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

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.

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

Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

PubMed Central

Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

2012-01-01

SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

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.

A nucleotide binding rectification Brownian ratchet model for translocation of Y-family DNA polymerases

PubMed Central

2011-01-01

Y-family DNA polymerases are characterized by low-fidelity synthesis on undamaged DNA and ability to catalyze translesion synthesis over the damaged DNA. Their translocation along the DNA template is an important event during processive DNA synthesis. In this work we present a Brownian ratchet model for this translocation, where the directed translocation is rectified by the nucleotide binding to the polymerase. Using the model, different features of the available structures for Dpo4, Dbh and polymerase ι in binary and ternary forms can be easily explained. Other dynamic properties of the Y-family polymerases such as the fast translocation event upon dNTP binding for Dpo4 and the considerable variations of the processivity among the polymerases can also be well explained by using the model. In addition, some predicted results of the DNA synthesis rate versus the external force acting on Dpo4 and Dbh polymerases are presented. Moreover, we compare the effect of the external force on the DNA synthesis rate of the Y-family polymerase with that of the replicative DNA polymerase. PMID:21699732

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

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.

Applications of adenine nucleotide measurements in oceanography

NASA Technical Reports Server (NTRS)

Holm-Hansen, O.; Hodson, R.; Azam, F.

1975-01-01

The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.

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.

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

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.

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)translocation through an electrode-equipped nanopore. The results represent interest for the theorists and practitioners in the field of third generation sequencing techniques as well as in the field of DNA chemistry. Copyright © 2015 Elsevier B.V. All rights reserved.

Ebselen induces mitochondrial permeability transition because of its interaction with adenine nucleotide translocase.

PubMed

Pavón, Natalia; Correa, Francisco; Buelna-Chontal, Mabel; Hernández-Esquivel, Luz; Chávez, Edmundo

2015-10-15

Mitochondrial permeability transition is a process established through massive Ca(2+) load in addition to an inducer reagent. Ebselen (Ebs), an antioxidant seleno compound, has been introduced as a reagent which inhibits mitochondrial dysfunction induced by permeability transition. Paradoxically enough, it has been shown that Ebs may also be able to induce the opening of the mitochondrial non-selective pores. This study was performed with the purpose of establishing the membrane system involved in Ebs-induced pore opening. Permeability transition was appraised by analyzing the following: i) matrix Ca(2+) release, and mitochondrial swelling, ii) efflux of cytochrome c, and iii) the inhibition of superoxide dismutase. All of these adverse reactions were inhibited by N-ethylmaleimide and cyclosporin A. At concentrations from 5 to 20 μM, we found that Ebs induces non-specific membrane permeability. Remarkably, Ebs blocks the binding of the fluorescent reagent eosin-5-maleimide to the thiol groups of the adenine nucleotide translocase. Based on the above, it is tempting to hypothesize that Ebs induces pore opening through its binding to the ADP/ATP carrier. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

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

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

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.

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.

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.

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

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.

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

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

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

PubMed Central

Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

2013-01-01

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

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.

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.

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

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.

Precise detection of chromosomal translocation or inversion breakpoints by whole-genome sequencing.

PubMed

Suzuki, Toshifumi; Tsurusaki, Yoshinori; Nakashima, Mitsuko; Miyake, Noriko; Saitsu, Hirotomo; Takeda, Satoru; Matsumoto, Naomichi

2014-12-01

Structural variations (SVs), including translocations, inversions, deletions and duplications, are potentially associated with Mendelian diseases and contiguous gene syndromes. Determination of SV-related breakpoints at the nucleotide level is important to reveal the genetic causes for diseases. Whole-genome sequencing (WGS) by next-generation sequencers is expected to determine structural abnormalities more directly and efficiently than conventional methods. In this study, 14 SVs (9 balanced translocations, 1 inversion and 4 microdeletions) in 9 patients were analyzed by WGS with a shallow (5 × ) to moderate read coverage (20 × ). Among 28 breakpoints (as each SV has two breakpoints), 19 SV breakpoints had been determined previously at the nucleotide level by any other methods and 9 were uncharacterized. BreakDancer and Integrative Genomics Viewer determined 20 breakpoints (16 translocation, 2 inversion and 2 deletion breakpoints), but did not detect 8 breakpoints (2 translocation and 6 deletion breakpoints). These data indicate the efficacy of WGS for the precise determination of translocation and inversion breakpoints.

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

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.

NOS1 mediates AP1 nuclear translocation and inflammatory response.

PubMed

Srivastava, Mansi; Baig, Mirza S

2018-06-01

A hallmark of the AP1 functioning is its nuclear translocation, which induces proinflammatory cytokine expression and hence the inflammatory response. After endotoxin shock AP1 transcription factor, which comprises Jun, ATF2, and Fos family of proteins, translocates into the nucleus and induces proinflammatory cytokine expression. In the current study, we found, NOS1 inhibition prevents nuclear translocation of the AP1 transcription factor subunits. Pharmacological inhibition of NOS1 impedes translocation of subunits into the nucleus, suppressing the transcription of inflammatory genes causing a diminished inflammatory response. In conclusion, the study shows the novel mechanism of NOS1- mediated AP1 nuclear translocation, which needs to be further explored. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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.

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

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. Electronic supplementary information (ESI) available: Details of the data analysis; Fig. S1-S5 histograms of rupture lengths; histograms for Au-adenine and Au-amine interactions; Force-extension curve for MCH-Au interactions; normalized force-extension curves; theoretical length of the DNA oligomers. See DOI: 10.1039/c5nr05695k

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

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.

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

Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells.

PubMed

Fredholm, Bertil B; Assender, Jean W; Irenius, Eva; Kodama, Noriko; Saito, Naoaki

2003-06-01

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP3), and on activation of protein kinase C (PKC) was studied in DDT1 MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A1 and some as yet unidentified P2Y receptor(s). 2. Activation of either receptor type stimulated the production of InsP3 and raised intracellular calcium in DDT1 MF2 cells. Similarly, the A1 selective agonist N6-cyclopentyladenosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP(4-14) and induced a PKC translocation to the plasma membrane as determined using [3H]-phorbol dibutyrate (PDBu) binding in DDT1 MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected gamma-PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of gamma-PKC-GFP and activation of PKC. 3. Doses of the A1 agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca2+ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 microM) and UTP (or ATP, 2.5 microM), which per se caused no detectable translocation of either gamma- or epsilon-PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A1 receptors synergistically increases Ca2+ transients and translocation of PKC in DDT1 MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.

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.

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

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).

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.

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

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.

cAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane.

PubMed

Consonni, Sarah V; Gloerich, Martijn; Spanjaard, Emma; Bos, Johannes L

2012-03-06

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.

The uncoupling of catalysis and translocation in the viral RNA-dependent RNA polymerase

PubMed Central

Shu, Bo; Gong, Peng

2017-01-01

ABSTRACT The nucleotide addition cycle of nucleic acid polymerases includes 2 major events: the pre-chemistry active site closure leading to the addition of one nucleotide to the product chain; the post-chemistry translocation step moving the polymerase active site one position downstream on its template. In viral RNA-dependent RNA polymerases (RdRPs), structural and biochemical evidences suggest that these 2 events are not tightly coupled, unlike the situation observed in A-family polymerases such as the bacteriophage T7 RNA polymerase. Recently, an RdRP translocation intermediate crystal structure of enterovirus 71 shed light on how translocation may be controlled by elements within RdRP catalytic motifs, and a series of poliovirus apo RdRP crystal structures explicitly suggest that a motif B loop may assist the movement of the template strand in late stages of transcription. Implications of RdRP catalysis-translocation uncoupling and the remaining challenges to further elucidate RdRP translocation mechanism are also discussed. PMID:28277928

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.

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 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

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

Number of blastocysts biopsied as a predictive indicator to obtain at least one normal/balanced embryo following preimplantation genetic diagnosis with single nucleotide polymorphism microarray in translocation cases.

PubMed

Wang, Yi-Zi; Ding, Chen-Hui; Wang, Jing; Zeng, Yan-Hong; Zhou, Wen; Li, Rong; Zhou, Can-Quan; Deng, Ming-Fen; Xu, Yan-Wen

2017-01-01

The aim of this study is to investigate the minimum number of blastocysts for biopsy to increase the likelihood of obtaining at least one normal/balanced embryo in preimplantation genetic diagnosis (PGD) for translocation carriers. This blinded retrospective study included 55 PGD cycles for Robertsonian translocation (RT) and 181 cycles for reciprocal translocation (rcp) to indicate when only one of the couples carried a translocation. Single-nucleotide polymorphism microarray after trophectoderm biopsy was performed. Reliable results were obtained for 355/379 (93.7 %) biopsied blastocysts in RT group and 986/1053 (93.6 %) in rcp group. Mean numbers of biopsied embryos per patient, normal/balanced embryos per patient, and mean normal/balanced embryo rate per patient were 7.4, 3.1, and 40.7 % in RT group and 8.0, 2.1, and 27.3 %, respectively, in rcp group. In a regression model, three factors significantly affected the number of genetically transferrable embryos: number of biopsied embryos (P = 0.001), basal FSH level (P = 0.040), and maternal age (P = 0.027). ROC analysis with a cutoff of 1.5 was calculated for the number of biopsied embryos required to obtain at least one normal/balanced embryo for RT carriers. For rcp carriers, the cutoff was 3.5. The clinical pregnancy rate per embryo transfer was 44.2 and 42.6 % in RT and rcp groups (P = 0.836). The minimum numbers of blastocysts to obtain at least one normal/balanced embryo for RT and rcp were 2 and 4 under the conditions of female age < 37 years with a basal FSH level < 11.4 IU/L.

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.

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.

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

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

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

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.

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).

Molecular events during translocation and proofreading extracted from 200 static structures of DNA polymerase.

PubMed

Ren, Zhong

2016-09-06

DNA polymerases in family B are workhorses of DNA replication that carry out the bulk of the job at a high speed with high accuracy. A polymerase in this family relies on a built-in exonuclease for proofreading. It has not been observed at the atomic resolution how the polymerase advances one nucleotide space on the DNA template strand after a correct nucleotide is incorporated, that is, a process known as translocation. It is even more puzzling how translocation is avoided after the primer strand is excised by the exonuclease and returned back to the polymerase active site once an error occurs. The structural events along the bifurcate pathways of translocation and proofreading have been unwittingly captured by hundreds of structures in Protein Data Bank. This study analyzes all available structures of a representative member in family B and reveals the orchestrated event sequence during translocation and proofreading. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

DOE PAGES

Dangkulwanich, Manchuta; Ishibashi, Toyotaka; Liu, Shixin; ...

2013-09-24

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limitingmore » steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation.« less

SESN2 facilitates mitophagy by helping Parkin translocation through ULK1 mediated Beclin1 phosphorylation.

PubMed

Kumar, Ashish; Shaha, Chandrima

2018-01-12

Mitophagy, the selective degradation of mitochondria by autophagy, is crucial for the maintenance of healthy mitochondrial pool in cells. The critical event in mitophagy is the translocation of cytosolic Parkin, a ubiquitin ligase, to the surface of defective mitochondria. This study elucidates a novel role of SESN2/Sestrin2, a stress inducible protein, in mitochondrial translocation of PARK2/Parkin during mitophagy. The data demonstrates that SESN2 downregulation inhibits BECN1/Beclin1 and Parkin interaction, thereby preventing optimum mitochondrial accumulation of Parkin. SESN2 interacts with ULK1 (unc-51 like kinase 1) and assists ULK1 mediated phosphorylation of Beclin1 at serine-14 position required for binding with Parkin prior to mitochondrial translocation. The trigger for SESN2 activation and regulation of Parkin translocation is the generation of mitochondrial superoxide. Scavenging of mitochondrial superoxide lower the levels of SESN2, resulting in retardation of Parkin translocation. Importantly, we observe that SESN2 mediated cytosolic interaction of Parkin and Beclin1 is PINK1 independent but mitochondrial translocation of Parkin is PINK1 dependent. Together, these findings suggest the role of SESN2 as a positive regulator of Parkin mediated mitophagy.

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

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

PubMed Central

Koslover, Daniel J.; Fazal, Furqan M.; Mooney, Rachel A.; Landick, Robert; Block, Steven M.

2012-01-01

Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single- molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho- utilization (rut) site of the ? tR1 terminator. Our results are consistent with Rho complexes adopting two states, one that binds 57 ±2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ±2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation, and establish a novel experimental approach that should facilitate additional single- molecule studies of RNA-binding proteins. PMID:22885804

Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.

PubMed

Russell, R R; Bergeron, R; Shulman, G I; Young, L H

1999-08-01

Insulin increases glucose uptake through the translocation of GLUT-4 via a pathway mediated by phosphatidylinositol 3-kinase (PI3K). In contrast, myocardial glucose uptake during ischemia and hypoxia is stimulated by the translocation of GLUT-4 to the surface of cardiac myocytes through a PI3K-independent pathway that has not been characterized. AMP-activated protein kinase (AMPK) activity is also increased by myocardial ischemia, and we examined whether AMPK stimulates glucose uptake and GLUT-4 translocation. In isolated rat ventricular papillary muscles, 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, as well as cyanide-induced chemical hypoxia and insulin, increased 2-[(3)H]deoxyglucose uptake two- to threefold. Wortmannin, a PI3K inhibitor, did not affect either the AICAR- or the cyanide-stimulated increase in deoxyglucose uptake but eliminated the insulin-stimulated increase in deoxyglucose uptake. Immunofluorescence studies demonstrated translocation of GLUT-4 to the myocyte sarcolemma in response to stimulation with AICAR, cyanide, or insulin. Preincubation of papillary muscles with the kinase inhibitor iodotubercidin or adenine 9-beta-D-arabinofuranoside (araA), a precursor of araATP (a competitive inhibitor of AMPK), decreased AICAR- and cyanide-stimulated glucose uptake but did not affect basal or insulin-stimulated glucose uptake. In vivo infusion of AICAR caused myocardial AMPK activation and GLUT-4 translocation in the rat. We conclude that AMPK activation increases cardiac muscle glucose uptake through translocation of GLUT-4 via a pathway that is independent of PI3K. These findings suggest that AMPK activation may be important in ischemia-induced translocation of GLUT-4 in the heart.

JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation

PubMed Central

Lu, Ben; Antoine, Daniel J.; Kwan, Kevin; Lundbäck, Peter; Wähämaa, Heidi; Schierbeck, Hanna; Robinson, Melissa; Van Zoelen, Marieke A. D.; Yang, Huan; Li, Jianhua; Erlandsson-Harris, Helena; Chavan, Sangeeta S.; Wang, Haichao; Andersson, Ulf; Tracey, Kevin J.

2014-01-01

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release. PMID:24469805

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.

G protein βγ11 complex translocation is induced by Gi, Gq and Gs coupling receptors and is regulated by the α subunit type

PubMed Central

Azpiazu, Inaki; Akgoz, Muslum; Kalyanaraman, Vani; Gautam, N.

2008-01-01

G protein activation by Gi/Go coupling M2 muscarinic receptors, Gq coupling M3 receptors and Gs coupling β2 adrenergic receptors causes rapid reversible translocation of the G protein γ11 subunit from the plasma membrane to the Golgi complex. Co-translocation of the β1 subunit suggests that γ11 translocates as a βγ complex. Pertussis toxin ADP ribosylation of the αi subunit type or substitution of the C terminal domain of αo with the corresponding region of αs inhibits γ11 translocation demonstrating that α subunit interaction with a receptor and its activation are requirements for the translocation. The rate of γ11 translocation is sensitive to the rate of activation of the G protein α subunit. α subunit types that show high receptor activated rates of guanine nucleotide exchange in vitro support high rates of γ11 translocation compared to α subunit types that have a relatively lower rate of guanine nucleotide exchange. The results suggest that the receptor induced translocation of γ11 is controlled by the rate of cycling of the G protein through active and inactive forms. They also demonstrate that imaging of γ11 translocation can be used as a non-invasive tool to measure the relative activities of wild type or mutant receptor and α subunit types in a live cell. PMID:16242307

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

Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

PubMed

Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

2016-08-01

This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation.

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

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.

Growth factor deprivation induces cytosolic translocation of SIRT1

NASA Astrophysics Data System (ADS)

Meng, Chengbo; Xing, Da; Wu, Shengnan; Huang, Lei

2010-02-01

Sirtuin type 1 (SIRT1), a NAD+-dependent histone deacetylases, plays a critical role in cellular senescence, aging and longevity. In general, SIRT1 is localized in nucleus and is believed as a nuclear protein. Though overexpression of SIRT1 delays senescence, SIRT1-protein levels decline naturally in thymus and heart during aging. In the present studies, we investigated the subcellular localization of SIRT1 in response to growth factor deprivation in African green monkey SV40-transformed kidney fibroblast cells (COS-7). Using SIRT1-EGFP fluorescence reporter, we found that SIRT1 localized to nucleus in physiological conditions. We devised a model enabling cell senescence via growth factor deprivation, and we found that SIRT1 partially translocated to cytosol under the treatment, suggesting a reduced level of SIRT1's activity. We found PI3K/Akt pathway was involved in the inhibition of SIRT1's cytosolic translocation, because inhibition of these kinases significantly decreased the amount of SIRT1 maintained in nucleus. Taken together, we demonstrated that growth factor deprivation induces cytosolic translocation of SIRT1, which suggesting a possible connection between cytoplasm-localized SIRT1 and the aging process.

A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes

PubMed Central

Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2016-01-01

Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity. PMID:27739494

Homoeologous cloning of omega-secalin gene family in a wheat 1BL/1RS translocation.

PubMed

Chai, Jian Fang; Liu, Xu; Jia, Ji Zeng

2005-08-01

Wheat 1BL/1RS translocations are widely planted in China as well as in most of the wheat producing area in the world for their good qualities of disease resistance and high yield. 1BL/1RS translocations are however poor in bread making, partially caused by a family of small monomeric proteins, omega-secalins, which are encoded by genes on 1RS. Based on published sequence of a rye omega-secalin gene we designed a pair of primers to cover the whole mature protein coding sequence. A major band could be amplified from 1BL/1RS translocations but not from euploid wheat. Using this primer set we conducted PCR amplification by using high fidelity Pfu polymerase on the genomic DNAs and cDNAs purified from a 1BL/1RS translocation Lankao 906. Sequencing analysis indicated that this gene family contains several members of 1150 bp, 1076 bp, 1075 bp, 1052 bp and 1004 bp genes, including two pseudogenes and three active genes. The gene transcripts were differentially expressed in developing seeds.

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.

Tributyltin interacts with mitochondria and induces cytochrome c release.

PubMed Central

Nishikimi, A; Kira, Y; Kasahara, E; Sato, E F; Kanno, T; Utsumi, K; Inoue, M

2001-01-01

Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis. PMID:11368793

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

PAK1 translocates into nucleus in response to prolactin but not to estrogen

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

Oladimeji, Peter, E-mail: Peter.Oladimeji@rockets.utoledo.edu; Diakonova, Maria, E-mail: mdiakon@utnet.utoledo.edu

2016-04-22

Tyrosyl phosphorylation of the p21-activated serine–threonine kinase 1 (PAK1) has an essential role in regulating PAK1 functions in breast cancer cells. We previously demonstrated that PAK1 serves as a common node for estrogen (E2)- and prolactin (PRL)-dependent pathways. We hypothesize herein that intracellular localization of PAK1 is affected by PRL and E2 treatments differently. We demonstrate by immunocytochemical analysis that PAK1 nuclear translocation is ligand-dependent: only PRL but not E2 stimulated PAK1 nuclear translocation. Tyrosyl phosphorylation of PAK1 is essential for this nuclear translocation because phospho-tyrosyl-deficient PAK1 Y3F mutant is retained in the cytoplasm in response to PRL. We confirmedmore » these data by Western blot analysis of subcellular fractions. In 30 min of PRL treatment, only 48% of pTyr-PAK1 is retained in the cytoplasm of PAK1 WT clone while 52% re-distributes into the nucleus and pTyr-PAK1 shuttles back to the cytoplasm by 60 min of PRL treatment. In contrast, PAK1 Y3F is retained in the cytoplasm. E2 treatment causes nuclear translocation of neither PAK1 WT nor PAK1 Y3F. Finally, we show by an in vitro kinase assay that PRL but not E2 stimulates PAK1 kinase activity in the nuclear fraction. Thus, PAK1 nuclear translocation is ligand-dependent: PRL activates PAK1 and induces translocation of activated pTyr-PAK1 into nucleus while E2 activates pTyr-PAK1 only in the cytoplasm. - Highlights: • Prolactin but not estrogen causes translocation of PAK1 into nucleus. • Tyrosyl phosphorylation of PAK1 is required for nuclear localization. • Prolactin but not estrogen stimulates PAK1 kinase activity in nucleus.« less

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

Production and identification of wheat - Agropyron cristatum (1.4P) alien translocation lines.

PubMed

Liu, Wei-Hua; Luan, Yang; Wang, Jing-Chang; Wang, Xiao-Guang; Su, Jun-Ji; Zhang, Jin-Peng; Yang, Xin-Ming; Gao, Ai-Nong; Li, Li-Hui

2010-06-01

The P genome of Agropyron Gaertn., a wild relative of wheat, contains an abundance of desirable genes that can be utilized as genetic resources to improve wheat. In this study, wheat - Aegilops cylindrica Host gametocidal chromosome 2C addition lines were crossed with wheat - Agropyron cristatum (L.) Gaertn. disomic addition line accession II-21 with alien recombinant chromosome (1.4)P. We successfully induced wheat - A. cristatum alien chromosomal translocations for the first time. The frequency of translocation in the progeny was 3.75%, which was detected by molecular markers and genomic in situ hybridization (GISH). The translocation chromosomes were identified by dual-color GISH /fluorescence in situ hybridization (FISH). The P genomic DNA was used as probe to detect the (1.4)P chromosome fragment, and pHvG39, pAs1, or pSc119.2 repeated sequences were used as probes to identify wheat translocated chromosomes. The results showed that six types of translocations were identified in the three wheat - A. cristatum alien translocation lines, including the whole arm or terminal portion of a (1.4)P chromosome. The (1.4)P chromosome fragments were translocated to wheat chromosomes 1B, 2B, 5B, and 3D. The breakpoints were located at the centromeres of 1B and 2B, the pericentric locations of 5BS, and the terminals of 5BL and 3DS. In addition, we obtained 12 addition-deletion lines that contained alien A. cristatum chromosome (1.4)P in wheat background. All of these wheat - A. cristatum alien translocation lines and addition-deletion lines would be valuable for identifying A. cristatum chromosome (1.4)P-related genes and providing genetic resources and new germplasm accessions for the genetic improvement of wheat. The specific molecular markers of A. cristatum (1.4)P chromosome have been developed and used to track the (1.4)P chromatin.

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

Carrier-mediated translocation of uridine diphosphate glucose into the lumen of endoplasmic reticulum-derived vesicles from rat liver.

PubMed Central

Vanstapel, F; Blanckaert, N

1988-01-01

Radiolabeled UDPGlc incubated with rough endoplasmic reticulum (RER)-derived microsomes from rat liver became associated with the vesicles. This microsomal uptake of nucleotide sugar was time and temperature dependent. Analysis of the molecular species containing radiolabel revealed that initial uptake represented entry of predominantly intact UDPGlc in the microsomes. Conclusive evidence for proper translocation of UDPGlc across the microsomal membrane into the intravesicular space was obtained by demonstrating that UDPGlc was transported into an osmotically sensitive compartment. Microsomal uptake of UDPGlc exhibited features characteristic of carrier-mediated transport including saturation, specificity, and countertransport. Inhibition and trans-stimulation studies showed that other uridine-containing nucleotide sugars and 5'-UMP were substrates of the postulated microsomal carrier system for UDPGlc, while cytosine- or guanosine-containing nucleotides and non-5'-uridine monophosphates were, at best, very poor substrates. UDPGlc translocation activities were lower in smooth microsomal fractions than in the RER-derived vesicles, indicating that contamination with Golgi membranes could not be responsible for microsomal transport of UDPGlc. Our findings suggest that rat liver endoplasmic reticulum possesses a carrier system mediating proper translocation of UDPGlc and 5'-uridine-substituted structural analogues across the membrane. PMID:3417868

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.

Featured Article: Effect of copper on nuclear translocation of copper chaperone for superoxide dismutase-1

PubMed Central

Wang, Lin; Ge, Yan

2016-01-01

Copper chaperone for superoxide dismutase-1 (CCS-1), facilitating copper insertion into superoxide dismutase 1 (SOD-1), is present in the nucleus. However, it is unknown how CCS-1 is translocated to the nucleus. The present study was undertaken to determine the effect of copper on nuclear translocation of CCS-1. Human umbilical vein endothelial cells (HUVECs) were subjected to hypoxia, causing an increase in both copper and CCS-1 in the nucleus. Treatment with tetraethylenepentamine (TEPA) not only decreased the total cellular concentration and the nuclear translocation of copper, but also completely suppressed the entry of CCS-1 to the nucleus. On the other hand, siRNA targeting CCS-1 neither inhibited the increase in total concentrations nor blocked the nuclear translocation of copper. This study thus demonstrates that under hypoxia condition, both copper and CCS-1 are transported to the nucleus. The nuclear translocation of CCS-1 is copper dependent, but the nuclear translocation of copper could take place alternatively in a CCS-1-independent pathway. PMID:27190267

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.

Molecular Mechanism of Processive 3' to 5' RNA Translocation in the Active Subunit of the RNA Exosome Complex.

PubMed

Vuković, Lela; Chipot, Christophe; Makino, Debora L; Conti, Elena; Schulten, Klaus

2016-03-30

Recent experimental studies revealed structural details of 3' to 5' degradation of RNA molecules, performed by the exosome complex. ssRNA is channeled through its multisubunit ring-like core into the active site tunnel of its key exonuclease subunit Rrp44, which acts both as an enzyme and a motor. Even in isolation, Rrp44 can pull and sequentially cleave RNA nucleotides, one at a time, without any external energy input and release a final 3-5 nucleotide long product. Using molecular dynamics simulations, we identify the main factors that control these processes. Our free energy calculations reveal that RNA transfer from solution into the active site of Rrp44 is highly favorable, but dependent on the length of the RNA strand. While RNA strands formed by 5 nucleotides or more correspond to a decreasing free energy along the translocation coordinate toward the cleavage site, a 4-nucleotide RNA experiences a free energy barrier along the same direction, potentially leading to incomplete cleavage of ssRNA and the release of short (3-5) nucleotide products. We provide new insight into how Rrp44 catalyzes a localized enzymatic reaction and performs an action distributed over several RNA nucleotides, leading eventually to the translocation of whole RNA segments into the position suitable for cleavage.

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

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.

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.

TGF-{beta} signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

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

Luciakova, Katarina, E-mail: katarina.luciakova@savba.sk; Kollarovic, Gabriel; Kretova, Miroslava

2011-08-05

Highlights: {yields} TGF-{beta} induces the formation of unique nuclear NF1/Smad4 complexes that repress expression of the ANT-2 gene. {yields} Repression is mediated through an NF1-dependent repressor element in the promoter. {yields} The formation of NF1/Smad4 complexes and the repression of ANT2 are prevented by inhibitors of p38 kinase and TGF-{beta} RI. {yields} NF1/Smad complexes implicate novel role for NF1 and Smad proteins in the regulation of growth. -- Abstract: We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G.more » Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-{beta}, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-{beta} is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-{beta} are prevented by inhibitors of TGF-{beta} RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-{beta} and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-{beta} signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.« less

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.

PubMed

Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid; Rapoport, Tom A

2017-05-12

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

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

Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, wemore » report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.« less

Slowing down DNA translocation by a nanofiber meshed layer

NASA Astrophysics Data System (ADS)

Zhao, Yue; Xie, Wanyi; Tian, Enling; Ren, Yiwei; Zhu, Jifeng; Deng, Yunsheng; He, Shixuan; Liang, Liyuan; Wang, Yunjiao; Zhou, Daming; Wang, Deqiang

2018-01-01

Due to the weak interaction between DNA molecules and the inner surface of nanopores, DNA translocation is very fast, just leaving a short current drop without sufficient information to recognise the nucleotide sequence in the strand. In this paper, we propose a nanopore-nanofiber mesh hybridized structure to decelerate DNA translocation speed. Experimental results reveal that due to hydrophobic interaction between the DNA fragments and the nanofibers, the DNA moving speed can be retarded to two orders of magnitude slower. Furthermore, according to theory simulations, the additional fiber layer will reduce the electric field in the channel but elongate the capture region at the pore orifice, which will be helpful for increasing the capture rate and extending the DNA dwelling time in the meanwhile.

Substrate-Dependence of Competitive Nucleotide Pyrophosphatase/Phosphodiesterase1 (NPP1) Inhibitors

PubMed Central

Lee, Sang-Yong; Sarkar, Soumya; Bhattarai, Sanjay; Namasivayam, Vigneshwaran; De Jonghe, Steven; Stephan, Holger; Herdewijn, Piet; El-Tayeb, Ali; Müller, Christa E.

2017-01-01

Nucleotide pyrophosphatase/phosphodiesterase type 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its major substrate is ATP which is converted to AMP and diphosphate. NPP1 was proposed as a new therapeutic target in brain cancer and immuno-oncology. Several NPP1 inhibitors have been reported to date, most of which were evaluated vs. the artificial substrate p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP). Recently, we observed large discrepancies in inhibitory potencies for a class of competitive NPP1 inhibitors when tested vs. the artificial substrate p-Nph-5′-TMP as compared to the natural substrate ATP. Therefore, the goal of the present study was to investigate whether inhibitors of human NPP1 generally display substrate-dependent inhibitory potency. Systematic evaluation of nucleotidic as well as non-nucleotidic NPP1 inhibitors revealed significant differences in determined Ki values for competitive, but not for non- and un-competitive inhibitors when tested vs. the frequently used artificial substrate p-Nph-5′-TMP as compared to ATP. Allosteric modulation of NPP1 by p-Nph-5′-TMP may explain these discrepancies. Results obtained using the AMP derivative p-nitrophenyl 5′-adenosine monophosphate (p-Nph-5′-AMP) as an alternative artificial substrate correlated much better with those employing the natural substrate ATP. PMID:28261095

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.

The effect of O-GlcNAcylation on hnRNP A1 translocation and interaction with transportin1.

PubMed

Roth, Shira; Khalaila, Isam

2017-01-01

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a major pre-mRNA binding protein involved in transcription and translation. Although predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytosol, delivering its anchored pre-mRNA for further processing. Translocation is important for hnRNP A1 to accomplish its transcriptional and translational roles. Transportin1 (Trn1), a translocation protein, facilitates the translocation of hnRNP A1 back to the nucleus. Moreover, phosphorylation of serine residues at hnRNP A1 C-terminal domain affects its translocation. In this study, we found that phosphorylation is not the only modification that hnRNP A1 undergoes, but also O-linked N-acetylglucosaminylation (O-GlcNAcylation) could occur. Several putative novel O-GlcNAcylation and phosphorylation sites in hnRNP A1 were mapped. Whereas enhanced O-GlcNAcylation increased hnRNP A1 interaction with Trn1, enhanced phosphorylation reduced the interaction between the proteins. In addition, elevated O-GlcNAcylation resulted in hnRNP A1 seclusion in the nucleus, whereas elevated phosphorylation resulted in its accumulation in the cytosol. These findings suggest that a new player, i.e., O-GlcNAcylation, regulates hnRNP A1 translocation and interaction with Trn1, possibly affecting its function. There is a need for further study, to elucidate the role of O-GlcNAcylation in the regulation of the specific activities of hnRNP A1 in transcription and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

BCL2 oncogene translocation is mediated by a chi-like consensus

PubMed Central

1992-01-01

Examination of 64 translocations involving the major breakpoint region (mbr) of the BCL2 oncogene and the immunoglobulin heavy chain locus identified three short (14, 16, and 18 bp) segments within the mbr at which translocations occurred with very high frequency. Each of these clusters was associated with a 15-bp region of sequence homology, the principal one containing an octamer related to chi, the procaryotic activator of recombination. The presence of short deletions and N nucleotide additions at the breakpoints, as well as involvement of JH and DH coding regions, suggested that these sequences served as signals capable of interacting with the VDJ recombinase complex, even though no homology with the traditional heptamer/spacer/nonamer (IgRSS) existed. Furthermore, the BCL2 signal sequences were employed in a bidirectional fashion and could mediate recombination of one mbr region with another. Segments homologous to the BCL2 signal sequences flanked individual members of the XP family of diversity gene segments, which were themselves highly overrepresented in the reciprocal products (18q-) of BCL2 translocation. We propose that the chi-like signal sequences of BCL2 represent a distinct class of recognition sites for the recombinase complex, responsible for initiating interactions between regions of DNA separated by great distances, and that BCL2 translocation begins by a recombination event between mbr and DXP chi signals. Since recombinant joints containing chi, not IgRSS, occur in brain cells expressing RAG-1 (Matsuoka, M., F. Nagawa, K. Okazaki, L. Kingsbury, K. Yoshida, U. Muller, D. T. Larue, J. A. Winer, and H. Sakano. 1991. Science [Wash. DC]. 254:81; reference 1), we further suggest that the product of this gene could mediate both BCL2 translocation and the first step of normal DJ assembly through the creation of chi joints, rather than signal or coding joints. PMID:1588282

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.

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.

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

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.

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

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.

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.

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.

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

Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation

PubMed Central

Jo, Stephanie Y.; Granowicz, Eric M.; Maillard, Ivan; Thomas, Dafydd

2011-01-01

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes. PMID:21398221

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

PubMed

Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

2014-08-01

Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

PubMed Central

Niu, Ying-Lin; Li, Ya-Jun; Wang, Jing-Bo; Lu, Yuan-Yuan; Liu, Zhen-Xiong; Feng, Shan-Shan; Hu, Jian-Guo; Zhai, Hui-Hong

2016-01-01

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27

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.

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.

Hydroquinone Increases 5-Hydroxymethylcytosine Formation through Ten Eleven Translocation 1 (TET1) 5-Methylcytosine Dioxygenase*

PubMed Central

Coulter, Jonathan B.; O'Driscoll, Cliona M.; Bressler, Joseph P.

2013-01-01

DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase. PMID:23940045

Hydroquinone increases 5-hydroxymethylcytosine formation through ten eleven translocation 1 (TET1) 5-methylcytosine dioxygenase.

PubMed

Coulter, Jonathan B; O'Driscoll, Cliona M; Bressler, Joseph P

2013-10-04

DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase.

Molecular targets and signaling pathways regulated by nuclear translocation of syndecan-1.

PubMed

Szatmári, Tünde; Mundt, Filip; Kumar-Singh, Ashish; Möbus, Lena; Ötvös, Rita; Hjerpe, Anders; Dobra, Katalin

2017-12-08

The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-β pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκβ, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix

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.

Nuclear EGFRvIII resists hypoxic microenvironment induced apoptosis via recruiting ERK1/2 nuclear translocation

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

Xie, Hui; Yang, Jinfeng; Xing, Wenjing

2016-02-05

Glioblastoma (GBM) is the most aggressive type of primary brain tumor. Its interaction with the tumor microenvironment promotes tumor progression. Furthermore, GBM bearing expression of EGFRvIII displays more adaptation to tumor microenvironment related stress. But the mechanisms were poorly understood. Here, we presented evidence that in the human U87MG glioblastoma tumor model, EGFRvIII overexpression led aberrant kinase activation and nuclear translocation of EGFRvIII/ERK1/2 under hypoxia, which induced growth advantage by resisting apoptosis. Additionally, EGFRvIII defective in nuclear entry impaired this capacity in hypoxia adaptation, and partially interrupted ERK1/2 nuclear translocation. Pharmacology or genetic interference ERK1/2 decreased hypoxia resistance triggered bymore » EGFRvIII expression, but not EGFRvIII nuclear translocation. In summary, this study identified a novel role for EGFRvIII in hypoxia tolerance, supporting an important link between hypoxia and subcellular localization alterations of the receptor. - Highlights: • Nuclear translocation of EGFRvIII contributes to GBM cell apoptotic resistance by hypoxia. • Nuclear ERK1/2 facilitates EGFRvIII in hypoxia resistance. • EGFRvIII nuclear translocation is not dependent on ERK1/2.« less

SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

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

Joo, Hyun-Yoo; Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-713; Woo, Seon Rang

2012-08-10

Highlights: Black-Right-Pointing-Pointer SIRT1 serves to retain GAPDH in the cytosol, preventing GAPDH nuclear translocation. Black-Right-Pointing-Pointer When SIRT1 is depleted, GAPDH translocation occurs even in the absence of stress. Black-Right-Pointing-Pointer Upon irradiation, SIRT1 interacts with GAPDH. Black-Right-Pointing-Pointer SIRT1 prevents irradiation-induced nuclear translocation of GAPDH. Black-Right-Pointing-Pointer SIRT1 presence rather than activity is essential for inhibiting GAPDH translocation. -- Abstract: Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggeredmore » nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.« less

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.

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.

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.

Phloem Water Relations and Translocation 1

PubMed Central

Kaufmann, Merrill R.; Kramer, Paul J.

1967-01-01

Satisfactory measurements of phloem water potential of trees can be obtained with the Richards and Ogata psychrometer and the vapor equilibration techniques, although corrections for loss of dry weight and for heating by respiration are required for the vapor equilibrium values. The psychrometer technique is the more satisfactory of the 2 because it requires less time for equilibration, less tissue, and less handling of tissue. Phloem water potential of a yellow-poplar tree followed a diurnal pattern quite similar to that of leaves, except that the values were higher (less negative) and changed less than in the leaves. The psychrometer technique permits a different approach to the study of translocation in trees. Measurements of water potential of phloem discs followed by freezing of samples and determination of osmotic potential allows estimation of turgor pressure in various parts of trees as the difference between osmotic potential and total water potential. This technique was used in evaluating gradients in water potential, osmotic potential, and turgor pressure in red maple trees. The expected gradients in osmotic potential were observed in the phloem, osmotic potential of the cell sap increasing (sap becoming more dilute) down the trunk. However, values of water potential were such that a gradient in turgor pressure apparently did not exist at a time when rate of translocation was expected to be high. These results do not support the mass flow theory of translocation favored by many workers. PMID:16656495

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.

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...

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.

Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation.

PubMed

Samih, N; Hovsepian, S; Aouani, A; Lombardo, D; Fayet, G

2000-11-01

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached

Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells

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

Voelker, D.R.

1989-12-01

Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({supmore » 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.« less

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.

Centromere structure and function analysis in wheat-rye translocation lines.

PubMed

Wang, Jing; Liu, Yalin; Su, Handong; Guo, Xianrui; Han, Fangpu

2017-07-01

1RS.1BL translocations are centric translocations formed by misdivision and have been used extensively in wheat breeding. However, the role that the centromere plays in the formation of 1RS.1BL translocations is still unclear. Fluorescence in situ hybridization (FISH) was applied to detect the fine structures of the centromeres in 130 1RS.1BL translocation cultivars. Immuno-FISH, chromatin immunoprecipitation (ChIP)-qPCR and RT-PCR were used to investigate the functions of the hybrid centromeres in 1RS.1BL translocations. New 1R translocations with different centromere structures were created by misdivision and pollen irradiation to elucidate the role that the centromere plays in the formation of 1RS.1BL translocations. We found that all of the 1RS.1BL translocations detected contained hybrid centromeres and that wheat-derived CENH3 bound to both the wheat and rye centromeres in the 1RS.1BL translocation chromosomes. Moreover, a rye centromere-specific retrotransposon was actively transcribed in 1RS.1BL translocations. The frequencies of new 1RS hybrid centromere translocations and group-1 chromosome translocations were higher during 1R misdivision. Our study demonstrates the hybrid nature of the centromere in 1RS.1BL translocations. New 1R translocations with different centromere structures were created to help understand the fusion centromere used for wheat breeding and for use as breeding material for the improvement of wheat. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1×1 Nucleotide UU Internal Loop Conformations⊥

PubMed Central

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D.

2011-01-01

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5′CUG/3′GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures are disclosed of a model DM1 triplet repeating construct, 5′r(UUGGGC(CUG)3GUCC)2, refined to 2.20 Å and 1.52 Å resolution. Here, differences in orientation of the 5′ dangling UU end between the two structures induce changes in the backbone groove width, which reveals that non-canonical 1×1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5′UU forms one hydrogen-bonded pairs with a 5′UU of a neighboring helix in the unit cell to form a pseudo-infinite helix. The central 1×1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1×1 nucleotide UU internal loops each form a one hydrogen-bonded pair. In the 1.52 Å structure, CUGb, the 5′ UU dangling end is tucked into the major groove of the duplex. While the canonical paired bases show no change in base pairing, in CUGb the terminal 1×1 nucleotide UU internal loops form now two hydrogen-bonded pairs. Thus, the shift in major groove induced by the 5′UU dangling end alters non-canonical base patterns. Collectively, these structures indicate that 1×1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands. PMID:21988728

Myotonic dystrophy type 1 RNA crystal structures reveal heterogeneous 1 × 1 nucleotide UU internal loop conformations.

PubMed

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W; Disney, Matthew D

2011-11-15

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[UUGGGC(CUG)(3)GUCC](2), refined to 2.20 and 1.52 Å resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 × 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 × 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 × 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 Å structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 × 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 × 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

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

Kumar, Amit; Park, HaJeung; Fang, Pengfei

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations.more » In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.« less

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

Familial translocation involving chromosomes 1 and 9 in a patient with Philadelphia-positive CML

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

Rehman, K.; Rosner, F.; Shanske, A.

1994-09-01

CML has provided a model for understanding the genetic basis of neoplasia. Approximately 5% of Philadelphia-positive patients have a variant chromosome rearrangement. We recently evaluated a patient with a previously unreported simple variant translocation that is part of a familial rearrangement. He had a constitutional translocation, t(1;9)(p21;p22), which was initially identified after his wife had a routine amniocentesis. Case report: K.H. was a 54-year-old male with CML for 4 years. He had been treated until recently with hydroxyurea. An abnormal male karyotype, 46,XY,t(1;9)(q21;p22),t(9;22)(q34;q11) was recorded from an unstimulated blood sample soon after diagnosis. Both translocations involved the same number 9more » homologue resulting in a derivative 9(1pter{r_arrow}1q21::9p22{r_arrow}9q34::22q11{r_arrow}22qter). A recent CT scan of the chest showed a lytic lesion of a rib with associated soft tissue mass in the right costo-vertebral angle. He was hospitalized for progressive pain in the right lower chest and fever, treated for a UTI, required multiple transfusions for declining hemoglobin and platelets and died shortly thereafter. Autopsy revealed widespread chloromas as part of terminal CML. At least 13 complex rearrangements involving chromosomes 1, 9 and 22 are known. Our case represents a unique rearrangement with a familial component and also unique breakpoints for a Philadelphia variant. In line with the current view of cancer as a clonal disorder, perhaps the constitutional translocation contributed to the multi-step nature of the malignant transformation. In fact, a number of cancer-specific breakpoints in both regions of 1p and 9p are involved in the familial translocation.« less

Role of CRTC1/MAML2 Translocation in the Prognosis and Clinical Outcomes of Mucoepidermoid Carcinoma.

PubMed

Saade, Rami E; Bell, Diana; Garcia, Joaquin; Roberts, Dianna; Weber, Randal

2016-03-01

The CRTC1/MAML2 fusion transcript, which arises from the CRTC1/MAML2 translocation, is a molecular marker unique to mucoepidermoid carcinoma (MEC), the most common malignant tumor of the salivary gland. The extent to which the transcript influences disease features and patient survival is unclear. To determine whether the CRTC1/MAML2 fusion transcript is associated with disease stage, tumor grade, or survival outcomes in patients with MEC. A retrospective medical record review was performed at a tertiary-care academic medical institution. The review included 90 patients with MEC who underwent treatment from January 1, 1995, to December 31, 2011, and for whom archived formalin-fixed, paraffin-embedded tumor specimens were available. Records were reviewed for clinical, demographic, and survival data. Tumor specimens underwent fluorescence in situ hybridization. Follow-up was completed on May 15, 2014, and data were analyzed from June 1 to July 1, 2014. CRTC1/MAML2 fusion transcript status. Statistical analysis determined whether transcript status was associated with disease stage, tumor grade, and/or overall and disease-free survival. Among the 90 eligible patients (median [range] age, 55.1 [7.8-89.2] years), 42 were female and 48 were male. Fluorescence in situ hybridization revealed a CRTC1/MAML2 translocation in 50 patients (56%). The translocations were more prevalent in intermediate-grade tumors (31 of 49 [63%]) than in high-grade (11 of 49 [22%]) and low-grade (7 of 49 [14%]) tumors; 1 tumor sample had no available grading. Similar proportions of patients with translocation-positive disease had T1 (13 of 49 [26%]), T2 (15 of 49 [31%]), T4a (14 of 49 [28%]), or T0 or Tx (8 of 49 [16%]) stages of disease. Thirty-eight of 49 patients with translocation-positive MEC (78%) had N0 stage of disease. Rates of 5-year overall survival were similar for patients with translocation-positive and translocation-negative disease (76.8% vs 75.5%, respectively; P = .17), as

KPNB1 mediates PER/CRY nuclear translocation and circadian clock function.

PubMed

Lee, Yool; Jang, A Reum; Francey, Lauren J; Sehgal, Amita; Hogenesch, John B

2015-08-29

Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

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.

Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

PubMed Central

Shaifta, Yasin; MacKay, Charles E.; Irechukwu, Nneka; O'Brien, Katie A.; Wright, David B.; Ward, Jeremy P. T.

2017-01-01

Key points Transforming growth‐factor‐β (TGF‐β) and RhoA/Rho‐kinase are independently implicated in the airway hyper‐responsiveness associated with asthma, but how these proteins interact is not fully understood.We examined the effects of pre‐treatment with TGF‐β on expression and activity of RhoA, Rho‐kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin‐induced contraction, in airway smooth muscle.TGF‐β enhanced bradykinin‐induced RhoA translocation, Rho‐kinase‐dependent phosphorylation and contraction, but partially suppressed bradykinin‐induced RhoA activity (RhoA‐GTP content).TGF‐β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF‐β on RhoA and Rho‐kinase activity and contraction, respectively.ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin‐sensitized mice.ARHGEF1 is a key TGF‐β target gene, an important regulator of Rho‐kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper‐responsiveness. Abstract Transforming growth factor‐β (TGF‐β), RhoA/Rho‐kinase and Src‐family kinases (SrcFK) have independently been implicated in airway hyper‐responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF‐β pre‐treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho‐kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF‐β pre‐treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT‐1 and the actin‐severing protein cofilin, but not of RhoA, ROCK2 or c‐Src. In hASMCs, acute treatment with BK triggered subcellular translocation

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.

Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining

PubMed Central

Soni, Aashish; Siemann, Maria; Grabos, Martha; Murmann, Tamara; Pantelias, Gabriel E.; Iliakis, George

2014-01-01

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors. PMID:24748665

Detection of zinc translocation into apical dendrite of CA1 pyramidal neuron after electrical stimulation.

PubMed

Suh, Sang Won

2009-02-15

Translocation of the endogenous cation zinc from presynaptic terminals to postsynaptic neurons after brain insult has been implicated as a potential neurotoxic event. Several studies have previously demonstrated that a brief electrical stimulation is sufficient to induce the translocation of zinc from presynaptic vesicles into the cytoplasm (soma) of postsynaptic neurons. In the present work I have extended those findings in three ways: (i) providing evidence that zinc translocation occurs into apical dendrites, (ii) presenting data that there is an apparent translocation into apical dendrites when only a zinc-containing synaptic input is stimulated, and (iii) presenting data that there is no zinc translocation into apical dendrite of ZnT3 KO mice following electrical stimulation. Hippocampal slices were preloaded with the "trappable" zinc fluorescent probe, Newport Green. After washout, a single apical dendrite in the stratum radiatum of hippocampal CA1 area was selected and focused on. Burst stimulation (100Hz, 500microA, 0.2ms, monopolar) was delivered to either the adjacent Schaffer-collateral inputs (zinc-containing) or to the adjacent temporo-ammonic inputs (zinc-free) to the CA1 dendrites. Stimulation of the Schaffer collaterals increased the dendritic fluorescence, which was blocked by TTX, low-Ca medium, or the extracellular zinc chelator, CaEDTA. Stimulation of the temporo-ammonic pathway caused no significant rise in the fluorescence. Genetic depletion of vesicular zinc by ZnT3 KO showed no stimulation-induced apical dendrite zinc rise. The present study provides evidence that synaptically released zinc translocates into postsynaptic neurons through the apical dendrites of CA1 pyramidal neurons during physiological synaptic activity.

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

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.

Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining.

PubMed

Soni, Aashish; Siemann, Maria; Grabos, Martha; Murmann, Tamara; Pantelias, Gabriel E; Iliakis, George

2014-06-01

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

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.

Stress and translocation: alterations in the stress physiology of translocated birds

PubMed Central

Dickens, Molly J.; Delehanty, David J.; Romero, L. Michael

2009-01-01

Translocation and reintroduction have become major conservation actions in attempts to create self-sustaining wild populations of threatened species. However, avian translocations have a high failure rate and causes for failure are poorly understood. While ‘stress’ is often cited as an important factor in translocation failure, empirical evidence of physiological stress is lacking. Here we show that experimental translocation leads to changes in the physiological stress response in chukar partridge, Alectoris chukar. We found that capture alone significantly decreased the acute glucocorticoid (corticosterone, CORT) response, but adding exposure to captivity and transport further altered the stress response axis (the hypothalamic–pituitary–adrenal axis) as evident from a decreased sensitivity of the negative feedback system. Animals that were exposed to the entire translocation procedure, in addition to the reduced acute stress response and disrupted negative feedback, had significantly lower baseline CORT concentrations and significantly reduced body weight. These data indicate that translocation alters stress physiology and that chronic stress is potentially a major factor in translocation failure. Under current practices, the restoration of threatened species through translocation may unwittingly depend on the success of chronically stressed individuals. This conclusion emphasizes the need for understanding and alleviating translocation-induced chronic stress in order to use most effectively this important conservation tool. PMID:19324794

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

Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma.

PubMed

Graham, Rondell P; Barr Fritcher, Emily G; Pestova, Ekaterina; Schulz, John; Sitailo, Leonid A; Vasmatzis, George; Murphy, Stephen J; McWilliams, Robert R; Hart, Steven N; Halling, Kevin C; Roberts, Lewis R; Gores, Gregory J; Couch, Fergus J; Zhang, Lizhi; Borad, Mitesh J; Kipp, Benjamin R

2014-08-01

Patients with cholangiocarcinoma often present with locally advanced or metastatic disease. There is a need for effective therapeutic strategies for advanced stage cholangiocarcinoma. Recently, FGFR2 translocations have been identified as a potential target for tyrosine kinase inhibitor therapies. This study evaluated 152 cholangiocarcinomas and 4 intraductal papillary biliary neoplasms of the bile duct for presence of FGFR2 translocations by fluorescence in situ hybridization and characterized the clinicopathologic features of cases with FGFR2 translocations. Thirteen (10 women, 3 men; 8%) of 156 biliary tumors harbored FGFR2 translocations, including 12 intrahepatic cholangiocarcinomas (12/96; 13%) and 1 intraductal papillary neoplasm of the bile duct. Histologically, cholangiocarcinomas with FGFR2 translocations displayed prominent intraductal growth (62%) or anastomosing tubular glands with desmoplasia (38%). Immunohistochemically, the tumors with FGFR2 translocations frequently showed weak and patchy expression of CK19 (77%). Markers of the stem cell phenotype in cholangiocarcinoma, HepPar1 and CK20, were negative in all cases. The median cancer-specific survival for patients whose tumors harbored FGFR2 translocations was 123 months compared to 37 months for cases without FGFR2 translocations (P = .039). This study also assessed 100 cholangiocarcinomas for ERBB2 amplification and ROS1 translocations. Of the cases tested, 3% and 1% were positive for ERBB2 amplification and ROS1 translocation, respectively. These results confirm that FGFR2, ERRB2, and ROS1 alterations are potential therapeutic targets for intrahepatic cholangiocarcinoma. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Identification of a t(3;4)(p1.3;q1.5) translocation breakpoint in pigs using somatic cell hybrid mapping and high-resolution mate-pair sequencing

PubMed Central

Fève, Katia; Foissac, Sylvain; Pinton, Alain; Mompart, Florence; Esquerré, Diane; Faraut, Thomas; Yerle, Martine

2017-01-01

Reciprocal translocations are the most frequently occurring constitutional structural rearrangements in mammalian genomes. In phenotypically normal pigs, an incidence of 1/200 is estimated for such rearrangements. Even if constitutional translocations do not necessarily induce defects and diseases, they are responsible for significant economic losses in domestic animals due to reproduction failures. Over the last 30 years, advances in molecular and cytogenetic technologies have led to major improvements in the resolution of the characterization of translocation events. Characterization of translocation breakpoints helps to decipher the mechanisms that lead to such rearrangements and the functions of the genes that are involved in the translocation. Here, we describe the fine characterization of a reciprocal translocation t(3;4) (p1.3;q1.5) detected in a pig line. The breakpoint was identified at the base-pair level using a positional cloning and chromosome walking strategy in somatic cell hybrids that were generated from an animal that carries this translocation. We show that this translocation occurs within the ADAMTSL4 gene and results in a loss of expression in homozygous carriers. In addition, by taking this translocation as a model, we used a whole-genome next-generation mate-pair sequencing approach on pooled individuals to evaluate this strategy for high-throughput screening of structural rearrangements. PMID:29121641

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.

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

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

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.

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

Induction of long interspersed nucleotide element-1 (L1) retrotransposition by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct

PubMed Central

Okudaira, Noriyuki; Iijima, Kenta; Koyama, Takayoshi; Minemoto, Yuzuru; Kano, Shigeyuki; Mimori, Akio; Ishizaka, Yukihito

2010-01-01

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80–100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive element-binding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed. PMID:20852066

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

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy.

PubMed

Gutiérrez-Sanz, Oscar; Forbrig, Enrico; Batista, Ana P; Pereira, Manuela M; Salewski, Johannes; Mroginski, Maria A; Götz, Robert; De Lacey, Antonio L; Kozuch, Jacek; Zebger, Ingo

2018-05-22

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [ Di Luca , Proc. Natl. Acad. Sci. U.S.A. , 2017 , 114 , E6314 - E6321 ].

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.

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

PubMed Central

Jagot-Lacoussiere, Léonard; Faye, Audrey; Bruzzoni-Giovanelli, Heriberto; Villoutreix, Bruno O; Rain, Jean-Christophe; Poyet, Jean-Luc

2015-01-01

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response. PMID:25695197

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

PubMed Central

Lill, R; Robertson, J M; Wintermeyer, W

1989-01-01

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA. PMID:2583120

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.

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...

Embryonal rhabdomyosarcoma with a der(16)t(1;16) translocation.

PubMed

Kapels, Kayla M; Nishio, Jun; Zhou, Ming; Qualman, Stephen J; Bridge, Julia A

2007-04-01

Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of RMS that predominantly involves the genitourinary tract and the head and neck regions in children younger than 10 years of age. Cytogenetically, ERMS is most frequently hyperdiploid, with extra copies of chromosomes 2, 7, 8, 11, 12, 13, and 20. No consistent structural chromosomal alteration has been identified in ERMS. In contrast, a t(2;13)(q35;q14) or t(1;13)(q36;q14) corresponding to PAX3-FOXO1A (previously FKHR) and PAX7-FOXO1A gene fusions are considered tumor-specific anomalies for alveolar RMS (ARMS). Occasionally, a recurrent secondary structural rearrangement involving chromosomes 1 and 16 is seen in translocation-positive ARMS, a der(16)t(1;16) resulting in an imbalance of 1q and 16q material. Conventional cytogenetic analysis of an ERMS arising in the urinary bladder of a 22-month-old male child revealed this nonrandom secondary chromosomal aberration, der(16)(1;16)(q22;q24), in a hyperdiploid complement with extra copies of chromosomes 2, 7, 8, 10, 12, 13, 19, and 20. Subsequent analyses showed tumor cells to be negative for FOXO1A, PAX3, or PAX7 gene locus rearrangements (by fluorescence in situ hybridization) and also negative for PAX3-FOXO1A and PAX7-FOXO1A fusion transcripts (by reverse transcriptase-polymerase chain reaction). These results suggest that the unbalanced t(1;16) translocation may be seen in RMSs lacking a primary genetic rearrangement.

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.

The interaction of the Eco R1 restriction enzyme E.coli with nucleotides

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

Hollis, Donald F.

1979-11-01

The Eco R1 restriction enzyme can be shown to be inhibited by nucleotides which correspond to any part of its known site of phosphodiesterase activity. A series of di-, tetra-, and hexa-nucleotide fragments were synthesized and their effect on the activity of the enzyme upon superhelical Co1 E1 DNA studied. The inhibition caused by the individual mononucleotides were also studied. In general all the nucleotide fragments showed some form of interaction with the enzyme system. Tetranucleotides were stronger inhibitors than dinucleotides, which in turn were stronger inhibitors than the mononucleotides. Within each category of inhibitors, those containing the phosphodiester bondmore » which is acted upon by the enzyme were the strongest inhibitors. Only those fragments which were consistent with the enzymes site of activity showed competitive inhibition kinetics. Nucleotides which do not fit within the site of phosphodiesterase activity show non-competitive inhibition kinetics.« less

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

Childhood pre-B cell acute lymphoblastic leukemia with translocation t(1;19)(q21.1;p13.3) and two additional chromosomal aberrations involving chromosomes 1, 6, and 13: a case report.

PubMed

Wafa, Abdulsamad; As'sad, Manar; Liehr, Thomas; Aljapawe, Abdulmunim; Al Achkar, Walid

2017-04-07

The translocation t(1;19)(q23;p13), which results in the TCF3-PBX1 chimeric gene, is one of the most frequent rearrangements observed in B cell acute lymphoblastic leukemia. It appears in both adult and pediatric patients with B cell acute lymphoblastic leukemia at an overall frequency of 3 to 5%. Most cases of pre-B cell acute lymphoblastic leukemia carrying the translocation t(1;19) have a typical immunophenotype with homogeneous expression of CD19, CD10, CD9, complete absence of CD34, and at least diminished CD20. Moreover, the translocation t(1;19) correlates with known clinical high risk factors, such as elevated white blood cell count, high serum lactate dehydrogenase levels, and central nervous system involvement; early reports indicated that patients with translocation t(1;19) had a poor outcome under standard treatment. We report the case of a 15-year-old Syrian boy with pre-B cell acute lymphoblastic leukemia with abnormal karyotype with a der(19)t(1;19)(q21.1;p13.3) and two yet unreported chromosomal aberrations: an interstitial deletion 6q12 to 6q26 and a der(13)t(1;13)(q21.1;p13). According to the literature, cases who are translocation t(1;19)-positive have a significantly higher incidence of central nervous system relapse than patients with acute lymphoblastic leukemia without the translocation. Of interest, central nervous system involvement was also seen in our patient. To the best of our knowledge, this is the first case of childhood pre-B cell acute lymphoblastic leukemia with an unbalanced translocation t(1;19) with two additional chromosomal aberrations, del(6)(q12q26) and t(1;13)(q21.3;p13), which seem to be recurrent and could influence clinical outcome. Also the present case confirms the impact of the translocation t(1;19) on central nervous system relapse, which should be studied for underlying mechanisms in future.

Respiration-linked proton translocation coupled to anaerobic reduction of manganese(IV) and iron(III) in Shewanella putrefaciens MR-1.

PubMed Central

Myers, C R; Nealson, K H

1990-01-01

An oxidant pulse technique, with lactate as the electron donor, was used to study respiration-linked proton translocation in the manganese- and iron-reducing bacterium Shewanella putrefaciens MR-1. Cells grown anaerobically with fumarate or nitrate as the electron acceptor translocated protons in response to manganese (IV), fumarate, or oxygen. Cells grown anaerobically with fumarate also translocated protons in response to iron(III) and thiosulfate, whereas those grown with nitrate did not. Aerobically grown cells translocated protons only in response to oxygen. Proton translocation with all electron acceptors was abolished in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (20 microM) and was partially to completely inhibited by the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (50 microM). PMID:2172208

Measuring peptide translocation into large unilamellar vesicles.

PubMed

Spinella, Sara A; Nelson, Rachel B; Elmore, Donald E

2012-01-27

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell

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.

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

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.

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.

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

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.

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found

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.

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.

Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2

PubMed Central

2004-01-01

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5′-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain. PMID:15096095

Physiology in conservation translocations

PubMed Central

Tarszisz, Esther; Dickman, Christopher R.; Munn, Adam J.

2014-01-01

Conservation translocations aim to restore species to their indigenous ranges, protect populations from threats and/or reinstate ecosystem functions. They are particularly important for the conservation and management of rare and threatened species. Despite tremendous efforts and advancement in recent years, animal conservation translocations generally have variable success, and the reasons for this are often uncertain. We suggest that when little is known about the physiology and wellbeing of individuals either before or after release, it will be difficult to determine their likelihood of survival, and this could limit advancements in the science of translocations for conservation. In this regard, we argue that physiology offers novel approaches that could substantially improve translocations and associated practices. As a discipline, it is apparent that physiology may be undervalued, perhaps because of the invasive nature of some physiological measurement techniques (e.g. sampling body fluids, surgical implantation). We examined 232 publications that dealt with translocations of terrestrial vertebrates and aquatic mammals and, defining ‘success’ as high or low, determined how many of these studies explicitly incorporated physiological aspects into their protocols and monitoring. From this review, it is apparent that physiological evaluation before and after animal releases could progress and improve translocation/reintroduction successes. We propose a suite of physiological measures, in addition to animal health indices, for assisting conservation translocations over the short term and also for longer term post-release monitoring. Perhaps most importantly, we argue that the incorporation of physiological assessments of animals at all stages of translocation can have important welfare implications by helping to reduce the total number of animals used. Physiological indicators can also help to refine conservation translocation methods. These approaches fall

Physiology in conservation translocations.

PubMed

Tarszisz, Esther; Dickman, Christopher R; Munn, Adam J

2014-01-01

Conservation translocations aim to restore species to their indigenous ranges, protect populations from threats and/or reinstate ecosystem functions. They are particularly important for the conservation and management of rare and threatened species. Despite tremendous efforts and advancement in recent years, animal conservation translocations generally have variable success, and the reasons for this are often uncertain. We suggest that when little is known about the physiology and wellbeing of individuals either before or after release, it will be difficult to determine their likelihood of survival, and this could limit advancements in the science of translocations for conservation. In this regard, we argue that physiology offers novel approaches that could substantially improve translocations and associated practices. As a discipline, it is apparent that physiology may be undervalued, perhaps because of the invasive nature of some physiological measurement techniques (e.g. sampling body fluids, surgical implantation). We examined 232 publications that dealt with translocations of terrestrial vertebrates and aquatic mammals and, defining 'success' as high or low, determined how many of these studies explicitly incorporated physiological aspects into their protocols and monitoring. From this review, it is apparent that physiological evaluation before and after animal releases could progress and improve translocation/reintroduction successes. We propose a suite of physiological measures, in addition to animal health indices, for assisting conservation translocations over the short term and also for longer term post-release monitoring. Perhaps most importantly, we argue that the incorporation of physiological assessments of animals at all stages of translocation can have important welfare implications by helping to reduce the total number of animals used. Physiological indicators can also help to refine conservation translocation methods. These approaches fall under a

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.

Fenofibrate inhibits atrial metabolic remodelling in atrial fibrillation through PPAR-α/sirtuin 1/PGC-1α pathway.

PubMed

Liu, Guang-Zhong; Hou, Ting-Ting; Yuan, Yue; Hang, Peng-Zhou; Zhao, Jing-Jing; Sun, Li; Zhao, Guan-Qi; Zhao, Jing; Dong, Jing-Mei; Wang, Xiao-Bing; Shi, Hang; Liu, Yong-Wu; Zhou, Jing-Hua; Dong, Zeng-Xiang; Liu, Yang; Zhan, Cheng-Chuang; Li, Yue; Li, Wei-Min

2016-03-01

Atrial metabolic remodelling is critical for the process of atrial fibrillation (AF). The PPAR-α/sirtuin 1 /PPAR co-activator α (PGC-1α) pathway plays an important role in maintaining energy metabolism. However, the effect of the PPAR-α agonist fenofibrate on AF is unclear. Therefore, the aim of this study was to determine the effect of fenofibrate on atrial metabolic remodelling in AF and explore its possible mechanisms of action. The expression of metabolic proteins was examined in the left atria of AF patients. Thirty-two rabbits were divided into sham, AF (pacing with 600 beats·min(-1) for 1 week), fenofibrate treated (pretreated with fenofibrate before pacing) and fenofibrate alone treated (for 2 weeks) groups. HL-1 cells were subjected to rapid pacing in the presence or absence of fenofibrate, the PPAR-α antagonist GW6471 or sirtuin 1-specific inhibitor EX527. Metabolic factors, circulating biochemical metabolites, atrial electrophysiology, adenine nucleotide levels and accumulation of glycogen and lipid droplets were assessed. The PPAR-α/sirtuin 1/PGC-1α pathway was significantly inhibited in AF patients and in the rabbit/HL-1 cell models, resulting in a reduction of key downstream metabolic factors; this effect was significantly restored by fenofibrate. Fenofibrate prevented the alterations in circulating biochemical metabolites, reduced the level of adenine nucleotides and accumulation of glycogen and lipid droplets, reversed the shortened atrial effective refractory period and increased risk of AF. Fenofibrate inhibited atrial metabolic remodelling in AF by regulating the PPAR-α/sirtuin 1/PGC-1α pathway. The present study may provide a novel therapeutic strategy for AF. © 2016 The British Pharmacological Society.

Role of Free Space in Translocation in Sugar Beet 1

PubMed Central

Geiger, Donald R.; Sovonick, Susan A.; Shock, Terri L.; Fellows, Robert J.

1974-01-01

The involvement of the free space in phloem loading of sucrose was studied in sugar beet source leaves (Beta vulgaris, L.). Sucrose, supplied exogenously to the abraded upper surface of leaves at a concentration of 20 mm, was available for translocation at rates similar to those obtained with photosynthesis. The exogenous sucrose substituted as a source of translocate for assimilate derived from photosynthesis when the latter process was disrupted by plasmolysis of the leaf with 0.8 M mannitol. The mesophyll symplast was not completely disrupted by this treatment, however. Data from the sugar uptake experiments indicate that phloem loading can occur from the free space. Isotope trapping of labeled sugars derived from 14CO2 was used to intercept and identify sugars passing through the free space prior to phloem loading. Increased translocation rates induced by 4 mm ATP or increased light intensity were accompanied by increased trapping of sucrose but not of glucose. The data support the view that sucrose passes into the free space prior to phloem loading. Images PMID:16658995

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

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.

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

PubMed

Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

2018-01-15

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in

Ewing's sarcoma: analysis of single nucleotide polymorphism in the EWS gene.

PubMed

Silva, Deborah S B S; Sawitzki, Fernanda R; De Toni, Elisa C; Graebin, Pietra; Picanco, Juliane B; Abujamra, Ana Lucia; de Farias, Caroline B; Roesler, Rafael; Brunetto, Algemir L; Alho, Clarice S

2012-11-10

We aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p=0.02; Chi Square Test). About 300 bp from the rs4820804 SNP lies a palindromic hexamer (5'-GCTAGC-3') and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility. Copyright © 2012 Elsevier B.V. All rights reserved.

A strategy for generation and balancing of autosome: Y chromosome translocations.

PubMed

Joshi, Sonal S; Cheong, Han; Meller, Victoria H

2014-01-01

We describe a method for generation and maintenance of translocations that move large autosomal segments onto the Y chromosome. Using this strategy we produced ( 2;Y) translocations that relocate between 1.5 and 4.8 Mb of the 2nd chromosome.. All translocations were easily balanced over a male-specific lethal 1 (msl-1) mutant chromosome. Both halves of the translocation carry visible markers, as well as P-element ends that enable molecular confirmation. Halves of these translocations can be separated to produce offspring with duplications and with lethal second chromosome deficiencies . Such large deficiencies are otherwise tedious to generate and maintain.

Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases.

PubMed

Vandenbol, M; Jauniaux, J C; Grenson, M

1989-11-15

The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

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.

Identification of PPAP2B as a novel recurrent translocation partner gene of HMGA2 in lipomas.

PubMed

Bianchini, Laurence; Birtwisle, Loïc; Saâda, Esma; Bazin, Audrey; Long, Elodie; Roussel, Jean-François; Michiels, Jean-François; Forest, Fabien; Dani, Christian; Myklebost, Ola; Birtwisle-Peyrottes, Isabelle; Pedeutour, Florence

2013-06-01

Most lipomas are characterized by translocations involving the HMGA2 gene in 12q14.3. These rearrangements lead to the fusion of HMGA2 with an ectopic sequence from the translocation chromosome partner. Only five fusion partners of HMGA2 have been identified in lipomas so far. The identification of novel fusion partners of HMGA2 is important not only for diagnosis in soft tissue tumors but also because these genes might have an oncogenic role in other tumors. We observed that t(1;12)(p32;q14) was the second most frequent translocation in our series of lipomas after t(3;12)(q28;q14.3). We detected overexpression of HMGA2 mRNA and protein in all t(1;12)(p32;q14) lipomas. We used a fluorescence in situ hybridization-based positional cloning strategy to characterize the 1p32 breakpoint. In 11 cases, we identified PPAP2B, a member of the lipid phosphate phosphatases family as the 1p32 target gene. Reverse transcription-polymerase chain reaction analysis followed by nucleotide sequencing of the fusion transcript indicated that HMGA2 3' untranslated region (3'UTR) fused with exon 6 of PPAP2B in one case. In other t(1;12) cases, the breakpoint was extragenic, located in the 3'region flanking PPAP2B 3'UTR. Moreover, in one case showing a t(1;6)(p32;p21) we observed a rearrangement of PPAP2B and HMGA1, which suggests that HMGA1 might also be a fusion partner for PPAP2B. Our results also revealed that adipocytic differentiation of human mesenchymal stem cells derived from adipose tissue was associated with a significant decrease in PPAP2B mRNA expression suggesting that PPAP2B might play a role in adipogenesis. Copyright © 2013 Wiley Periodicals, Inc.

Non-genomic effects of the NR4A1/Nur77/TR3/NGFIB orphan nuclear receptor.

PubMed

Pawlak, Alicja; Strzadala, Leon; Kalas, Wojciech

2015-03-01

The orphan nuclear receptor NR4A1/Nur77/TR3/NGFIB acts primarily as a transcription factor to regulate the expression of multiple genes. However, increasing research attention has recently been given to non-genomic activities of NR4A1. The first description of a non-genomic action of NR4A1 referred to the conversion of anti-apoptotic Bcl-2 into a pro-apoptotic protein by direct interaction with NR4A1. In response to certain apoptotic stimuli, NR4A1 translocates from the nucleus to the mitochondrial outer membrane (MOM) where it associates with Bcl-2 and thereby causes apoptosis. Afterwards, it appeared that NR4A1 could also bind and convert other anti-apoptotic Bcl-2 family members. The latest studies indicate a significant role of NR4A1 in the process of autophagy. For example, a new NR4A1-mediated pathway specific for melanoma cells has been described where NR4A1 interacts with the adenine nucleotide translocase 1 (ANT1) on the mitochondrial inner membrane (MIM) leading to induction of the autophagy pathway. Moreover, NR4A1 interaction with cytoplasmic p53 may also contribute to the induction of autophagy. In addition to mitochondria, NR4A1 could be translocated to the outer membrane of the endoplasmic reticulum (ER) and associate with Bcl-2 or translocon-associated protein subunit γ (TRAPγ) causing ER stress-induced apoptosis. NR4A1 also contributes to the proteasomal degradation of β-catenin in colon cancer cells in vitro and in vivo, as well as to the stabilization of hypoxia-inducible factor-1α (HIF-1α) under non-hypoxic conditions. This review summarizes research findings on non-genomic effects of NR4A1 in normal and cancer cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Slowing DNA Translocation in a Nanofluidic Field-Effect Transistor.

PubMed

Liu, Yifan; Yobas, Levent

2016-04-26

Here, we present an experimental demonstration of slowing DNA translocation across a nanochannel by modulating the channel surface charge through an externally applied gate bias. The experiments were performed on a nanofluidic field-effect transistor, which is a monolithic integrated platform featuring a 50 nm-diameter in-plane alumina nanocapillary whose entire length is surrounded by a gate electrode. The field-effect transistor behavior was validated on the gating of ionic conductance and protein transport. The gating of DNA translocation was subsequently studied by measuring discrete current dips associated with single λ-DNA translocation events under a source-to-drain bias of 1 V. The translocation speeds under various gate bias conditions were extracted by fitting event histograms of the measured translocation time to the first passage time distributions obtained from a simple 1D biased diffusion model. A positive gate bias was observed to slow the translocation of single λ-DNA chains markedly; the translocation speed was reduced by an order of magnitude from 18.4 mm/s obtained under a floating gate down to 1.33 mm/s under a positive gate bias of 9 V. Therefore, a dynamic and flexible regulation of the DNA translocation speed, which is vital for single-molecule sequencing, can be achieved on this device by simply tuning the gate bias. The device is realized in a conventional semiconductor microfabrication process without the requirement of advanced lithography, and can be potentially further developed into a compact electronic single-molecule sequencer.

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.

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

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys.

PubMed

Marq, Jean-Baptiste; Hausmann, Stéphane; Veillard, Nicolas; Kolakofsky, Daniel; Garcin, Dominique

2011-02-25

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.

The Chromobacterium violaceum type III effector CopE, a guanine nucleotide exchange factor for Rac1 and Cdc42, is involved in bacterial invasion of epithelial cells and pathogenesis.

PubMed

Miki, Tsuyoshi; Akiba, Kinari; Iguchi, Mirei; Danbara, Hirofumi; Okada, Nobuhiko

2011-06-01

The type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS- and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen. © 2011 Blackwell Publishing Ltd.

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.

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

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.

PubMed

Mor, Adam; Wynne, Joseph P; Ahearn, Ian M; Dustin, Michael L; Du, Guangwei; Philips, Mark R

2009-06-01

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.

A t(1;11) translocation linked to schizophrenia and affective disorders gives rise to aberrant chimeric DISC1 transcripts that encode structurally altered, deleterious mitochondrial proteins

PubMed Central

Eykelenboom, Jennifer E.; Briggs, Gareth J.; Bradshaw, Nicholas J.; Soares, Dinesh C.; Ogawa, Fumiaki; Christie, Sheila; Malavasi, Elise L.V.; Makedonopoulou, Paraskevi; Mackie, Shaun; Malloy, Mary P.; Wear, Martin A.; Blackburn, Elizabeth A.; Bramham, Janice; McIntosh, Andrew M.; Blackwood, Douglas H.; Muir, Walter J.; Porteous, David J.; Millar, J. Kirsty

2012-01-01

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a risk factor for psychiatric illness through its disruption by a balanced chromosomal translocation, t(1;11)(q42.1;q14.3), that co-segregates with schizophrenia, bipolar disorder and depression. We previously reported that the translocation reduces DISC1 expression, consistent with a haploinsufficiency disease model. Here we report that, in lymphoblastoid cell lines, the translocation additionally results in the production of abnormal transcripts due to the fusion of DISC1 with a disrupted gene on chromosome 11 (DISC1FP1/Boymaw). These chimeric transcripts encode abnormal proteins, designated CP1, CP60 and CP69, consisting of DISC1 amino acids 1–597 plus 1, 60 or 69 amino acids, respectively. The novel 69 amino acids in CP69 induce increased α-helical content and formation of large stable protein assemblies. The same is predicted for CP60. Both CP60 and CP69 exhibit profoundly altered functional properties within cell lines and neurons. Both are predominantly targeted to mitochondria, where they induce clustering and loss of membrane potential, indicative of severe mitochondrial dysfunction. There is currently no access to neural material from translocation carriers to confirm these findings, but there is no reason to suppose that these chimeric transcripts will not also be expressed in the brain. There is thus potential for the production of abnormal chimeric proteins in the brains of translocation carriers, although at substantially lower levels than for native DISC1. The mechanism by which inheritance of the translocation increases risk of psychiatric illness may therefore involve both DISC1 haploinsufficiency and mitochondrial deficiency due to the effects of abnormal chimeric protein expression. GenBank accession numbers: DISC1FP1 (EU302123), Boymaw (GU134617), der 11 chimeric transcript DISC1FP1 exon 2 to DISC1 exon 9 (JQ650115), der 1 chimeric transcript DISC1 exon 4 to DISC1FP1 exon 4 (JQ650116

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.

37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

Code of Federal Regulations, 2011 CFR

2011-07-01

... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data shall...

β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

PubMed Central

Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P.; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-01-01

Hypoxia-inducible factor-1α (HIF-1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF-1α remain unclear. β-catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF-1α and β-catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4-2B, were grouped as follows: Negative control (no treatment), HIF-1α overexpression group (transfected with HIF-1α overexpression plasmid) and β-catenin silenced group (transfected with HIF-1α plasmids and β-catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4-2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4-2B cells, transfection with HIF-1α overexpression plasmid led to an enhanced β-catenin nuclear translocation, while β-catenin silencing inhibited β-catenin nuclear translocation. The enhanced β-catenin nuclear translocation induced by HIF-1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non-homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF-1α overexpression enhanced β-catenin nuclear translocation, which led to the activation of the β-catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF-1α overexpression promotes the radioresistance of PCa cells. PMID:29658569

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.

Variant translocation partners of the anaplastic lymphoma kinase (ALK) gene in two cases of anaplastic large cell lymphoma, identified by inverse cDNA polymerase chain reaction.

PubMed

Takeoka, Kayo; Okumura, Atsuko; Honjo, Gen; Ohno, Hitoshi

2014-01-01

In anaplastic large cell lymphoma (ALCL), the anaplastic lymphoma kinase (ALK) gene is rearranged with diverse partners due to variant translocations/inversions. Case 1 was a 39-year-old man who developed multiple tumors in the mediastinum, psoas muscle, lung, and lymph nodes. A biopsy specimen of the inguinal node was effaced by large tumor cells expressing CD30, epithelial membrane antigen, and cytoplasmic ALK, which led to a diagnosis of ALK(+) ALCL. Case 2 was a 51-year-old man who was initially diagnosed with undifferentiated carcinoma. He developed multiple skin tumors eight years after his initial presentation, and was finally diagnosed with ALK(+) ALCL. He died of therapy-related acute myeloid leukemia. G-banding and fluorescence in situ hybridization using an ALK break-apart probe revealed the rearrangement of ALK and suggested variant translocation in both cases. We applied an inverse cDNA polymerase chain reaction (PCR) strategy to identify the partner of ALK. Nucleotide sequencing of the PCR products and a database search revealed that the sequences of ATIC in case 1 and TRAF1 in case 2 appeared to follow those of ALK. We subsequently confirmed ATIC-ALK and TRAF1-ALK fusions by reverse transcriptase PCR and nucleotide sequencing. We successfully determined the partner gene of ALK in two cases of ALK(+) ALCL. ATIC is the second most common partner of variant ALK rearrangements, while the TRAF1-ALK fusion gene was first reported in 2013, and this is the second reported case of ALK(+) ALCL carrying TRAF1-ALK.

Nuclear translocation of HIF-1α induced by influenza A (H1N1) infection is critical to the production of proinflammatory cytokines.

PubMed

Guo, Xinkun; Zhu, Zhaoqin; Zhang, Wanju; Meng, Xiaoxiao; Zhu, Yong; Han, Peng; Zhou, Xiaohui; Hu, Yunwen; Wang, Ruilan

2017-05-24

Infection with the influenza A (H1N1) virus is a major challenge for public health because it can cause severe morbidity and even mortality in humans. The over-secretion of inflammatory cytokines (cytokine storm) is considered to be a key contributor to the severe pneumonia caused by H1N1 infection. It has been reported that hypoxia-inducible factor 1-alpha (HIF-1α) is associated with the production of proinflammatory molecules, but whether HIF-1α participates in the acute inflammatory responses against H1N1 infection is still unclear. To investigate the role of HIF-1α in H1N1 infection, the expression and nuclear translocation of HIF-1α in A549 and THP-1 cell lines infected with H1N1 virus were observed. The results showed that without altering the intracellular mRNA or protein expression of HIF-1α, H1N1 infection only induced nuclear translocation of HIF-1α under normal oxygen concentrations. The use of 2-methoxyestradiol (2ME2), a HIF-1α inhibitor that blocks HIF-1α nuclear accumulation, in H1N1-infected cells decreased the mRNA and protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and increased the levels of IL-10. In contrast, H1N1-infected cells under hypoxic conditions had increased HIF-1α nuclear accumulation, increased expression of TNF-α and IL-6 and decreased levels of IL-10. In conclusion, our data implied that in vitro H1N1 infection induced nuclear translocation of HIF-1α without altering the expression of HIF-1α, which may promote the secretion of proinflammatory cytokines during H1N1 infection.

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.

Gender and chronological age affect erythrocyte membrane oxidative indices in citrate phosphate dextrose adenine-formula 1 (CPDA-1) blood bank storage condition.

PubMed

Erman, Hayriye; Aksu, Uğur; Belce, Ahmet; Atukeren, Pınar; Uzun, Duygu; Cebe, Tamer; Kansu, Ahmet D; Gelişgen, Remisa; Uslu, Ezel; Aydın, Seval; Çakatay, Ufuk

2016-07-01

It is well known that in vitro storage lesions lead to membrane dysfunction and decreased number of functional erythrocytes. As erythrocytes get older, in storage media as well as in peripheral circulation, they undergo a variety of biochemical changes. In our study, the erythrocytes with different age groups in citrate phosphate dextrose adenine-formula 1 (CPDA-1) storage solution were used in order to investigate the possible effect of gender factor on oxidative damage. Oxidative damage biomarkers in erythrocyte membranes such as ferric reducing antioxidant power, pro-oxidant-antioxidant balance, protein-bound advance glycation end products, and sialic acid were analyzed. Current study reveals that change in membrane redox status during blood-bank storage condition also depends on both gender depended homeostatic factors and the presence of CPDA-1. During the storage period in CPDA-1, erythrocytes from the male donors are mostly affected by free radical-mediated oxidative stress but erythrocytes obtained from females are severely affected by glyoxidative stress.

Financial costs of large carnivore translocations--accounting for conservation.

PubMed

Weise, Florian J; Stratford, Ken J; van Vuuren, Rudolf J

2014-01-01

Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars). Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23), and $2,108 per leopard (n = 6). One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%), followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4%) of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown) with a strong species bias. Four leopards (66.7%) were successfully translocated but only eight of the 20 cheetahs (40.0%) with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC) and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

The inside and outside: topological issues in plant cell wall biosynthesis and the roles of nucleotide sugar transporters.

PubMed

Temple, Henry; Saez-Aguayo, Susana; Reyes, Francisca C; Orellana, Ariel

2016-09-01

The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

PubMed Central

Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

2015-01-01

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

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.

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.

S100A11 protects against neuronal cell apoptosis induced by cerebral ischemia via inhibiting the nuclear translocation of annexin A1.

PubMed

Xia, Qian; Li, Xing; Zhou, Huijuan; Zheng, Lu; Shi, Jing

2018-05-29

The subcellular location of annexin A1 (ANXA1) determines the ultimate fate of neurons after ischemic stroke. ANXA1 nuclear translocation is involved in neuronal apoptosis after cerebral ischemia, and extracellular ANXA1 is also associated with regulation of inflammatory responses. As the factors and mechanism that influence ANXA1 subcellular translocation remain unclear, studies aiming to determine and clarify the role of ANXA1 as a cell fate 'regulator' within cells are critically needed. In this study, we found that intracerebroventricular injection of the recombinant adenovirus vector Ad-S100A11 (carrying S100A11) strongly improved cognitive function and induced robust neuroprotective effects after ischemic stroke in vivo. Furthermore, upregulation of S100A11 protected against neuronal apoptosis induced by oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. Surprisingly, S100A11 overexpression markedly decreased ANXA1 nuclear translocation and subsequently alleviated OGD/R-induced neuronal apoptosis. Notably, S100A11 exerted its neuroprotective effect by directly binding ANXA1. Importantly, S100A11 directly interacted with ANXA1 through the nuclear translocation signal (NTS) of ANXA1, which is essential for ANXA1 to import into the nucleus. Consistent with our previous studies, ANXA1 nuclear translocation after OGD/R promoted p53 transcriptional activity, induced mRNA expression of the pro-apoptotic Bid gene, and activated the caspase-3 apoptotic pathway, which was almost completely reversed by S100A11 overexpression. Thus, S100A11 protects against cell apoptosis by inhibiting OGD/R-induced ANXA1 nuclear translocation. This study provides a novel mechanism whereby S100A11 protects against neuronal cells apoptosis, suggesting the potential for a previously unidentified treatment strategy in minimizing apoptosis after ischemic stroke.

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.

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

Melanotic Translocation Renal Cell Carcinoma With a Novel ARID1B-TFE3 Gene Fusion.

PubMed

Antic, Tatjana; Taxy, Jerome B; Alikhan, Mir; Segal, Jeremy

2017-11-01

A 36-year-old male was found to have a 7.0 cm left upper pole renal mass on renal ultrasound. Following nephrectomy, the mass was grossly ill-demarcated, friable and red-brown, invading renal parenchyma, hilar fat and the renal vein. Microscopically, the tumor had a nested and papillary architecture. The cells demonstrated abundant clear and eosinophilic cytoplasm and focal intracytoplasmic melanin pigment. Nucleoli were prominent. By immunohistochemistry, the tumor was positive for TFE3; HMB-45 stained approximately 5% of tumor cells corresponding to the histologic melanin pigment, which was confirmed with Fontana-Masson stain with bleach. Immunostains for PAX8, CD10, MiTF, and CAIX were negative; keratins Cam 5.2 and AE1/AE3 were focally positive. Targeted next-generation sequencing revealed an ARID1B-TFE3 gene fusion. Melanotic Xp11 renal cell carcinoma is a rare, pigment containing translocation variant demonstrating overlapping features with melanoma and is usually associated with an SFPQ-TFE3 gene fusion. The patient is alive and without evidence of disease 7 years after his diagnosis. The combination of high grade histopathology, the presence of melanin, absent PAX8, keratin positivity, and relatively indolent clinical behavior with a unique translocation may warrant recognition as a distinct renal cell carcinoma translocation subtype.

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

Effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora and bacterial translocation in rats with experimental cirrhosis.

PubMed

Chiva, Maite; Soriano, Germán; Rochat, Isabelle; Peralta, Carmen; Rochat, Florence; Llovet, Teresa; Mirelis, Beatriz; Schiffrin, Eduardo J; Guarner, Carlos; Balanzó, Joaquim

2002-10-01

Probiotics and antioxidants could be alternatives to antibiotics in the prevention of bacterial infections in cirrhosis. The aim of the present study was to determine the effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora, endotoxemia, and bacterial translocation in cirrhotic rats. Twenty-nine Sprague-Dawley rats with cirrhosis induced by CCl(4) and ascites received Lactobacillus johnsonii La1 10(9)cfu/day in vehicle (antioxidants: vitamin C+glutamate) (n=10), vehicle alone (n=11), or water (n=8) by gavage. Another eight non-cirrhotic rats formed the control group. After 10 days of treatment, a laparotomy was performed to determine microbiological study of ileal and cecal feces, bacterial translocation, endotoxemia, and intestinal malondialdehyde (MDA) levels as index of intestinal oxidative damage. Intestinal enterobacteria and enterococci, bacterial translocation (0/11 and 0/10 vs. 5/8, P<0.01), and ileal MDA levels (P<0.01) were lower in cirrhotic rats treated with antioxidants alone or in combination with Lactobacillus johnsonii La1 compared to cirrhotic rats receiving water. Only rats treated with antioxidants and Lactobacillus johnsonii La1 showed a decrease in endotoxemia with respect to cirrhotic rats receiving water (P<0.05). Antioxidants alone or in combination with Lactobacillus johnsonii La1 can be useful in preventing bacterial translocation in cirrhosis.

A fluorescence assay for peptide translocation into mitochondria.

PubMed

Martinez-Caballero, Sonia; Peixoto, Pablo M V; Kinnally, Kathleen W; Campo, María Luisa

2007-03-01

Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.

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)

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

PubMed

Hayama, Yoshitomo; Kimura, Tetsuya; Takeda, Yoshito; Nada, Shigeyuki; Koyama, Shohei; Takamatsu, Hyota; Kang, Sujin; Ito, Daisuke; Maeda, Yohei; Nishide, Masayuki; Nojima, Satoshi; Sarashina-Kida, Hana; Hosokawa, Takashi; Kinehara, Yuhei; Kato, Yasuhiro; Nakatani, Takeshi; Nakanishi, Yoshimitsu; Tsuda, Takeshi; Koba, Taro; Okada, Masato; Kumanogoh, Atsushi

2018-06-01

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses. Copyright © 2018 by The American Association of Immunologists, Inc.

The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coli RNA polymerase without abolishing its catalytic activities

PubMed Central

Hung, Siu Chun; Gottesman, Max E.

1997-01-01

Bacteriophage HK022 Nun protein blocks transcription elongation by Escherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3′-OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex. PMID:9334329

Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo

PubMed Central

Berkowitz, Bruce A.; Gorgis, Jawan; Patel, Ankit; Baameur, Faiza; Gurevich, Vsevolod V.; Craft, Cheryl M.; Kefalov, Vladimir J.; Roberts, Robin

2015-01-01

Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark- and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina Δ1/T1ρ was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark-to-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo.—Berkowitz, B. A., Gorgis, J., Patel, A., Baameur, F., Gurevich, V. V., Craft, C. M., Kefalov, V. J., Roberts, R. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo. PMID:25351983

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

PubMed

Pany, Satyabrata; Ghosh, Anamitra; You, Youngki; Nguyen, Nga; Das, Joydip

2017-11-01

Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca 2+ -sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca 2+ -sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1 H567K , a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural rearrangements at the translocation pore of the human glutamate transporter, EAAT1.

PubMed

Leighton, Barbara H; Seal, Rebecca P; Watts, Spencer D; Skyba, Mary O; Amara, Susan G

2006-10-06

Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-beta-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport.

β‑catenin nuclear translocation induced by HIF‑1α overexpression leads to the radioresistance of prostate cancer.

PubMed

Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-06-01

Hypoxia-inducible factor‑1α (HIF‑1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF‑1α remain unclear. β‑catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF‑1α and β‑catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4‑2B, were grouped as follows: Negative control (no treatment), HIF‑1α overexpression group (transfected with HIF‑1α overexpression plasmid) and β‑catenin silenced group (transfected with HIF‑1α plasmids and β‑catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4‑2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4‑2B cells, transfection with HIF‑1α overexpression plasmid led to an enhanced β‑catenin nuclear translocation, while β‑catenin silencing inhibited β‑catenin nuclear translocation. The enhanced β‑catenin nuclear translocation induced by HIF‑1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non‑homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF‑1α overexpression enhanced β‑catenin nuclear translocation, which led to the activation of the β‑catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF‑1α overexpression promotes the radioresistance of PCa cells.

Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

PubMed

Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

2016-04-10

Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Translocation of a heterogeneous polymer

PubMed Central

Mirigian, Stephen; Wang, Yanbo; Muthukumar, Murugappan

2012-01-01

We present results on the sequence dependence of translocation kinetics for a partially charged heteropolymer moving through a very thin pore using theoretical tools and Langevin dynamics simulational techniques. The chain is composed of two types of monomers of differing frictional interaction with the pore and charge. We present exact analytical expressions for passage probability, mean first passage time, and mean successful passage times for both reflecting/absorbing and absorbing/absorbing boundary conditions, showing rich and unexpected dependence of translocation behavior on charge fraction, distribution along the chain, and electric field configuration. We find excellent qualitative and good quantitative agreement between theoretical and simulation results. Surprisingly, there emerges a threshold charge fraction of a diblock copolymer beyond which the success rate of translocation is independent of charge fraction. Also, the mean successful translocation time of a diblock copolymer displays non-monotonic behavior with increasing length of the charged block; there is an optimum length of the charged block where the mean translocation rate is the slowest; and there can be a substantial range of higher charge fractions which make the translocation slower than even a minimally charged chain. Additionally, we find for a fixed total charge on the chain, finer distribution along the backbone significantly decreases mean translocation time. PMID:22897308

Problems with mitigation translocation of herpetofauna.

PubMed

Sullivan, Brian K; Nowak, Erika M; Kwiatkowski, Matthew A

2015-02-01

Mitigation translocation of nuisance animals is a commonly used management practice aimed at resolution of human-animal conflict by removal and release of an individual animal. Long considered a reasonable undertaking, especially by the general public, it is now known that translocated subjects are negatively affected by the practice. Mitigation translocation is typically undertaken with individual adult organisms and has a much lower success rate than the more widely practiced conservation translocation of threatened and endangered species. Nonetheless, the public and many conservation practitioners believe that because population-level conservation translocations have been successful that mitigation translocation can be satisfactorily applied to a wide variety of human-wildlife conflict situations. We reviewed mitigation translocations of reptiles, including our own work with 3 long-lived species (Gila monsters [Heloderma suspectum], Sonoran desert tortoises [Gopherus morafkai], and western diamond-backed rattlesnakes [Crotalus atrox]). Overall, mitigation translocation had a low success rate when judged either by effects on individuals (in all studies reviewed they exhibited increased movement or increased mortality) or by the success of the resolution of the human-animal conflict (translocated individuals often returned to the capture site). Careful planning and identification of knowledge gaps are critical to increasing success rates in mitigation translocations in the face of increasing pressure to find solutions for species threatened by diverse anthropogenic factors, including climate change and exurban and energy development. © 2014 Society for Conservation Biology.

Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

PubMed Central

Lagutina, Irina V.; Valentine, Virginia; Picchione, Fabrizio; Harwood, Frank; Valentine, Marcus B.; Villarejo-Balcells, Barbara; Carvajal, Jaime J.; Grosveld, Gerard C.

2015-01-01

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice. PMID:25659124

Financial Costs of Large Carnivore Translocations – Accounting for Conservation

PubMed Central

Weise, Florian J.; Stratford, Ken J.; van Vuuren, Rudolf J.

2014-01-01

Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars). Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23), and $2,108 per leopard (n = 6). One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%), followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4%) of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown) with a strong species bias. Four leopards (66.7%) were successfully translocated but only eight of the 20 cheetahs (40.0%) with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC) and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis. PMID

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.

Does translocation influence physiological stress in the desert tortoise?

USGS Publications Warehouse

Drake, K.K.; Nussear, K.E.; Esque, T.C.; Barber, A.M.; Vittum, K.M.; Medica, P.A.; Tracy, C.R.; Hunter, K.W.

2012-01-01

Wildlife translocation is increasingly used to mitigate disturbances to animals or habitat due to human activities, yet little is known about the extent to which translocating animals causes stress. To understand the relationship between physiological stress and translocation, we conducted a multiyear study (2007–2009) using a population of desert tortoises (Gopherus agassizii) near Fort Irwin, California. Blood samples were collected from adult tortoises in three treatment groups (resident, translocated and control) for 1 year prior to and 2 years after translocation. Samples were analyzed by radioimmunoassay for plasma total corticosterone (CORT), a glucocorticoid hormone commonly associated with stress responses in reptiles. CORT values were analyzed in relation to potential covariates (animal sex, date, behavior, treatment, handling time, air temperature, home-range size, precipitation and annual plant production) among seasons and years. CORT values in males were higher than in females, and values for both varied monthly throughout the activity season and among years. Year and sex were strong predictors of CORT, and translocation explained little in terms of CORT. Based on these results, we conclude that translocation does not elicit a physiological stress response in desert tortoises.

Septin 9 isoform 1 (SEPT9_i1) specifically interacts with importin-α7 to drive hypoxia-inducible factor (HIF)-1α nuclear translocation.

PubMed

Tazat, Keren; Schindler, Susanne; Deppeing, Reinhard; Mabjeesh, Nicola J

2018-05-09

We have shown previously that septin 9 isoform 1 (SEPT9_i1) protein associates with hypoxia-inducible factor (HIF)-1α to augment HIF-1 transcriptional activity by driving its importin-α-mediated nuclear translocation. Using in vitro and in vivo binding assays we identified that HIF-1α interacts with importin-α5 and importin-α7 in prostate cancer cells but only importin-α7 interacts with SEPT9_i1. The interaction with importin-α7 was dependent on the first 25 amino acids of SEPT9_i1 that are unique compared to other members of the mammalian septin family. Depletion of endogenous importin-α7 reduced nuclear HIF-1α levels in the nucleus. Our results provide evidence that there are importin-α specificities in the cytosolic/nuclear translocation process of HIF-1α protein, which may act differently under certain pathophysiological circumstances where SEPT9_i1 is overexpressed. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Phenotypic consequences of gene disruption by a balanced de novo translocation involving SLC6A1 and NAA15.

PubMed

Pesz, Karolina; Pienkowski, Victor Murcia; Pollak, Agnieszka; Gasperowicz, Piotr; Sykulski, Maciej; Kosińska, Joanna; Kiszko, Magdalena; Krzykwa, Bogusława; Bartnik-Głaska, Magdalena; Nowakowska, Beata; Rydzanicz, Małgorzata; Sasiadek, Maria Małgorzata; Płoski, Rafał

2018-04-03

Mapping of de novo balanced chromosomal translocations (BCTs) in patients with sporadic poorly characterized disease(s) is an unbiased method of finding candidate gene(s) responsible for the observed symptoms. We present a paediatric patient suffering from epilepsy, developmental delay (DD) and atrial septal defect IIº (ASD) requiring surgery. Karyotyping indicated an apparently balanced de novo reciprocal translocation 46,XX,t(3;4)(p25.3;q31.1), whereas aCGH did not reveal any copy number changes. Using shallow mate-pair whole genome sequencing and direct Sanger sequencing of breakpoint regions we found that translocation disrupted SLC6A1 and NAA15 genes. Our results confirm two previous reports indicating that loss of function of a single allele of SLC6A1 causes epilepsy. In addition, we extend existing evidence that disruption of NAA15 is associated with DD and with congenital heart defects. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Imidazopyridine- and purine-thioacetamide derivatives: potent inhibitors of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).

PubMed

Chang, Lei; Lee, Sang-Yong; Leonczak, Piotr; Rozenski, Jef; De Jonghe, Steven; Hanck, Theodor; Müller, Christa E; Herdewijn, Piet

2014-12-11

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) belongs to the family of ecto-nucleotidases, which control extracellular nucleotide, nucleoside, and (di)phosphate levels. To study the (patho)physiological roles of NPP1 potent and selective inhibitors with drug-like properties are required. Therefore, a compound library was screened for NPP1 inhibitors using a colorimetric assay with p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as a hit compound with a Ki value of 217 nM. Subsequent structure-activity relationship studies led to the development of purine and imidazo[4,5-b]pyridine analogues with high inhibitory potency (Ki values of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5'-TMP as a substrate. Surprisingly, the compounds were significantly less potent when tested versus ATP as a substrate, with Ki values in the low micromolar range. A prototypic inhibitor was investigated for its mechanism of inhibition and found to be competitive versus both substrates.

Interactions between Kar2p and Its Nucleotide Exchange Factors Sil1p and Lhs1p Are Mechanistically Distinct*

PubMed Central

Hale, Sarah J.; Lovell, Simon C.; de Keyzer, Jeanine; Stirling, Colin J.

2010-01-01

Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p. In contrast, the IIB domain of Kar2p is sufficient for binding of Sil1p, and point mutations within IIB specifically blocked Sil1p-dependent activation while remaining competent for activation by Lhs1p. Taken together, these results demonstrate that the interactions between Kar2p and its two nucleotide exchange factors can be functionally resolved and are thus mechanistically distinct. PMID:20430899

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction.

PubMed

Ivanova, Christa; Ramoni, Jonas; Aouam, Thiziri; Frischmann, Alexa; Seiboth, Bernhard; Baker, Scott E; Le Crom, Stéphane; Lemoine, Sophie; Margeot, Antoine; Bidard, Frédérique

2017-01-01

The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei , the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1 , and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose

Cellular and molecular effects of nonreciprocal chromosome translocations in Saccharomyces cerevisiae

PubMed Central

Nikitin, Dmitri; Tosato, Valentina; Zavec, Apolonija Bedina; Bruschi, Carlo V.

2008-01-01

Saccharomyces cerevisiae strains harboring a nonreciprocal, bridge-induced translocation (BIT) between chromosomes VIII and XV exhibited an abnormal phenotype comprising elongated buds and multibudded, unevenly nucleated pseudohyphae. In these cells, we found evidence of molecular effects elicited by the translocation event and specific for its particular genomic location. Expression of genes flanking both translocation breakpoints increased up to five times, correlating with an increased RNA polymerase II binding to their promoters and with their histone acetylation pattern. Microarray data, CHEF, and quantitative PCR confirmed the data on the dosage of genes present on the chromosomal regions involved in the translocation, indicating that telomeric fragments were either duplicated or integrated mostly on chromosome XI. FACS analysis revealed that the majority of translocant cells were blocked in G1 phase and a few of them in G2. Some cells showed a posttranslational decrease of cyclin B1, in agreement with elongated buds diagnostic of a G2/M phase arrest. The actin1 protein was in some cases modified, possibly explaining the abnormal morphology of the cells. Together with the decrease in Rad53p and the lack of its phosphorylation, these results indicate that these cells have undergone adaptation after checkpoint-mediated G2/M arrest after chromosome translocation. These BIT translocants could serve as model systems to understand further the cellular and molecular effects of chromosome translocation and provide fundamental information on its etiology of neoplastic transformation in mammals. PMID:18599460

Sequencing and Analyzing the "t" (1;7) Reciprocal Translocation Breakpoints Associated with a Case of Childhood-Onset Schizophrenia/Autistic Disorder

ERIC Educational Resources Information Center

Idol, Jacquelyn R.; Addington, Anjene M.; Long, Robert T.; Rapoport, Judith L.; Green, Eric D.

2008-01-01

We characterized a "t"(1;7)(p22;q21) reciprocal translocation in a patient with childhood-onset schizophrenia (COS) and autism using genome mapping and sequencing methods. Based on genomic maps of human chromosome 7 and fluorescence in situ hybridization (FISH) studies, we delimited the region of 7q21 harboring the translocation breakpoint to a…

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

PubMed Central

García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

2015-01-01

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo.

PubMed

Berkowitz, Bruce A; Gorgis, Jawan; Patel, Ankit; Baameur, Faiza; Gurevich, Vsevolod V; Craft, Cheryl M; Kefalov, Vladimir J; Roberts, Robin

2015-02-01

Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark- and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina Δ1/T1ρ was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark-to-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo. © FASEB.

Moderate Light-Induced Degeneration of Rod Photoreceptors with Delayed Transducin Translocation in shaker1 Mice

PubMed Central

Zallocchi, Marisa; Wang, Wei-Min; Delimont, Duane; Cosgrove, Dominic

2011-01-01

Purpose. Usher syndrome is characterized by congenital deafness associated with retinitis pigmentosa (RP). Mutations in the myosin VIIa gene (MYO7A) cause a common and severe subtype of Usher syndrome (USH1B). Shaker1 mice have mutant MYO7A. They are deaf and have vestibular dysfunction but do not develop photoreceptor degeneration. The goal of this study was to investigate abnormalities of photoreceptors in shaker1 mice. Methods. Immunocytochemistry and hydroethidine-based detection of intracellular superoxide production were used. Photoreceptor cell densities under various conditions of light/dark exposures were evaluated. Results. In shaker1 mice, the rod transducin translocation is delayed because of a shift of its light activation threshold to a higher level. Even moderate light exposure can induce oxidative damage and significant rod degeneration in shaker1 mice. Shaker1 mice reared under a moderate light/dark cycle develop severe retinal degeneration in less than 6 months. Conclusions. These findings show that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation. Importantly, USH1B animal models are likely vulnerable to light-induced photoreceptor damage, even under moderate light. PMID:21447681

Suitability of amphibians and reptiles for translocation.

PubMed

Germano, Jennifer M; Bishop, Phillip J

2009-02-01

Translocations are important tools in the field of conservation. Despite increased use over the last few decades, the appropriateness of translocations for amphibians and reptiles has been debated widely over the past 20 years. To provide a comprehensive evaluation of the suitability of amphibians and reptiles for translocation, we reviewed the results of amphibian and reptile translocation projects published between 1991 and 2006. The success rate of amphibian and reptile translocations reported over this period was twice that reported in an earlier review in 1991. Success and failure rates were independent of the taxonomic class (Amphibia or Reptilia) released. Reptile translocations driven by human-wildlife conflict mitigation had a higher failure rate than those motivated by conservation, and more recent projects of reptile translocations had unknown outcomes. The outcomes of amphibian translocations were significantly related to the number of animals released, with projects releasing over 1000 individuals being most successful. The most common reported causes of translocation failure were homing and migration of introduced individuals out of release sites and poor habitat. The increased success of amphibian and reptile translocations reviewed in this study compared with the 1991 review is encouraging for future conservation projects. Nevertheless, more preparation, monitoring, reporting of results, and experimental testing of techniques and reintroduction questions need to occur to improve translocations of amphibians and reptiles as a whole.

Probiotic Mixture Golden Bifido Prevents Neonatal Escherichia coli K1 Translocation via Enhancing Intestinal Defense

PubMed Central

Zeng, Qing; He, Xiaolong; Puthiyakunnon, Santhosh; Xiao, Hansen; Gong, Zelong; Boddu, Swapna; Chen, Lecheng; Tian, Huiwen; Huang, Sheng-He; Cao, Hong

2017-01-01

Escherichia coli (E. coli) K1 sepsis and meningitis is a severe infection characterized by high mortality in neonates. Successful colonization and translocation across the intestinal mucosa have been regarded as the critical steps for E. coli K1 sepsis and meningitis. We recently reported that the probiotic mixture, Golden Bifido (containing live Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus, LBS) has a preventive role against neonatal E. coli K1 bacteremia and meningitis. However, the interaction between the neonatal gut barrier, probiotics and E. coli K1 is still not elucidated. The present study aims to investigate how LBS exerts its protective effects on neonatal gut barrier during E. coli K1 infection. The beneficial effects of LBS were explored in vitro and in vivo using human colon carcinoma cell lines HT-29 and rat model of neonatal E. coli K1 infection, respectively. Our results showed that stimulation with E. coli K1 was able to cause intestinal barrier dysfunction, which were reflected by E. coli K1-induced intestinal damage and apoptosis of intestinal epithelial cells, reduction of mucin, immunoglobulin A (IgA) and tight junction proteins expression, as well as increase in intestinal permeability, all these changes facilitate E. coli K1 intestinal translocation. However, these changes were alleviated when HT-29 cells were treated with LBS before E. coli K1 infection. Furthermore, we found that LBS-treated neonatal rats (without E. coli K1 infection) have showed higher production of mucin, ZO-1, IgA, Ki67 in intestinal mucosa as well as lower intestinal permeability than that of non-treated rats, indicating that LBS could accelerate the development of neonatal intestinal defense. Taken together, our results suggest that enhancement of the neonatal intestinal defense to fight against E. coli K1 translocation could be the potential mechanism to elucidate how LBS confers a protective effect against neonatal E. coli K1 bacteremia

Probiotic Mixture Golden Bifido Prevents Neonatal Escherichia coli K1 Translocation via Enhancing Intestinal Defense.

PubMed

Zeng, Qing; He, Xiaolong; Puthiyakunnon, Santhosh; Xiao, Hansen; Gong, Zelong; Boddu, Swapna; Chen, Lecheng; Tian, Huiwen; Huang, Sheng-He; Cao, Hong

2017-01-01

Escherichia coli ( E. coli ) K1 sepsis and meningitis is a severe infection characterized by high mortality in neonates. Successful colonization and translocation across the intestinal mucosa have been regarded as the critical steps for E. coli K1 sepsis and meningitis. We recently reported that the probiotic mixture, Golden Bifido (containing live Lactobacillus bulgaricus, Bifidobacterium , and Streptococcus thermophilus , LBS) has a preventive role against neonatal E. coli K1 bacteremia and meningitis. However, the interaction between the neonatal gut barrier, probiotics and E. coli K1 is still not elucidated. The present study aims to investigate how LBS exerts its protective effects on neonatal gut barrier during E. coli K1 infection. The beneficial effects of LBS were explored in vitro and in vivo using human colon carcinoma cell lines HT-29 and rat model of neonatal E. coli K1 infection, respectively. Our results showed that stimulation with E. coli K1 was able to cause intestinal barrier dysfunction, which were reflected by E. coli K1-induced intestinal damage and apoptosis of intestinal epithelial cells, reduction of mucin, immunoglobulin A (IgA) and tight junction proteins expression, as well as increase in intestinal permeability, all these changes facilitate E. coli K1 intestinal translocation. However, these changes were alleviated when HT-29 cells were treated with LBS before E. coli K1 infection. Furthermore, we found that LBS-treated neonatal rats (without E. coli K1 infection) have showed higher production of mucin, ZO-1, IgA, Ki67 in intestinal mucosa as well as lower intestinal permeability than that of non-treated rats, indicating that LBS could accelerate the development of neonatal intestinal defense. Taken together, our results suggest that enhancement of the neonatal intestinal defense to fight against E. coli K1 translocation could be the potential mechanism to elucidate how LBS confers a protective effect against neonatal E. coli K1

MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

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

Yu, Teng, E-mail: tengyu33@yahoo.com; Ji, Jiang; Guo, Yong-li

2013-11-08

Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen speciesmore » (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.« less

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.

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.

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.

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.

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.

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.

Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress

PubMed Central

Picard, Martin; McManus, Meagan J.; Gray, Jason D.; Nasca, Carla; Moffat, Cynthia; Kopinski, Piotr K.; Seifert, Erin L.; McEwen, Bruce S.; Wallace, Douglas C.

2015-01-01

The experience of psychological stress triggers neuroendocrine, inflammatory, metabolic, and transcriptional perturbations that ultimately predispose to disease. However, the subcellular determinants of this integrated, multisystemic stress response have not been defined. Central to stress adaptation is cellular energetics, involving mitochondrial energy production and oxidative stress. We therefore hypothesized that abnormal mitochondrial functions would differentially modulate the organism’s multisystemic response to psychological stress. By mutating or deleting mitochondrial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine nucleotide translocator 1 (ANT1) and nicotinamide nucleotide transhydrogenase (NNT)], we selectively impaired mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance in mice. The resulting impact on physiological reactivity and recovery from restraint stress were then characterized. We show that mitochondrial dysfunctions altered the hypothalamic–pituitary–adrenal axis, sympathetic adrenal–medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress. Each mitochondrial defect generated a distinct whole-body stress-response signature. These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease. This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases. PMID:26627253

Cloning a balanced t(9;11)(p24;q23.1) chromosomal translocation breakpoint segregating with bipolar affective disorder in a small pedigree

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

Duggan, D.J.; Baysal, B.E.; Gollin, S.M.

A small multigenerational pedigree was previously identified in which a balanced 9;11 chromosomal translocation was cosegregating with bipolar affective disorder. We hypothesize that genes or gene regulatory sequences disrupted by the translocation are contributing to bipolar affective disorder in a dominant fashion. The general strategy involves (1) using somatic cell hybrids containing the derivative 9 or 11 chromosomes to identify the closest chromosome 9 and 11 flanking markers, (2) using the nearest markers as PCR and hybridization probes to isolate both normal DNA (YAC) and patient DNA (cosmid) adjacent to and incorporating the translocation breakpoint, and (3) identifying expressed sequencesmore » in the genomic DNA that may be disrupted by the translocation. From a fusion of the translocation patient cell line and a recipient hamster cell line, somatic cell hybrids were isolated which contain either the human derivative 9 or derivative 11 chromosome. Using PCR-based STS assays with these hybrids, the location of the translocation breakpoint was localized to an estimated 500 kb region at chromosome 11 band q23.1 and a 1 cM region in 9 band p24 (more telomeric than originally reported). From a large set of CEPH and Roswell Park yeast artificial chromosomes (YACs), six chromosome 11 YACs spanning the 11q23.1 breakpoint have now been identified. A combination of pulsed field gel eletrophoresis and YAC mapping has narrowed the chromosome 11 region to less than 430 kb. Current efforts are focused on generating new chromosome 11 probes within the flanking markers, mapping these probes back to the der(9) and der(11) containing hybrids and the chromosome 11 YAC mapping panel. As the region is physically narrowed, we will identify candidate genes whose expression may be altered by this t(9:11) translocation.« less

CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

PubMed

Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

2017-01-10

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis.

PubMed

Raad, Houssam; Serrano-Sanchez, Martin; Harfouche, Ghida; Mahfouf, Walid; Bortolotto, Doriane; Bergeron, Vanessa; Kasraian, Zeinab; Dousset, Lea; Hosseini, Mohsen; Taieb, Alain; Rezvani, Hamid Reza

2017-06-01

The nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes are involved in several physiological functions. However, their roles in keratinocyte responses to UV radiation have not been clearly elucidated. This study shows that, among other NOX family members, UVB irradiation results in a biphasic activation of NOX1 that plays a critical role in defining keratinocyte fate through the modulation of the DNA damage response network. Indeed, suppression of both bursts of UVB-induced NOX1 activation by using a specific peptide inhibitor of NOX1 (InhNOX1) is associated with increased nucleotide excision repair efficiency and reduction of apoptosis, which is finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation is blocked, both nucleotide excision repair efficiency and apoptosis are decreased. Our results show that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced skin cancer in nucleotide excision repair-proficient and -deficient patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

PubMed

Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

2016-05-16

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

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

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

DOE PAGES

Gleghorn, Michael L.; Zhao, Jianbo; Turner, Douglas H.; ...

2016-06-10

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA 7, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA 7 forms well-ordered crystals, whereas rA 6 forms fragile crystalline-like structures, and rA 5, rA 8 and rA 11 fail to crystallize. Our findings support studies from ~50 years ago: one showed usingmore » spectroscopic methods that duplex formation at pH 4.5 largely starts with rA 7 and begins to plateau with rA 8; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP–rAMP helix base pair. Lastly, our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.« less

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

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

Gleghorn, Michael L.; Zhao, Jianbo; Turner, Douglas H.

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA 7, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA 7 forms well-ordered crystals, whereas rA 6 forms fragile crystalline-like structures, and rA 5, rA 8 and rA 11 fail to crystallize. Our findings support studies from ~50 years ago: one showed usingmore » spectroscopic methods that duplex formation at pH 4.5 largely starts with rA 7 and begins to plateau with rA 8; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP–rAMP helix base pair. Lastly, our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.« less

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.

High-speed detection of DNA translocation in nanopipettes.

PubMed

Fraccari, Raquel L; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

2016-04-14

We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

PubMed Central

Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

2016-01-01

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

Chromosomal translocations and palindromic AT-rich repeats

PubMed Central

Kato, Takema; Kurahashi, Hiroki; Emanuel1, Beverly S.

2012-01-01

Repetitive DNA sequences constitute 30% of the human genome, and are often sites of genomic rearrangement. Recently, it has been found that several constitutional translocations, especially those that involve chromosome 22, take place utilizing palindromic sequences on 22q11 and on the partner chromosome. Analysis of translocation junction fragments shows that the breakpoints of such palindrome-mediated translocations are localized at the center of palindromic AT-rich repeats (PATRRs). The presence of PATRRs at the breakpoints, indicates a palindrome-mediated mechanism involved in the generation of these constitutional translocations. Identification of these PATRR-mediated translocations suggests a universal pathway for gross chromosomal rearrangement in the human genome. De novo occurrences of PATRR-mediated translocations can be detected by PCR in normal sperm samples but not somatic cells. Polymorphisms of various PATRRs influence their propensity for adopting a secondary structure, which in turn affects de novo translocation frequency. We propose that the PATRRs form an unstable secondary structure, which leads to double-strand breaks at the center of the PATRR. The double-strand breaks appear to be followed by a non-homologous end-joining repair pathway, ultimately leading to the translocations. This review considers recent findings concerning the mechanism of meiosis-specific, PATRR-mediated translocations. PMID:22402448

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.

High-speed detection of DNA translocation in nanopipettes

NASA Astrophysics Data System (ADS)

Fraccari, Raquel L.; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

2016-03-01

We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface. Electronic supplementary information (ESI) available: Gel electrophoresis confirming lengths and purity of DNA samples, comparison between Axopatch 200B and custom-built setup, comprehensive low-noise amplifier characterization, representative I-V curves of nanopipettes used, typical scatter plots of τ vs. peak amplitude for the four LDNA's used, table of most probable τ values, a comparison between different fitting models for the DNA translocation time distribution, further details on the stochastic numerical simulation of the scaling statistics and the derivation of the extended

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).

Minimizing the cost of translocation failure with decision-tree models that predict species' behavioral response in translocation sites.

PubMed

Ebrahimi, Mehregan; Ebrahimie, Esmaeil; Bull, C Michael

2015-08-01

The high number of failures is one reason why translocation is often not recommended. Considering how behavior changes during translocations may improve translocation success. To derive decision-tree models for species' translocation, we used data on the short-term responses of an endangered Australian skink in 5 simulated translocations with different release conditions. We used 4 different decision-tree algorithms (decision tree, decision-tree parallel, decision stump, and random forest) with 4 different criteria (gain ratio, information gain, gini index, and accuracy) to investigate how environmental and behavioral parameters may affect the success of a translocation. We assumed behavioral changes that increased dispersal away from a release site would reduce translocation success. The trees became more complex when we included all behavioral parameters as attributes, but these trees yielded more detailed information about why and how dispersal occurred. According to these complex trees, there were positive associations between some behavioral parameters, such as fight and dispersal, that showed there was a higher chance, for example, of dispersal among lizards that fought than among those that did not fight. Decision trees based on parameters related to release conditions were easier to understand and could be used by managers to make translocation decisions under different circumstances. © 2015 Society for Conservation Biology.

Increased frequency of chromosome translocations in airline pilots with long-term flying experience.

PubMed

Yong, L C; Sigurdson, A J; Ward, E M; Waters, M A; Whelan, E A; Petersen, M R; Bhatti, P; Ramsey, M J; Ron, E; Tucker, J D

2009-01-01

Chromosome translocations are an established biomarker of cumulative exposure to external ionising radiation. Airline pilots are exposed to cosmic ionising radiation, but few flight crew studies have examined translocations in relation to flight experience. We determined the frequency of translocations in the peripheral blood lymphocytes of 83 airline pilots and 50 comparison subjects (mean age 47 and 46 years, respectively). Translocations were scored in an average of 1039 cell equivalents (CE) per subject using fluorescence in situ hybridisation (FISH) whole chromosome painting and expressed per 100 CE. Negative binomial regression models were used to assess the relationship between translocation frequency and exposure status and flight years, adjusting for age, diagnostic x ray procedures, and military flying. There was no significant difference in the adjusted mean translocation frequency of pilots and comparison subjects (0.37 (SE 0.04) vs 0.38 (SE 0.06) translocations/100 CE, respectively). However, among pilots, the adjusted translocation frequency was significantly associated with flight years (p = 0.01) with rate ratios of 1.06 (95% CI 1.01 to 1.11) and 1.81 (95% CI 1.16 to 2.82) for a 1- and 10-year incremental increase in flight years, respectively. The adjusted rate ratio for pilots in the highest compared to the lowest quartile of flight years was 2.59 (95% CI 1.26 to 5.33). Our data suggests that pilots with long-term flying experience may be exposed to biologically significant doses of ionising radiation. Epidemiological studies with longer follow-up of larger cohorts of pilots with a wide range of radiation exposure levels are needed to clarify the relationship between cosmic radiation exposure and cancer risk.

Production and Identification of Wheat-Agropyron cristatum 2P Translocation Lines

PubMed Central

Li, Huanhuan; Lv, Mingjie; Song, Liqiang; Zhang, Jinpeng; Gao, Ainong; Li, Lihui; Liu, Weihua

2016-01-01

Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), a wild relative of common wheat, possesses many potentially valuable traits that can be transferred to common wheat through breeding programs. The wheat-A. cristatum disomic addition and translocation lines can be used as bridge materials to introduce alien chromosomal segments to wheat. Wheat-A. cristatum 2P disomic addition line II-9-3 was highly resistant to powdery mildew and leaf rust, which was reported in our previous study. However, some translocation lines induced from II-9-3 have not been reported. In this study, some translocation lines were induced from II-9-3 by 60Co-γ irradiation and gametocidal chromosome 2C and then identified by cytological methods. Forty-nine wheat-A. cristatum translocation lines were obtained and various translcoation types were identified by GISH (genomic in situ hybridization), such as whole-arm, segmental and intercalary translocations. Dual-color FISH (fluorescent in situ hybridization) was applied to identify the wheat chromosomes involved in the translocations, and the results showed that A. cristatum 2P chromosome segments were translocated to the different wheat chromosomes, including 1A, 2A, 3A, 4A, 5A, 6A, 7A, 3B, 5B, 7B, 1D, 4D and 6D. Many different types of wheat-A. cristatum alien translocation lines would be valuable for not only identifying and cloning A. cristatum 2P-related genes and understanding the genetics and breeding effects of the translocation between A. cristatum chromosome 2P and wheat chromosomes, but also providing new germplasm resources for the wheat genetic improvement. PMID:26731742

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

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

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

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

PubMed

Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

2011-01-01

Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Synchrotron Protein Footprinting Supports Substrate Translocation by ClpA via ATP-Induced Movements of the D2 Loop

PubMed Central

Bohon, Jen; Jennings, Laura D.; Phillips, Christine M.; Licht, Stuart; Chance, Mark R.

2010-01-01

SUMMARY Synchrotron x-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation. PMID:18682217

Aminocyclopyrachlor absorption, translocation and metabolism in field 1 bindweed (convolulus arvensis)

USDA-ARS?s Scientific Manuscript database

Field bindweed (Convolvulus arvensis L.) is extremely susceptible to aminocyclopyrachlor compared to other weed species. Laboratory studies were conducted to determine if absorption, translocation, and metabolism of aminocyclopyrachlor in field bindweed differs from other, less susceptible species....

Clinicopathological features of Xp11.2 translocation renal cell carcinoma.

PubMed

Lim, Bumjin; You, Dalsan; Jeong, In Gab; Kwon, Taekmin; Hong, Sungwoo; Song, Cheryn; Cho, Yong Mee; Hong, Bumsik; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung Soo

2015-03-01

Xp11.2 translocation renal cell carcinoma (RCC) is characterized by various translocations of the TFE3 transcription factor gene. These rare cancers occur predominantly in children and young adults. Here, we review the clinicopathological features of Xp11.2 translocation RCC. We identified 21 patients with Xp11.2 translocation RCC. We retrospectively analyzed patient characteristics, clinical manifestations, and specific pathological features to assess definitive diagnosis, surgical and systemic treatments, and clinical outcomes. The mean age at diagnosis was 43.4±20.0 years (range, 8-80 years; 8 males and 13 females). Eleven patients were incidentally diagnosed, nine patients presented with local symptoms, and one patient presented with systemic symptoms. The mean tumor size was 6.2±3.8 cm (range, 1.9-14 cm). At the time of diagnosis, 11, 1, and 5 patients showed stage I, II, and III, respectively. Four patients showed distant metastasis. At analysis, 15 patients were disease-free after a median follow-up period of 30.0 months. Four patients received target therapy but not effectively. Xp11 translocation RCC tends to develop in young patients with lymph node metastasis. Targeted therapy did not effectively treat our patients. Surgery is the only effective therapy for Xp11 translocation RCC, and further studies are needed to assess systemic therapy and long-term prognosis.

Clinicopathological features of Xp11.2 translocation renal cell carcinoma

PubMed Central

Lim, Bumjin; You, Dalsan; Jeong, In Gab; Kwon, Taekmin; Hong, Sungwoo; Song, Cheryn; Cho, Yong Mee; Hong, Bumsik; Hong, Jun Hyuk; Ahn, Hanjong

2015-01-01

Purpose Xp11.2 translocation renal cell carcinoma (RCC) is characterized by various translocations of the TFE3 transcription factor gene. These rare cancers occur predominantly in children and young adults. Here, we review the clinicopathological features of Xp11.2 translocation RCC. Materials and Methods We identified 21 patients with Xp11.2 translocation RCC. We retrospectively analyzed patient characteristics, clinical manifestations, and specific pathological features to assess definitive diagnosis, surgical and systemic treatments, and clinical outcomes. Results The mean age at diagnosis was 43.4±20.0 years (range, 8-80 years; 8 males and 13 females). Eleven patients were incidentally diagnosed, nine patients presented with local symptoms, and one patient presented with systemic symptoms. The mean tumor size was 6.2±3.8 cm (range, 1.9-14 cm). At the time of diagnosis, 11, 1, and 5 patients showed stage I, II, and III, respectively. Four patients showed distant metastasis. At analysis, 15 patients were disease-free after a median follow-up period of 30.0 months. Four patients received target therapy but not effectively. Conclusions Xp11 translocation RCC tends to develop in young patients with lymph node metastasis. Targeted therapy did not effectively treat our patients. Surgery is the only effective therapy for Xp11 translocation RCC, and further studies are needed to assess systemic therapy and long-term prognosis. PMID:25763125

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Crystal Structures of MEK1 Binary and Ternary Complexes with Nucleotides and Inhibitors

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

Fischmann, Thierry O.; Smith, Catherine K.; Mayhood, Todd W.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35-382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-{gamma}S determined at 2.1 {angstrom}. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow formore » the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 {angstrom} resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-{gamma}S with compound 1 and ADP with either U0126 or the MEK1 clinical candidate PD325089 at 1.8, 2.0, and 2.5 {angstrom}, respectively. Compound 1 is structurally similar to PD325901. These structures illustrate fundamental differences among various mechanisms of inhibition at the molecular level. Residues 44-51 have previously been shown to play a negative regulatory role in MEK1 activity. The crystal structure of the integral kinase domain provides a structural rationale for the role of these residues. They form helix A and repress enzymatic activity by stabilizing an inactive conformation in which helix C is displaced from its active state position. Finally, the structure provides for the first time a molecular rationale that explains how mutations in MEK may lead to the cardio-facio-cutaneous syndrome.« less

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein modulates ABCA1 trafficking and function

PubMed Central

Lin, Sisi; Zhou, Chun; Neufeld, Edward; Wang, Yu-Hua; Xu, Suo-Wen; Lu, Liang; Wang, Ying; Liu, Zhi-Ping; Li, Dong; Li, Cuixian; Chen, Shaorui; Le, Kang; Huang, Heqing; Liu, Peiqing; Moss, Joel; Vaughan, Martha; Shen, Xiaoyan

2013-01-01

Objective Cell surface localization and intracellular trafficking of ATP-binding cassette transporter A-1 (ABCA1) are essential for its function. However, regulation of these activities is still largely unknown. Brefeldin A (BFA), a uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. Methods and Results By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibited by 60% cholesterol release. In contrast, BIG1 over-expression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through over-expression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 siRNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1 (ARF1). Conclusions BIG1, through its ability to activate ARF1, regulates cell surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action. PMID:23220274

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.

Increased frequency of chromosome translocations in airline pilots with long-term flying experience

PubMed Central

Yong, L C; Sigurdson, A J; Ward, E M; Waters, M A; Whelan, E A; Petersen, M R; Bhatti, P; Ramsey, M J; Ron, E; Tucker, J D

2008-01-01

Background Chromosome translocations are an established biomarker of cumulative exposure to external ionising radiation. Airline pilots are exposed to cosmic ionising radiation, but few flight crew studies have examined translocations in relation to flight experience. Methods We determined the frequency of translocations in the peripheral blood lymphocytes of 83 airline pilots and 50 comparison subjects (mean age 47 and 46 years, respectively). Translocations were scored in an average of 1039 cell equivalents (CE) per subject using fluorescence in situ hybridisation (FISH) whole chromo-some painting and expressed per 100 CE. Negative binomial regression models were used to assess the relationship between translocation frequency and exposure status and flight years, adjusting for age, diagnostic x ray procedures, and military flying. Results There was no significant difference in the adjusted mean translocation frequency of pilots and comparison subjects (0.37 (SE 0.04) vs 0.38 (SE 0.06) translocations/100 CE, respectively). However, among pilots, the adjusted translocation frequency was significantly associated with flight years (p = 0.01) with rate ratios of 1.06 (95% CI 1.01 to 1.11) and 1.81 (95% CI 1.16 to 2.82) for a 1- and 10-year incremental increase in flight years, respectively. The adjusted rate ratio for pilots in the highest compared to the lowest quartile of flight years was 2.59 (95% CI 1.26 to 5.33). Conclusions This data suggests that pilots with long-term flying experience may be exposed to biologically significant doses of ionising radiation. Epidemiological studies with longer follow-up of larger cohorts of pilots with a wide range of radiation exposure levels are needed to clarify the relationship between cosmic radiation exposure and cancer risk. PMID:19074211

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation.

PubMed

Willett-Brozick, J E; Savul, S A; Richey, L E; Baysal, B E

2001-08-01

Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.

Human X-Linked genes regionally mapped utilizing X-autosome translocations and somatic cell hybrids.

PubMed Central

Shows, T B; Brown, J A

1975-01-01

Human genes coding for hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49; D-glucose-6-phosphate:NADP+ 1-oxidoreductase), and phosphoglycerate kinase (PGK, EC 2.7.2.3; ATP:3-phospho-D-glycerate 1-phosphotransferase) have been assigned to specific regions on the long arm of the X chromosome by somatic cell gentic techniques. Gene assignment and linear order were determined by employing human somatic cells possessing an X/9 translocation or an X/22 translocation in man-mouse cell hybridization studies. The X/9 translocation involved the majority of the X long arm translocated to chromosome 9 and the X/22 translocation involved the distal half of the X long arm translocated to 22. In each case these rearrangements appeared to be reciprocal. Concordant segregation of X-linked enzymes and segments of the X chromosome generated by the translocations indicated assignment of the PGK gene to a proximal long arm region (q12-q22) and the HPRT and G6PD genes to the distal half (q22-qter) of the X long arm. Further evidence suggests a gene order on the X long arm of centromere-PGK-HPRT-G6PD. Images PMID:1056018

PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum

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

Ode, Takashi; Research Fellow of the Japan Society for the Promotion of Science; Podyma-Inoue, Katarzyna A.

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, wemore » found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER. - Highlights: • The ceramide analogue, PDMP, suppressed the activation of mTORC1. • PDMP induced the translocation of mTOR from lysosomes to ER. • PDMP led to the dissociation of mTOR from its activator Rheb. • PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation.« less

Yeast one-hybrid system used to identify the binding proteins for rat glutathione S-transferase P enhancer I.

PubMed

Liao, Ming-Xiang; Liu, Dong-Yuan; Zuo, Jin; Fang, Fu-De

2002-03-01

To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI). Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI. cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. The binding of c-Jun and ANT to GPEI core sequence were confirmed. Rat c-jun transcriptional factor and ANT may interact with cGPEI. They could play an important role in the induced expression of GST-P gene.

Polymer translocation under a pulling force: Scaling arguments and threshold forces

NASA Astrophysics Data System (ADS)

Menais, Timothée

2018-02-01

DNA translocation through nanopores is one of the most promising strategies for next-generation sequencing technologies. Most experimental and numerical works have focused on polymer translocation biased by electrophoresis, where a pulling force acts on the polymer within the nanopore. An alternative strategy, however, is emerging, which uses optical or magnetic tweezers. In this case, the pulling force is exerted directly at one end of the polymer, which strongly modifies the translocation process. In this paper, we report numerical simulations of both linear and structured (mimicking DNA) polymer models, simple enough to allow for a statistical treatment of the pore structure effects on the translocation time probability distributions. Based on extremely extended computer simulation data, we (i) propose scaling arguments for an extension of the predicted translocation times τ ˜N2F-1 over the moderate forces range and (ii) analyze the effect of pore size and polymer structuration on translocation times τ .

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

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.

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

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

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

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.

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

Movements of translocated wolves in Minnesota

USGS Publications Warehouse

Fritts, S.H.; Paul, W.J.; Mech, L.D.

1984-01-01

From Feb. 1975-May 1978, 104 wolves (C. lupus) captured at or near Minnesota farms where depredations on livestock had been reported were translocated northward and eastward for 50-317 km into extensive forests; 3 others were released westward. Nine wolves were translocated twice, and 1 three times. Information on movements was obtained by radiotracking 17 wolves and by recovery of 16 others. All radio-tagged wolves left the release areas; adults left quickly, but pups generally remained longer, behaved less decisively, and settled nearby temporarily. Wolves released together did not remain together. Initial travel of most radio-tagged adults was between south and west, the general direction to their original location. Final directions were primarily to the west and northwest, due in part to physiographic barriers. Eight adults homed, 1 twice, to capture areas that were < 64 km from release sites. Nonhoming wolves were radiotracked and/or recovered 32-351 km from their capture sites and 23-302 km from their release sites. Translocation was largely unsuccessful at keeping problem wolves out of lifestock production areas. The problem of initial travel away from release sites in wolf reintroductions probably could be minimized by transport and release of 6-9 mo. old wolves.

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.

NIP1;2 is a plasma membrane-localized transporter mediating aluminum uptake, translocation, and tolerance in Arabidopsis

PubMed Central

Wang, Yuqi; Li, Ruihong; Li, Demou; Jia, Xiaomin; Zhou, Dangwei; Li, Jianyong; Lyi, Sangbom M.; Hou, Siyu; Huang, Yulan

2017-01-01

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis. We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis. We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis. PMID:28439024

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.

Current concepts on Escherichia coli K1 translocation of the blood-brain barrier.

PubMed

Xie, Yi; Kim, Kee Jun; Kim, Kwang Sik

2004-11-01

The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively. With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E. coli requires a high-degree of bacteremia, E. coli binding to and invasion of HBMEC, and E. coli traversal of the BBB as live bacteria. Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E. coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo. We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E. coli translocation of the BBB is the result of specific pathogen-host cell interactions. Recent studies using functional genomics techniques have identified additional E. coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis. In this review, we summarize the current knowledge on the mechanisms underlying the successful E. coli translocation of the BBB.

Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation.

PubMed

Leng, Yan; Zhang, Jinyi; Badour, Karen; Arpaia, Enrico; Freeman, Spencer; Cheung, Pam; Siu, Michael; Siminovitch, Katherine

2005-01-25

WAVE2 is a member of the Wiskott-Aldrich syndrome protein family of cytoskeletal regulatory proteins shown to link Rac activation to actin remodeling via induction of Arp 2/3 activity. WAVE2 is thought to be regulated by its positioning in a macromolecular complex also containing the Abelson-(Abl) interactor-1 (Abi-1) adaptor, but the molecular basis and biologic relevance of WAVE2 inclusion in this complex are ill defined. Here we show that Abi-1 binding to WAVE2 is mediated by discrete motifs in the Abi-1 coiled-coil and WAVE2 WAVE-homology domains and increases markedly in conjunction with Abi-1-WAVE2 translocation and colocalization at the leading edge in B16F1 cells after fibronectin stimulation. Abi-1 also couples WAVE2 to Abl after cell stimulation, an interaction that triggers Abl membrane translocation with WAVE2, Abi-1, and activated Rac, as well as Abl-mediated tyrosine phosphorylation and WAVE2 activation. By contrast, mutation of tyrosine residue Y150, identified here as the major site of Abl-mediated WAVE2 tyrosine phosphorylation, as well as disruption of WAVE2-Abi-1 binding, impairs induction of WAVE2-driven actin polymerization and its membrane translocation in association with activated Rac. Similarly, WAVE2 tyrosine phosphorylation and induction of membrane actin rearrangement are abrogated in fibroblasts lacking the Abl family kinase. Together, these data reveal that Abi-1-mediated coupling of Abl to WAVE2 promotes Abl-evoked WAVE2 tyrosine phosphorylation required to link WAVE2 with activated Rac and with actin polymerization and remodeling at the cell periphery.

The Unexplored Mechanisms and Regulatory Functions of Ribosomal Translocation

NASA Astrophysics Data System (ADS)

Alejo, Jose Luis

In every cell, protein synthesis is carried out by the ribosome, a complex macromolecular RNA-protein assembly. Decades of structural and kinetic studies have increased our understanding of ribosome initiation, decoding, translocation and termination. Yet, the underlying mechanism of these fundamental processes has yet to be fully delineated. Hence, the molecular basis of regulation remains obscure. Here, single-molecule fluorescence methods are applied to decipher the mechanism and regulatory roles of the multi-step process of directional substrate translocation on the ribosome that accompanies every round of protein synthesis. In Chapter 1, single-molecule fluorescence resonance energy transfer (smFRET) is introduced as a tool for studying bacterial ribosome translocation. Chapter 2 details the experimental methods. In Chapter 3, the elongation factor G(EF-G)-catalyzed movement of substrates through the ribosome is examined from several perspectives or signals reporting on various degrees of freedom of ribosome dynamics. Two ribosomal states interconvert in the presence of EF-G(GDP), displaying novel head domain motions, until relocking takes place. In Chapter 4, in order to test if the mentioned fluctuations leading to relocking are correlated to the engagement of the P-site by the peptidyl-tRNA, the translocation of miscoded tRNAs is studied. Severe defects in the relocking stages of translocation reveal the correlation between this new stage of translocation and P-site tRNA engagement.

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.

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

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

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.

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

C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

PubMed Central

Ng, Chai Ann; Ke, Ying; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Vandenberg, Jamie I.

2013-01-01

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

Cancer translocations in human cells induced by zinc finger and TALE nucleases

PubMed Central

Piganeau, Marion; Ghezraoui, Hind; De Cian, Anne; Guittat, Lionel; Tomishima, Mark; Perrouault, Loic; René, Oliver; Katibah, George E.; Zhang, Lei; Holmes, Michael C.; Doyon, Yannick; Concordet, Jean-Paul; Giovannangeli, Carine; Jasin, Maria; Brunet, Erika

2013-01-01

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1–FLI1 and NPM1–ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell–derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases. PMID:23568838

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.

An integrated model of cardiac mitochondrial energy metabolism and calcium dynamics.

PubMed

Cortassa, Sonia; Aon, Miguel A; Marbán, Eduardo; Winslow, Raimond L; O'Rourke, Brian

2003-04-01

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an

An Integrated Model of Cardiac Mitochondrial Energy Metabolism and Calcium Dynamics

PubMed Central

Cortassa, Sonia; Aon, Miguel A.; Marbán, Eduardo; Winslow, Raimond L.; O'Rourke, Brian

2003-01-01

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca2+ handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH2, which in turn are used by the electron transport chain to establish a proton motive force (ΔμH), driving the F1F0-ATPase. In addition, mitochondrial matrix Ca2+, determined by Ca2+ uniporter and Na+/Ca2+ exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (ΔΨm), and matrix concentrations of Ca2+, NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca2+. The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca2+ dynamics, and respiratory control. Significant increases in oxygen consumption (VO2), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca2+, are obtained when the Ca2+-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca2+ plays an important role in matching energy supply with demand in

Uptake and translocation of imidacloprid, clothianidin and flupyradifurone in seed-treated soybeans.

PubMed

Stamm, Mitchell D; Heng-Moss, Tiffany M; Baxendale, Frederick P; Siegfried, Blair D; Blankenship, Erin E; Nauen, Ralf

2016-06-01

Seed treatment insecticides have become a popular management option for early-season insect control. This study investigated the total uptake and translocation of seed-applied [(14) C]imidacloprid, [(14) C]clothianidin and [(14) C]flupyradifurone into different plant parts in three soybean vegetative stages (VC, V1 and V2). The effects of soil moisture stress on insecticide uptake and translocation were also assessed among treatments. We hypothesized that (1) uptake and translocation would be different among the insecticides owing to differences in water solubility, and (2) moisture stress would increase insecticide uptake and translocation. Uptake and translocation did not follow a clear trend in the three vegetative stages. Initially, flupyradifurone uptake was greater than clothianidin uptake in VC soybeans. In V1 soybeans, differences in uptake among the three insecticides were not apparent and unaffected by soil moisture stress. Clothianidin was negatively affected by soil moisture stress in V2 soybeans, while imidacloprid and flupyradifurone were unaffected. Specifically, soil moisture stress had a positive effect on the distribution of flupyradifurone in leaves. This was not observed with the neonicotinoids. This study enhances our understanding of the uptake and distribution of insecticides used as seed treatments in soybean. The uptake and translocation of these insecticides differed in response to soil moisture stress. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

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.

P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1.

PubMed

Hinze, Annette Viktoria; Mayer, Peter; Harst, Anja; von Kügelgen, Ivar

2013-12-01

Adenine nucleotides acting at P2X1 receptors are potent vasoconstrictors. Recently, we demonstrated that activation of adenosine A2B receptors on human coronary smooth muscle cells inhibits cell proliferation by the induction of the nuclear receptor subfamily 4, group A, member 1 (NR4A1; alternative notation Nur77). In the present study, we searched for long-term effects mediated by P2X1 receptors by analyzing receptor-mediated changes in cell proliferation and in the expression of NR4A1. Cultured human coronary smooth muscle cells were treated with selective receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The P2X receptor agonist α,β-methylene-ATP and its analog β,γ-methylene-ATP inhibited cell proliferation by about 50 % after 5 days in culture with half-maximal concentrations of 0.3 and 0.08 μM, respectively. The effects were abolished or markedly attenuated by the P2X1 receptor antagonist NF449 (carbonylbis-imino-benzene-triylbis-(carbonylimino)tetrakis-benzene-1,3-disulfonic acid; 100 nM and 1 μM). α,β-methylene-ATP and β,γ-methylene-ATP applied for 30 min to 4 h increased the expression of NR4A1; NF449 blocked or attenuated this effect. Small interfering RNA directed against NR4A1 diminished the antiproliferative effects of α,β-methylene-ATP and β,γ-methylene-ATP. α,β-methylene-ATP (0.1 to 30 μM) decreased migration of cultured human coronary smooth muscle cells in a chamber measuring changes in impedance; NF449 blocked the effect. In conclusion, our results demonstrate for the first time that adenine nucleotides acting at P2X1 receptors inhibit the proliferation of human coronary smooth muscle cells via the induction of the early gene NR4A1.

Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome

PubMed Central

Schüle, Birgitt; Albalwi, Mohammed; Northrop, Emma; Francis, David I; Rowell, Margaret; Slater, Howard R; Gardner, RJ McKinlay; Francke, Uta

2005-01-01

Background Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15)(q27;q11.2). Methods We used metaphase FISH to narrow the breakpoint region and molecular analyses to map the breakpoints on both chromosomes at the nucleotide level. The expression of genes on chromosome 15 on both sides of the breakpoint was determined by RT-PCR analyses. Results Pertinent clinical features include neonatal hypotonia with feeding difficulties, hypogonadism, short stature, late-onset obesity, learning difficulties, abnormal social behavior and marked tolerance to pain, as well as sticky saliva and narcolepsy. Relative macrocephaly and facial features are not typical for PWS. The translocation breakpoints were identified within SNRPN intron 17 and intron 10 of a spliced non-coding transcript in band 4q27. LINE and SINE sequences at the exchange points may have contributed to the translocation event. By RT-PCR of lymphoblasts and fibroblasts, we find that upstream SNURF/SNRPN exons and snoRNAs HBII-437 and HBII-13 are expressed, but the downstream snoRNAs PWCR1/HBII-85 and HBII-438A/B snoRNAs are not. Conclusion As part of the PWCR1/HBII-85 snoRNA cluster is highly conserved between human and mice, while no copy of HBII-438 has been found in mouse, we conclude that PWCR1/HBII-85 snoRNAs is likely to play a major role in the PWS- phenotype. PMID:15877813

Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome.

PubMed

Schüle, Birgitt; Albalwi, Mohammed; Northrop, Emma; Francis, David I; Rowell, Margaret; Slater, Howard R; Gardner, R J McKinlay; Francke, Uta

2005-05-06

Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15)(q27;q11.2). We used metaphase FISH to narrow the breakpoint region and molecular analyses to map the breakpoints on both chromosomes at the nucleotide level. The expression of genes on chromosome 15 on both sides of the breakpoint was determined by RT-PCR analyses. Pertinent clinical features include neonatal hypotonia with feeding difficulties, hypogonadism, short stature, late-onset obesity, learning difficulties, abnormal social behavior and marked tolerance to pain, as well as sticky saliva and narcolepsy. Relative macrocephaly and facial features are not typical for PWS. The translocation breakpoints were identified within SNRPN intron 17 and intron 10 of a spliced non-coding transcript in band 4q27. LINE and SINE sequences at the exchange points may have contributed to the translocation event. By RT-PCR of lymphoblasts and fibroblasts, we find that upstream SNURF/SNRPN exons and snoRNAs HBII-437 and HBII-13 are expressed, but the downstream snoRNAs PWCR1/HBII-85 and HBII-438A/B snoRNAs are not. As part of the PWCR1/HBII-85 snoRNA cluster is highly conserved between human and mice, while no copy of HBII-438 has been found in mouse, we conclude that PWCR1/HBII-85 snoRNAs is likely to play a major role in the PWS- phenotype.

Human Lymphoid Translocation Fragile Zones Are Hypomethylated and Have Accessible Chromatin

PubMed Central

Lu, Zhengfei; Tsai, Albert G.; Pardo, Carolina E.; Müschen, Markus; Kladde, Michael P.

2015-01-01

Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions. PMID:25624348

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.

Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

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

Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engagesmore » a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.« less

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).

Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation

PubMed Central

Hsu, Chih‐Kai; Lin, Chih‐Chung; Hsiao, Li‐Der

2015-01-01

Background and Purpose Sphingosine 1‐phosphate (S1P), an important inflammatory mediator, has been shown to regulate COX‐2 production and promote various cellular responses such as cell migration. Mevastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CoA), effectively inhibits inflammatory responses. However, the mechanisms underlying S1P‐evoked COX‐2‐dependent cell migration, which is modulated by mevastatin in human tracheal smooth muscle cells (HTSMCs) remain unclear. Experimental Approach The expression of COX‐2 was determined by Western blotting, real time‐PCR and promoter analyses. The signalling molecules were investigated by pretreatment with respective pharmacological inhibitors or transfection with siRNAs. The interaction between COX‐2 promoter and transcription factors was determined by chromatin immunoprecipitation assay. Finally, the effect of mevastatin on HTSMC migration and leukocyte counts in BAL fluid and COX‐2 expression induced by S1P was determined by a cell migration assay, cell counting and Western blot. Key Results S1P stimulated mTOR activation through the Nox2/ROS and PI3K/Akt pathways, which can further stimulate FoxO1 phosphorylation and translocation to the cytosol. We also found that S1P induced CREB activation and translocation via an mTOR‐independent signalling pathway. Finally, we showed that pretreatment with mevastatin markedly reduced S1P‐induced cell migration and COX‐2/PGE2 production via a PPARγ‐dependent signalling pathway. Conclusions and Implications Mevastatin attenuates the S1P‐induced increased expression of COX‐2 and cell migration via the regulation of FoxO1 and CREB phosphorylation and translocation by PPARγ in HTSMCs. Mevastatin could be beneficial for prevention of airway inflammation in the future. PMID:26359950

Runx1t1 (Runt-Related Transcription Factor 1; Translocated to, 1) Epigenetically Regulates the Proliferation and Nitric Oxide Production of Microglia

PubMed Central

Baby, Nimmi; Li, Yali; Ling, Eng-Ang; Lu, Jia; Dheen, S. Thameem

2014-01-01

Background Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions. Methods and Findings siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2. Conclusion/Interpretation In conclusion

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)