined by their genetic code encrypted in their DNA. DNA polymerases are the enzymes that are responsible for all DNA syntheses occurring in nature. For DNA replication, repair and recombination these enzymes have to read the parental DNA and recognize the complementary nucleotide out of a pool of four structurally similar deoxynucleotide triphosphates (dNTPs) for a given template. The selection of the nucleotide is in accordance with the Watson-Crick rule. In this process the accuracy of DNA synthesis is crucial for the maintenance of the genome stability. However, to spur evolution a certain degree of freedom must be allowed. This brief review highlights the mechanistic basis for selecting the right nucleotide by DNA polymerases.

Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1.

PubMed Central

Le Gall, O; Candresse, T; Brault, V; Dunez, J

1989-01-01

The nucleotide sequence of the RNA1 of hungarian grapevine chrome mosaic virus, a nepovirus very closely related to tomato black ring virus, has been determined from cDNA clones. It is 7212 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame extending from nucleotides 216 to 6971. The presumably encoded polyprotein is 2252 amino acids in length with a molecular weight of 250 kDa. The primary structure of the polyprotein was compared with that of other viral polyproteins, revealing the same general genetic organization as that of other picorna-like viruses (comoviruses, potyviruses and picornaviruses), except that an additional protein is suspected to occupy the N-terminus of the polyprotein. PMID:2798128

Long (27-nucleotides) small inhibitory RNAs targeting E6 protein eradicate effectively the cervical cancer cells harboring human papilloma virus.

PubMed

Cho, Jun Sik; Lee, Shin-Wha; Kim, Yong-Man; Kim, Dongho; Kim, Dae-Yeon; Kim, Young-Tak

2015-05-01

This study was to identify small inhibitory RNAs (siRNAs) that are effective in inhibiting growth of cervical cancer cell lines harboring human papilloma virus (HPV) and to examine how siRNAs interact with interferon beta (IFN-β) and thimerosal. The HPV18-positive HeLa and C-4I cell lines were used. Four types of siRNAs were designed according to their target (both E6 and E7 vs. E6 only) and sizes (21- vs. 27-nucleotides); Ex-18E6/21, Ex-18E6/27, Sp-18E6/21, and Sp-18E6/27. Each siRNA-transfected cells were cultured with or without IFN-b and thimerosal and their viability was measured. The viabilities of HPV18-positive tumor cells were reduced by 21- and 27-nucleotide siRNAs in proportion to the siRNA concentrations. Of the two types of siRNAs, the 27-nucleotide siRNA constructs showed greater inhibitory efficacy. Sp-18E6 siRNAs, which selectively downregulates E6 protein only, were more effective than the E6- and E7-targeting Ex-18E6 siRNAs. siRNAs and IFN-β showed the synergistic effect to inhibit HeLa cell survival and the effect was proportional to both siRNA and IFN-β concentrations. Thimerosal in the presence of siRNA exerted a dose-dependent inhibition of C-4I cell survival. Finally, co-treatment with siRNA, IFN-β, and thimerosal induced the most profound decrease in the viability of both cell lines. Long (27-nucleotides) siRNAs targeting E6-E7 mRNAs effectively reduce the viability of HPV18-positive cervical cancer cells and show the synergistic effect in combination with IFN-b and thimerosal. It is necessary to find the rational design of siRNAs and effective co-factors to eradicate particular cervical cancer.

VDAC electronics: 3. VDAC-Creatine kinase-dependent generation of the outer membrane potential in respiring mitochondria.

PubMed

Lemeshko, Victor V

2016-07-01

Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space. An inhibition by creatine of Ca(2+)-induced swelling of isolated mitochondria and other protective effects of creatine reported in the literature might be explained by generated positive OMP. We suggest that VDAC-creatine kinase-dependent generation of OMP represents a novel physiological factor controlling metabolic state of mitochondria, cell energy channeling and resistance to death. Copyright © 2016 Elsevier B.V. All rights reserved.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

PubMed

Moulin, Morgane; Alguacil, Javier; Gu, Siyi; Mehtougui, Asmaa; Adams, Erin J; Peyrottes, Suzanne; Champagne, Eric

2017-12-01

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Vacuum ultraviolet photoionization of carbohydrates and nucleotides

NASA Astrophysics Data System (ADS)

Shin, Joong-Won; Bernstein, Elliot R.

2014-01-01

Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5'-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

Vacuum ultraviolet photoionization of carbohydrates and nucleotides.

PubMed

Shin, Joong-Won; Bernstein, Elliot R

2014-01-28

Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5(')-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

Vacuum ultraviolet photoionization of carbohydrates and nucleotides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Joong-Won, E-mail: jshin@govst.edu; Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872; Bernstein, Elliot R., E-mail: erb@lamar.colostate.edu

Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate,more » rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.« less

Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome.

PubMed

Dresch, Jacqueline M; Zellers, Rowan G; Bork, Daniel K; Drewell, Robert A

2016-01-01

A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.

Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome

PubMed Central

Dresch, Jacqueline M.; Zellers, Rowan G.; Bork, Daniel K.; Drewell, Robert A.

2016-01-01

A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development. PMID:27330274

Synthesis of Purine Nucleoside and Nucleotide Analogs as Antiparasitic Agents.

DTIC Science & Technology

1979-09-01

was to conduct studies on the synthesis of purine nucleoside and nucleotide analogs as anti- parasitic agents. The primary target compounds were 5...antiparasitic agents. - Jaffe has proposed that the susceptibility of pathogenic helminths and protozoa to fraudulent purine, in contrast to pyrimidine...8217-substituted derivatives are thus designed to inhibit nucleoside and nucleotide kinases as well as other parasitic enzymes. Mammalian cells, onthe

Acidity and complex formation studies of 3-(adenine-9-yl)-propionic and 3-(thymine-1-yl)-propionic acids in ethanol-water media

NASA Astrophysics Data System (ADS)

Hammud, Hassan H.; El Shazly, Shawky; Sonji, Ghassan; Sonji, Nada; Bouhadir, Kamal H.

2015-05-01

The ligands 3-(adenine-9-yl)propionic acid (AA) and 3-(thymine-1-yl)propionic acid (TA) were prepared by N9-alkylation of adenine and N1-alkylation of thymine with ethylacrylate in presence of a base catalyst, followed by acid hydrolysis of the formed ethyl esters to give the corresponding propionic acid derivatives. The products were characterized by spectral methods (FTIR, 1H NMR and 13C NMR), which confirm their structures. The dissociation constants of ligands, were potentiometrically determined in 0.3 M KCl at 20-50 °C temperature range. The work was extended to study complexation behavior of AA and TA with various biologically important divalent metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Mn2+ and Pb2+) in 50% v/v water-ethanol medium at four different temperatures, keeping ionic strength constant (0.3 M KCl). The order of the stability constants of the formed complexes decreases in the sequence Cu2+ > Pb2+ > Zn2+ > Ni2+ > Co2+ > Mn2+ > Cd2+ for both ligands. The effect of temperature was also studied and the corresponding thermodynamic functions (ΔG, ΔH, ΔS) were derived and discussed. The formation of metal complexes has been found to be spontaneous, and the stability constants were dependant markedly on the basicity of the ligands.

Nucleotide sequences specific to Yersinia pestis and methods for the detection of Yersinia pestis

DOEpatents

McCready, Paula M [Tracy, CA; Radnedge, Lyndsay [San Mateo, CA; Andersen, Gary L [Berkeley, CA; Ott, Linda L [Livermore, CA; Slezak, Thomas R [Livermore, CA; Kuczmarski, Thomas A [Livermore, CA; Motin, Vladinir L [League City, TX

2009-02-24

Nucleotide sequences specific to Yersinia pestis that serve as markers or signatures for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

Extracellular nucleotides potentiate the cytosolic Ca2+, but not cyclic adenosine 3', 5'-monophosphate response to parathyroid hormone in rat osteoblastic cells.

PubMed

Kaplan, A D; Reimer, W J; Feldman, R D; Dixon, S J

1995-04-01

Binding to PTH to its cell surface receptor activates both adenylyl cyclase and phospholipase-C, leading to elevation of cytosolic cAMP and free Ca2+. We have shown previously that extracellular nucleotides interact with P2U and P2Y subtypes of purinoceptor on osteoblastic cells, both linked to Ca2+ mobilization. In the present study, we investigated possible interactions between nucleotide and PTH signaling pathways in osteoblastic cells. The cytosolic free Ca2+ concentration ([Ca2+]i) of UMR-106 osteoblastic cells was monitored by fluorescence spectrophotometry. PTH (0.01-1 microM; bovine 1-84 or human 1-34) induced a small transient elevation of [Ca2+]i, lasting less than 1 min. A number of nucleotides, including ATP, UTP, and UDP, induced transient elevation of [Ca2+]i and potentiated the subsequent Ca2+ response to PTH. Of the nucleotides tested, UDP was the most effective at potentiating the PTH-induced Ca2+ transient. Treatment of cells with UDP (100 microM for 2.5 min), but not inorganic phosphate or uridine, reversibly potentiated the Ca2+ response to PTH (0.1 microM) by 11 +/- 2-fold (mean +/- SEM; n = 39). In contrast, UDP did not affect the cAMP response to PTH, indicating a selective action on Ca2+ signaling. Potentiation of the Ca2+ signal was still observed in the absence of extracellular Ca2+, establishing that nucleotides enhance PTH-induced release of Ca2+ from intracellular stores. Studies using selective purinoceptor agonists suggest that potentiation of PTH signaling is mediated by the P2U receptor subtype. In vivo, nucleotides released during trauma or inflammation may modulate PTH-induced Ca2+ signaling in osteoblasts.

Mechanism of nucleotide sensing in group II chaperonins.

PubMed

Pereira, Jose H; Ralston, Corie Y; Douglas, Nicholai R; Kumar, Ramya; Lopez, Tom; McAndrew, Ryan P; Knee, Kelly M; King, Jonathan A; Frydman, Judith; Adams, Paul D

2012-02-01

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.

The Single Nucleotide Polymorphism Consortium

NASA Technical Reports Server (NTRS)

Morgan, Michael

2003-01-01

I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

Code of Federal Regulations, 2014 CFR

2014-07-01

...” means those amino acids other than “Xaa” and those nucleotide bases other than “n”defined in accordance... 37 Patents, Trademarks, and Copyrights 1 2014-07-01 2014-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences...

37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

Code of Federal Regulations, 2013 CFR

2013-07-01

...” means those amino acids other than “Xaa” and those nucleotide bases other than “n”defined in accordance... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences...

37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

Code of Federal Regulations, 2012 CFR

2012-07-01

...” means those amino acids other than “Xaa” and those nucleotide bases other than “n”defined in accordance... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences...

Prokaryotic nucleotide composition is shaped by both phylogeny and the environment.

PubMed

Reichenberger, Erin R; Rosen, Gail; Hershberg, Uri; Hershberg, Ruth

2015-04-09

The causes of the great variation in nucleotide composition of prokaryotic genomes have long been disputed. Here, we use extensive metagenomic and whole-genome data to demonstrate that both phylogeny and the environment shape prokaryotic nucleotide content. We show that across environments, various phyla are characterized by different mean guanine and cytosine (GC) values as well as by the extent of variation on that mean value. At the same time, we show that GC-content varies greatly as a function of environment, in a manner that cannot be entirely explained by disparities in phylogenetic composition. We find environmentally driven differences in nucleotide content not only between highly diverged environments (e.g., soil, vs. aquatic vs. human gut) but also within a single type of environment. More specifically, we demonstrate that some human guts are associated with a microbiome that is consistently more GC-rich across phyla, whereas others are associated with a more AT-rich microbiome. These differences appear to be driven both by variations in phylogenetic composition and by environmental differences-which are independent of these phylogenetic composition differences. Combined, our results demonstrate that both phylogeny and the environment significantly affect nucleotide composition and that the environmental differences affecting nucleotide composition are far subtler than previously appreciated. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Pd-catalyzed versus uncatalyzed, PhI(OAc)2-mediated cyclization reactions of N6-([1,1'-biaryl]-2-yl)adenine nucleosides.

PubMed

Satishkumar, Sakilam; Poudapally, Suresh; Vuram, Prasanna K; Gurram, Venkateshwarlu; Pottabathini, Narender; Sebastian, Dellamol; Yang, Lijia; Pradhan, Padmanava; Lakshman, Mahesh K

2017-11-09

In this work we have assessed reactions of N 6 -([1,1'-biaryl]-2-yl)adenine nucleosides with Pd(OAc) 2 and PhI(OAc) 2 , via a Pd II /Pd IV redox cycle. The substrates are readily obtained by Pd/Xantphos-catalyzed reaction of adenine nucleosides with 2-bromo-1,1'-biaryls. In PhMe, the N 6 -biarylyl nucleosides gave C6-carbazolyl nucleoside analogues by C-N bond formation with the exocyclic N 6 nitrogen atom. In the solvent screening for the Pd-catalyzed reactions, an uncatalyzed process was found to be operational. It was observed that the carbazolyl products could also be obtained in the absence of a metal catalyst by reaction with PhI(OAc) 2 in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Thus, under Pd catalysis and in HFIP, reactions proceed to provide carbazolyl nucleoside analogues, with some differences. If reactions of N 6 -biarylyl nucleoside substrates were conducted in MeCN, formation of aryl benzimidazopurinyl nucleoside derivatives was observed in many cases by C-N bond formation with the N 1 ring nitrogen atom of the purine (carbazole and benzimidazole isomers are readily separated by chromatography). Whereas Pd II /Pd IV redox is responsible for carbazole formation under the metal-catalyzed conditions, in HFIP and MeCN radical cations and/or nitrenium ions can be intermediates. An extensive set of radical inhibition experiments was conducted and the data are presented.

Statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza A viruses.

PubMed Central

Ina, Y; Gojobori, T

1994-01-01

To examine whether positive selection operates on the hemagglutinin 1 (HA1) gene of human influenza A viruses (H1 subtype), 21 nucleotide sequences of the HA1 gene were statistically analyzed. The nucleotide sequences were divided into antigenic and nonantigenic sites. The nucleotide diversities for antigenic and nonantigenic sites of the HA1 gene were computed at synonymous and nonsynonymous sites separately. For nonantigenic sites, the nucleotide diversities were larger at synonymous sites than at nonsynonymous sites. This is consistent with the neutral theory of molecular evolution. For antigenic sites, however, the nucleotide diversities at nonsynonymous sites were larger than those at synonymous sites. These results suggest that positive selection operates on antigenic sites of the HA1 gene of human influenza A viruses (H1 subtype). PMID:8078892

Crystal structure of an intermolecular 2:1 complex between adenine and thymine. Evidence for both Hoogsteen and 'quasi-Watson-Crick' interactions.

PubMed

Chandrasekhar, Sosale; Naik, Tangali R Ravikumar; Nayak, Susanta K; Row, Tayur N Guru

2010-06-15

The titled complex, obtained by co-crystallization (EtOH/25 degrees C), is apparently the only known complex of the free bases. Its crystal structure, as determined by X-ray diffraction at both 90 K and 313 K, showed that one A-T pair involves a Hoogsteen interaction, and the other a Watson-Crick interaction but only with respect to the adenine unit. The absence of a clear-cut Watson-Crick base pair raises intriguing questions about the basis of the DNA double helix. Copyright 2010 Elsevier Ltd. All rights reserved.

The role of free radicals in cold injuries

NASA Astrophysics Data System (ADS)

Bhaumik, G.; Srivastava, K. K.; Selvamurthy, W.; Purkayastha, S. S.

1995-12-01

Cold injury is a tissue trauma produced by exposure to freezing temperatures and even brief exposure to a severely cold and windy environment. Rewarming of frozen tissue is associated with blood reperfusion and the simultaneous generation of free oxygen radicals. In this review is discussed the current understanding of the mechanism of action of free oxygen radicals as related to cold injury during rewarming. Decreased energy stores during ischaemia lead to the accumulation of adenine nucleotides and liberation of free fatty acids due to the breakdown of lipid membranes. On rewarming, free fatty acids are metabolized via cyclo-oxygenase and adenine nucleotides are metabolized via the xanthine oxidase pathway. These may be the source of free oxygen radicals. Leukocytes may also play a major role in the pathogenesis of cold injury. Oxygen radical scavengers, such as superoxide dismutase and catalase, may help to reduce the cold induced injury but their action is limited due to the inability readily to cross the plasma membrane. Lipid soluble antioxidants are likely to be more effective scavengers because of their presence in membranes where peroxidative reactions can be arrested.

Impaired mitochondrial Ca{sup 2+} homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kleist-Retzow, Juergen-Christoph von; Hue-Tran Hornig-Do; Schauen, Matthias

2007-08-15

Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca{sup 2+} homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNA{sup Leu(UUR)}, as well as Rho{sup 0} cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decreasemore » of cellular total adenine nucleotide pools and a concomitant shift in the AMP + ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.« less

Metabolic intervention to affect myocardial recovery following ischemia.

PubMed Central

Pasque, M K; Wechsler, A S

1984-01-01

Myocardial recovery during reperfusion following ischemia is critical to patient survival in a broad spectrum of clinical settings. Myocardial functional recovery following ischemia correlates well with recovery of myocardial adenosine triphosphate (ATP). Adenosine triphosphate recovery is uniformly incomplete during reperfusion following moderate ischemic injury and is therefore subject to manipulation by metabolic intervention. By definition ATP recovery is limited either by (1) energy availability and application in the phosphorylation of adenosine monophosphate (AMP) to ATP or (2) availability of AMP for this conversion. Experimental data suggest that substrate energy and the mechanisms required for its application in the creation of high energy phosphate bonds (AMP conversion to ATP) are more than adequate during reperfusion following moderate ischemic injury. Adenosine monophosphate availability, however, is inadequate following ischemia due to loss of diffusable adenine nucleotide purine metabolites. These purine precursors are necessary to fuel adenine nucleotide salvage pathways. Metabolic interventions that enhance AMP recovery rather than those that improve substrate energy availability during reperfusion are therefore recommended. The mechanisms of various metabolic interventions are discussed in this framework along with the rationale for or against their clinical application. PMID:6428332