Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

PubMed Central

Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

2008-01-01

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe

PubMed Central

De Groodt, A.; Whitehead, E. P.; Heslot, H.; Poirier, L.

1971-01-01

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine–xanthine–guanine phosphoribosyltransferase produced by this organism. PMID:5123876

Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

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

Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engendersmore » parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.« less

Metabolomics analysis: Finding out metabolic building blocks

PubMed Central

2017-01-01

In this paper we propose a new methodology for the analysis of metabolic networks. We use the notion of strongly connected components of a graph, called in this context metabolic building blocks. Every strongly connected component is contracted to a single node in such a way that the resulting graph is a directed acyclic graph, called a metabolic DAG, with a considerably reduced number of nodes. The property of being a directed acyclic graph brings out a background graph topology that reveals the connectivity of the metabolic network, as well as bridges, isolated nodes and cut nodes. Altogether, it becomes a key information for the discovery of functional metabolic relations. Our methodology has been applied to the glycolysis and the purine metabolic pathways for all organisms in the KEGG database, although it is general enough to work on any database. As expected, using the metabolic DAGs formalism, a considerable reduction on the size of the metabolic networks has been obtained, specially in the case of the purine pathway due to its relative larger size. As a proof of concept, from the information captured by a metabolic DAG and its corresponding metabolic building blocks, we obtain the core of the glycolysis pathway and the core of the purine metabolism pathway and detect some essential metabolic building blocks that reveal the key reactions in both pathways. Finally, the application of our methodology to the glycolysis pathway and the purine metabolism pathway reproduce the tree of life for the whole set of the organisms represented in the KEGG database which supports the utility of this research. PMID:28493998

THE PURINERGIC NEUROTRANSMITTER REVISITED: A SINGLE SUBSTANCE OR MULTIPLE PLAYERS?

PubMed Central

Mutafova-Yambolieva, Violeta N.; Durnin, Leonie

2014-01-01

The past half century has witnessed tremendous advances in our understanding of extracellular purinergic signaling pathways. Purinergic neurotransmission, in particular, has emerged as a key contributor in the efficient control mechanisms in the nervous system. The identity of the purine neurotransmitter, however, remains controversial. Identifying it is difficult because purines are present in all cell types, have a large variety of cell sources, and are released via numerous pathways. Moreover, studies on purinergic neurotransmission have relied heavily on indirect measurements of integrated postjunctional responses that do not provide direct information for neurotransmitter identity. This paper discusses experimental support for adenosine 5′-triphosphate (ATP) as a neurotransmitter and recent evidence for possible contribution of other purines, in addition to or instead of ATP, in chemical neurotransmission in the peripheral, enteric and central nervous systems. Sites of release and action of purines in model systems such as vas deferens, blood vessels, urinary bladder and chromaffin cells are discussed. This is preceded by a brief discussion of studies demonstrating storage of purines in synaptic vesicles. We examine recent evidence for cell type targets (e.g., smooth muscle cells, interstitial cells, neurons and glia) for purine neurotransmitters in different systems. This is followed by brief discussion of mechanisms of terminating the action of purine neurotransmitters, including extracellular nucleotide hydrolysis and possible salvage and reuptake in the cell. The significance of direct neurotransmitter release measurements is highlighted. Possibilities for involvement of multiple purines (e.g., ATP, ADP, NAD+, ADP-ribose, adenosine, and diadenosine polyphosphates) in neurotransmission are considered throughout. PMID:24887688

Parallel Prebiotic Origin of Canonical and Non-Canonical Purine Nucleosides

NASA Astrophysics Data System (ADS)

Becker, S.; Carell, T.

2017-07-01

RNA of all living organisms is highly modified. It is unclear if these non-canonical bases are ancestors of an early Earth or biological inventions. We investigated a prebiotic pathway that leads to canonical and non-canonical purine nucleosides.

Pediatric neurological syndromes and inborn errors of purine metabolism.

PubMed

Camici, Marcella; Micheli, Vanna; Ipata, Piero Luigi; Tozzi, Maria Grazia

2010-02-01

This review is devised to gather the presently known inborn errors of purine metabolism that manifest neurological pediatric syndromes. The aim is to draw a comprehensive picture of these rare diseases, characterized by unexpected and often devastating neurological symptoms. Although investigated for many years, most purine metabolism disorders associated to psychomotor dysfunctions still hide the molecular link between the metabolic derangement and the neurological manifestations. This basically indicates that many of the actual functions of nucleosides and nucleotides in the development and function of several organs, in particular central nervous system, are still unknown. Both superactivity and deficiency of phosphoribosylpyrophosphate synthetase cause hereditary disorders characterized, in most cases, by neurological impairments. The deficiency of adenylosuccinate lyase and 5-amino-4-imidazolecarboxamide ribotide transformylase/IMP cyclohydrolase, both belonging to the de novo purine synthesis pathway, is also associated to severe neurological manifestations. Among catabolic enzymes, hyperactivity of ectosolic 5'-nucleotidase, as well as deficiency of purine nucleoside phosphorylase and adenosine deaminase also lead to syndromes affecting the central nervous system. The most severe pathologies are associated to the deficiency of the salvage pathway enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase: the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to a clear impairment of mitochondrial functions. The assessment of hypo- or hyperuricemic conditions is suggestive of purine enzyme dysfunctions, but most disorders of purine metabolism may escape the clinical investigation because they are not associated to these metabolic derangements. This review may represent a starting point stimulating both scientists and physicians involved in the study of neurological dysfunctions caused by inborn errors of purine metabolism with the aim to find novel therapeutical approaches. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum.

PubMed

El Bissati, Kamal; Zufferey, Rachel; Witola, William H; Carter, Nicola S; Ullman, Buddy; Ben Mamoun, Choukri

2006-06-13

The human malaria parasite Plasmodium falciparum relies on the acquisition of host purines for its survival within human erythrocytes. Purine salvage by the parasite requires specialized transporters at the parasite plasma membrane (PPM), but the exact mechanism of purine entry into the infected erythrocyte, and the primary purine source used by the parasite, remain unknown. Here, we report that transgenic parasites lacking the PPM transporter PfNT1 (P. falciparum nucleoside transporter 1) are auxotrophic for hypoxanthine, inosine, and adenosine under physiological conditions and are viable only if these normally essential nutrients are provided at excess concentrations. Transport measurements across the PPM revealed a severe reduction in hypoxanthine uptake in the knockout, whereas adenosine and inosine transport were only partially affected. These data provide compelling evidence for a sequential pathway for exogenous purine conversion into hypoxanthine using host enzymes followed by PfNT1-mediated transport into the parasite. The phenotype of the conditionally lethal mutant establishes PfNT1 as a critical component of purine salvage in P. falciparum and validates PfNT1 as a potential therapeutic target.

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

PubMed Central

Worrell, V E; Nagle, D P

1990-01-01

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

Origin, Utilization, and Recycling of Nucleosides in the Central Nervous System

ERIC Educational Resources Information Center

Ipata, Piero Luigi

2011-01-01

The brain relies on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides, to maintain its nucleotide pool in the proper qualitative and quantitative balance. The transport of nucleosides from blood into neurons and glia is considered to be an essential prerequisite to enter their metabolic utilization in the…

Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

PubMed

Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

2016-10-01

Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

PubMed

Goswami, Moloy T; Chen, Guoan; Chakravarthi, Balabhadrapatruni V S K; Pathi, Satya S; Anand, Sharath K; Carskadon, Shannon L; Giordano, Thomas J; Chinnaiyan, Arul M; Thomas, Dafydd G; Palanisamy, Nallasivam; Beer, David G; Varambally, Sooryanarayana

2015-09-15

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway

PubMed Central

Torini, Juliana Roberta; Brandão-Neto, José; DeMarco, Ricardo; Pereira, Humberto D'Muniz

2016-01-01

Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity. PMID:27935959

Regulation of uric acid metabolism and excretion.

PubMed

Maiuolo, Jessica; Oppedisano, Francesca; Gratteri, Santo; Muscoli, Carolina; Mollace, Vincenzo

2016-06-15

Purines perform many important functions in the cell, being the formation of the monomeric precursors of nucleic acids DNA and RNA the most relevant one. Purines which also contribute to modulate energy metabolism and signal transduction, are structural components of some coenzymes and have been shown to play important roles in the physiology of platelets, muscles and neurotransmission. All cells require a balanced quantity of purines for growth, proliferation and survival. Under physiological conditions the enzymes involved in the purine metabolism maintain in the cell a balanced ratio between their synthesis and degradation. In humans the final compound of purines catabolism is uric acid. All other mammals possess the enzyme uricase that converts uric acid to allantoin that is easily eliminated through urine. Overproduction of uric acid, generated from the metabolism of purines, has been proven to play emerging roles in human disease. In fact the increase of serum uric acid is inversely associated with disease severity and especially with cardiovascular disease states. This review describes the enzymatic pathways involved in the degradation of purines, getting into their structure and biochemistry until the uric acid formation. Copyright © 2015. Published by Elsevier Ireland Ltd.

Alterations in metabolic pathways and networks in Alzheimer's disease

PubMed Central

Kaddurah-Daouk, R; Zhu, H; Sharma, S; Bogdanov, M; Rozen, S G; Matson, W; Oki, N O; Motsinger-Reif, A A; Churchill, E; Lei, Z; Appleby, D; Kling, M A; Trojanowski, J Q; Doraiswamy, P M; Arnold, S E

2013-01-01

The pathogenic mechanisms of Alzheimer's disease (AD) remain largely unknown and clinical trials have not demonstrated significant benefit. Biochemical characterization of AD and its prodromal phase may provide new diagnostic and therapeutic insights. We used targeted metabolomics platform to profile cerebrospinal fluid (CSF) from AD (n=40), mild cognitive impairment (MCI, n=36) and control (n=38) subjects; univariate and multivariate analyses to define between-group differences; and partial least square-discriminant analysis models to classify diagnostic groups using CSF metabolomic profiles. A partial correlation network was built to link metabolic markers, protein markers and disease severity. AD subjects had elevated methionine (MET), 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid, xanthosine and glutathione versus controls. MCI subjects had elevated 5-HIAA, MET, hypoxanthine and other metabolites versus controls. Metabolite ratios revealed changes within tryptophan, MET and purine pathways. Initial pathway analyses identified steps in several pathways that appear altered in AD and MCI. A partial correlation network showed total tau most directly related to norepinephrine and purine pathways; amyloid-β (Ab42) was related directly to an unidentified metabolite and indirectly to 5-HIAA and MET. These findings indicate that MCI and AD are associated with an overlapping pattern of perturbations in tryptophan, tyrosine, MET and purine pathways, and suggest that profound biochemical alterations are linked to abnormal Ab42 and tau metabolism. Metabolomics provides powerful tools to map interlinked biochemical pathway perturbations and study AD as a disease of network failure. PMID:23571809

Alterations in metabolic pathways and networks in Alzheimer's disease.

PubMed

Kaddurah-Daouk, R; Zhu, H; Sharma, S; Bogdanov, M; Rozen, S G; Matson, W; Oki, N O; Motsinger-Reif, A A; Churchill, E; Lei, Z; Appleby, D; Kling, M A; Trojanowski, J Q; Doraiswamy, P M; Arnold, S E

2013-04-09

The pathogenic mechanisms of Alzheimer's disease (AD) remain largely unknown and clinical trials have not demonstrated significant benefit. Biochemical characterization of AD and its prodromal phase may provide new diagnostic and therapeutic insights. We used targeted metabolomics platform to profile cerebrospinal fluid (CSF) from AD (n=40), mild cognitive impairment (MCI, n=36) and control (n=38) subjects; univariate and multivariate analyses to define between-group differences; and partial least square-discriminant analysis models to classify diagnostic groups using CSF metabolomic profiles. A partial correlation network was built to link metabolic markers, protein markers and disease severity. AD subjects had elevated methionine (MET), 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid, xanthosine and glutathione versus controls. MCI subjects had elevated 5-HIAA, MET, hypoxanthine and other metabolites versus controls. Metabolite ratios revealed changes within tryptophan, MET and purine pathways. Initial pathway analyses identified steps in several pathways that appear altered in AD and MCI. A partial correlation network showed total tau most directly related to norepinephrine and purine pathways; amyloid-β (Ab42) was related directly to an unidentified metabolite and indirectly to 5-HIAA and MET. These findings indicate that MCI and AD are associated with an overlapping pattern of perturbations in tryptophan, tyrosine, MET and purine pathways, and suggest that profound biochemical alterations are linked to abnormal Ab42 and tau metabolism. Metabolomics provides powerful tools to map interlinked biochemical pathway perturbations and study AD as a disease of network failure.

Structural Determinants of the 5′-Methylthioinosine Specificity of Plasmodium Purine Nucleoside Phosphorylase

PubMed Central

Donaldson, Teraya M.; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C.; Kim, Kami

2014-01-01

Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5′-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5′-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5′-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5′-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

Structural characterization of purine nucleoside phosphorylase from human pathogen Helicobacter pylori.

PubMed

Štefanić, Zoran; Mikleušević, Goran; Luić, Marija; Bzowska, Agnieszka; Leščić Ašler, Ivana

2017-08-01

Microaerophilic bacterium Helicobacer pylori is a well known human pathogen involved in the development of many diseases. Due to the evergrowing infection rate and increase of H. pylori antibiotic resistence, it is of utmost importance to find a new way to attack and eradicate H. pylori. The purine metabolism in H. pylori is solely dependant on the salvage pathway and one of the key enzymes in this pathway is purine nucleoside phosphorylase (PNP). In this timely context, we report here the basic biochemical and structural characterization of recombinant PNP from the H. pylori clinical isolate expressed in Escherichia coli. Structure of H. pylori PNP is typical for high molecular mass PNPs. However, its activity towards adenosine is very low, thus resembling more that of low molecular mass PNPs. Understanding the molecular mechanism of this key enzyme may lead to the development of new drug strategies and help in the eradication of H. pylori. Copyright © 2017 Elsevier B.V. All rights reserved.

Current Therapy and New Directions in the Treatment of Hairy Cell Leukemia: A Review.

PubMed

Sarvaria, Aditya; Topp, Zheng; Saven, Alan

2016-01-01

Hairy cell leukemia (HCL) is a chronic B-cell leukemia noted for an indolent course that ultimately results in cytopenias and massive splenomegaly. Whereas treatment with the nucleoside purine analogues cladribine and pentostatin results in lengthy remissions in nearly all patients with HCL, most patients will experience relapse while a small percentage of patients' disease fails to respond to therapy in the first place. Retreatment with a purine nucleoside analogue often leads to an effective but limited response. For decades, few other viable therapeutic options were available to these patients who required retreatment. Recently, new insights into the mechanism of disease of HCL have led to research in new potential treatment agents, either alone or with a purine nucleoside analogue. Clinical trials with rituximab, bendamustine, and conjugate immunotoxins will reveal what role these therapies will have in HCL treatment. A better understanding of the BRAF/MEK/ERK pathway and the B-cell signaling pathway has allowed further exploration into the novel drugs vemurafenib, dabrafenib, trametinib, and ibrutinib.

A Canonical Correlation Analysis of AIDS Restriction Genes and Metabolic Pathways Identifies Purine Metabolism as a Key Cooperator.

PubMed

Ye, Hanhui; Yuan, Jinjin; Wang, Zhengwu; Huang, Aiqiong; Liu, Xiaolong; Han, Xiao; Chen, Yahong

2016-01-01

Human immunodeficiency virus causes a severe disease in humans, referred to as immune deficiency syndrome. Studies on the interaction between host genetic factors and the virus have revealed dozens of genes that impact diverse processes in the AIDS disease. To resolve more genetic factors related to AIDS, a canonical correlation analysis was used to determine the correlation between AIDS restriction and metabolic pathway gene expression. The results show that HIV-1 postentry cellular viral cofactors from AIDS restriction genes are coexpressed in human transcriptome microarray datasets. Further, the purine metabolism pathway comprises novel host factors that are coexpressed with AIDS restriction genes. Using a canonical correlation analysis for expression is a reliable approach to exploring the mechanism underlying AIDS.

Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

PubMed

Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

2009-01-02

NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.

Purines: forgotten mediators in traumatic brain injury.

PubMed

Jackson, Edwin K; Boison, Detlev; Schwarzschild, Michael A; Kochanek, Patrick M

2016-04-01

Recently, the topic of traumatic brain injury has gained attention in both the scientific community and lay press. Similarly, there have been exciting developments on multiple fronts in the area of neurochemistry specifically related to purine biology that are relevant to both neuroprotection and neurodegeneration. At the 2105 meeting of the National Neurotrauma Society, a session sponsored by the International Society for Neurochemistry featured three experts in the field of purine biology who discussed new developments that are germane to both the pathomechanisms of secondary injury and development of therapies for traumatic brain injury. This included presentations by Drs. Edwin Jackson on the novel 2',3'-cAMP pathway in neuroprotection, Detlev Boison on adenosine in post-traumatic seizures and epilepsy, and Michael Schwarzschild on the potential of urate to treat central nervous system injury. This mini review summarizes the important findings in these three areas and outlines future directions for the development of new purine-related therapies for traumatic brain injury and other forms of central nervous system injury. In this review, novel therapies based on three emerging areas of adenosine-related pathobiology in traumatic brain injury (TBI) were proposed, namely, therapies targeting 1) the 2',3'-cyclic adenosine monophosphate (cAMP) pathway, 2) adenosine deficiency after TBI, and 3) augmentation of urate after TBI. © 2016 International Society for Neurochemistry.

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica.

PubMed

Nazario, G. M.; Lovatt, C. J.

1993-12-01

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.

Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.

PubMed

Kyoung, Minjoung; Russell, Sarah J; Kohnhorst, Casey L; Esemoto, Nopondo N; An, Songon

2015-01-27

Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly.

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells

PubMed Central

Perez Bay, Andres E.; Schreiner, Ryan; Benedicto, Ignacio; Paz Marzolo, Maria; Banfelder, Jason; Weinstein, Alan M.; Rodriguez-Boulan, Enrique J.

2016-01-01

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station. PMID:27180806

Folate restriction and methylenetetrahydrofolate reductase 677T polymorphism decreases adoMet synthesis via folate-dependent remethylation in human-transformed lymphoblasts.

PubMed

Chiang, E-P; Wang, Y-C; Tang, F-Y

2007-04-01

The homozygous mutation (677TT) in the methylenetetrahydrofolate reductase (MTHFR) gene reduces enzyme activity and alters cellular folate composition. Previous epidemiological studies reported a potential protective effect of MTHFR677C --> T against acute lymphocytic leukemia and malignant lymphoma, but the mechanism remains to be determined. We investigated the biochemical impacts of MTHFR677C --> T on cellular S-adenosyl methionine (adoMet) synthesis, global DNA methylation, and de novo purine synthesis, all of which are potential regulatory pathways involved in tumorigenesis. Metabolic fluxes of homocysteine remethylation and de novo purine synthesis were compared between Epstein-Barr virus-transformed lymphoblasts expressing MTHFR 677C and MTHFR 677T using stable isotopic tracers and GCMS. MTHFR TT genotype significantly reduced folate-dependent remethylation under folate restriction, reflecting limited methylated folates under folate restriction. Data also suggested increased formylated folate pool and increased purine synthesis when folate is adequate. The impacts of MTHFR 677T polymorphism appeared closely related to folate status, and such alterations may modulate metabolic pathways involved in cancer onset/progression. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in hematological malignancies. These transformed cells are potential models for studying the consequences of human genetic variation and cancer pathogenesis.

Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

PubMed

López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

2014-05-01

Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris.

PubMed

Coleto, Inmaculada; Trenas, Almudena T; Erban, Alexander; Kopka, Joachim; Pineda, Manuel; Alamillo, Josefa M

2016-08-01

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full-length sequences, which are intron-less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi-silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules. © 2016 John Wiley & Sons Ltd.

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

PubMed Central

Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

2016-01-01

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages. PMID:27768715

Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

PubMed

Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

2016-09-01

Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.

Synaptic activity regulates AMPA receptor trafficking through different recycling pathways

PubMed Central

Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

2015-01-01

Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. DOI: http://dx.doi.org/10.7554/eLife.06878.001 PMID:25970033

[Purine and pyrimidine nucleoside phosphorylases - remarkable enzymes still not fully understood].

PubMed

Bzowska, Agnieszka

2015-01-01

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

Molecular dynamics studies of a hexameric purine nucleoside phosphorylase.

PubMed

Zanchi, Fernando Berton; Caceres, Rafael Andrade; Stabeli, Rodrigo Guerino; de Azevedo, Walter Filgueira

2010-03-01

Purine nucleoside phosphorylase (PNP) (EC.2.4.2.1) is an enzyme that catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is involved in purine-salvage pathway and has been proposed as a promising target for design and development of antimalarial and antibacterial drugs. Recent elucidation of the three-dimensional structure of PNP by X-ray protein crystallography left open the possibility of structure-based virtual screening initiatives in combination with molecular dynamics simulations focused on identification of potential new antimalarial drugs. Most of the previously published molecular dynamics simulations of PNP were carried out on human PNP, a trimeric PNP. The present article describes for the first time molecular dynamics simulations of hexameric PNP from Plasmodium falciparum (PfPNP). Two systems were simulated in the present work, PfPNP in ligand free form, and in complex with immucillin and sulfate. Based on the dynamical behavior of both systems the main results related to structural stability and protein-drug interactions are discussed.

Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

PubMed

Pérez-Torrado, Roberto; Llopis, Silvia; Perrone, Benedetta; Gómez-Pastor, Rocío; Hube, Bernhard; Querol, Amparo

2015-01-01

In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

The Recycling Endosome of Madin-Darby Canine Kidney Cells Is a Mildly Acidic Compartment Rich in Raft Components

PubMed Central

Gagescu, Raluca; Demaurex, Nicolas; Parton, Robert G.; Hunziker, Walter; Huber, Lukas A.; Gruenberg, Jean

2000-01-01

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway. PMID:10930469

Gene set analysis of purine and pyrimidine antimetabolites cancer therapies.

PubMed

Fridley, Brooke L; Batzler, Anthony; Li, Liang; Li, Fang; Matimba, Alice; Jenkins, Gregory D; Ji, Yuan; Wang, Liewei; Weinshilboum, Richard M

2011-11-01

Responses to therapies, either with regard to toxicities or efficacy, are expected to involve complex relationships of gene products within the same molecular pathway or functional gene set. Therefore, pathways or gene sets, as opposed to single genes, may better reflect the true underlying biology and may be more appropriate units for analysis of pharmacogenomic studies. Application of such methods to pharmacogenomic studies may enable the detection of more subtle effects of multiple genes in the same pathway that may be missed by assessing each gene individually. A gene set analysis of 3821 gene sets is presented assessing the association between basal messenger RNA expression and drug cytotoxicity using ethnically defined human lymphoblastoid cell lines for two classes of drugs: pyrimidines [gemcitabine (dFdC) and arabinoside] and purines [6-thioguanine and 6-mercaptopurine]. The gene set nucleoside-diphosphatase activity was found to be significantly associated with both dFdC and arabinoside, whereas gene set γ-aminobutyric acid catabolic process was associated with dFdC and 6-thioguanine. These gene sets were significantly associated with the phenotype even after adjusting for multiple testing. In addition, five associated gene sets were found in common between the pyrimidines and two gene sets for the purines (3',5'-cyclic-AMP phosphodiesterase activity and γ-aminobutyric acid catabolic process) with a P value of less than 0.0001. Functional validation was attempted with four genes each in gene sets for thiopurine and pyrimidine antimetabolites. All four genes selected from the pyrimidine gene sets (PSME3, CANT1, ENTPD6, ADRM1) were validated, but only one (PDE4D) was validated for the thiopurine gene sets. In summary, results from the gene set analysis of pyrimidine and purine therapies, used often in the treatment of various cancers, provide novel insight into the relationship between genomic variation and drug response.

Pharmacometabolomic Signature of Ataxia SCA1 Mouse Model and Lithium Effects

PubMed Central

Wikoff, William R.; Gatchel, Jennifer R.; Wang, Lu; Barupal, Dinesh K.; Crespo-Barreto, Juan; Fiehn, Oliver

2013-01-01

We have shown that lithium treatment improves motor coordination in a spinocerebellar ataxia type 1 (SCA1) disease mouse model (Sca1154Q/+). To learn more about disease pathogenesis and molecular contributions to the neuroprotective effects of lithium, we investigated metabolomic profiles of cerebellar tissue and plasma from SCA1-model treated and untreated mice. Metabolomic analyses of wild-type and Sca1154Q/+ mice, with and without lithium treatment, were performed using gas chromatography time-of-flight mass spectrometry and BinBase mass spectral annotations. We detected 416 metabolites, of which 130 were identified. We observed specific metabolic perturbations in Sca1154Q/+ mice and major effects of lithium on metabolism, centrally and peripherally. Compared to wild-type, Sca1154Q/+ cerebella metabolic profile revealed changes in glucose, lipids, and metabolites of the tricarboxylic acid cycle and purines. Fewer metabolic differences were noted in Sca1154Q/+ mouse plasma versus wild-type. In both genotypes, the major lithium responses in cerebellum involved energy metabolism, purines, unsaturated free fatty acids, and aromatic and sulphur-containing amino acids. The largest metabolic difference with lithium was a 10-fold increase in ascorbate levels in wild-type cerebella (p<0.002), with lower threonate levels, a major ascorbate catabolite. In contrast, Sca1154Q/+ mice that received lithium showed no elevated cerebellar ascorbate levels. Our data emphasize that lithium regulates a variety of metabolic pathways, including purine, oxidative stress and energy production pathways. The purine metabolite level, reduced in the Sca1154Q/+ mice and restored upon lithium treatment, might relate to lithium neuroprotective properties. PMID:23936457

Consortium analysis of gene and gene-folate interactions in purine and pyrimidine metabolism pathways with ovarian carcinoma risk

PubMed Central

Kelemen, Linda E.; Terry, Kathryn L.; Goodman, Marc T.; Webb, Penelope M.; Bandera, Elisa V.; McGuire, Valerie; Rossing, Mary Anne; Wang, Qinggang; Dicks, Ed; Tyrer, Jonathan P.; Song, Honglin; Kupryjanczyk, Jolanta; Dansonka-Mieszkowska, Agnieszka; Plisiecka-Halasa, Joanna; Timorek, Agnieszka; Menon, Usha; Gentry-Maharaj, Aleksandra; Gayther, Simon A.; Ramus, Susan J.; Narod, Steven A.; Risch, Harvey A.; McLaughlin, John R.; Siddiqui, Nadeem; Glasspool, Rosalind; Paul, James; Carty, Karen; Gronwald, Jacek; Lubiński, Jan; Jakubowska, Anna; Cybulski, Cezary; Kiemeney, Lambertus A.; Massuger, Leon F. A. G.; van Altena, Anne M.; Aben, Katja K. H.; Olson, Sara H.; Orlow, Irene; Cramer, Daniel W.; Levine, Douglas A.; Bisogna, Maria; Giles, Graham G.; Southey, Melissa C.; Bruinsma, Fiona; Kjær, Susanne Krüger; Høgdall, Estrid; Jensen, Allan; Høgdall, Claus K.; Lundvall, Lene; Engelholm, Svend-Aage; Heitz, Florian; du Bois, Andreas; Harter, Philipp; Schwaab, Ira; Butzow, Ralf; Nevanlinna, Heli; Pelttari, Liisa M.; Leminen, Arto; Thompson, Pamela J.; Lurie, Galina; Wilkens, Lynne R.; Lambrechts, Diether; Van Nieuwenhuysen, Els; Lambrechts, Sandrina; Vergote, Ignace; Beesley, Jonathan; Fasching, Peter A.; Beckmann, Matthias W.; Hein, Alexander; Ekici, Arif B.; Doherty, Jennifer A.; Wu, Anna H.; Pearce, Celeste L.; Pike, Malcolm C.; Stram, Daniel; Chang-Claude, Jenny; Rudolph, Anja; Dörk, Thilo; Dürst, Matthias; Hillemanns, Peter; Runnebaum, Ingo B.; Bogdanova, Natalia; Antonenkova, Natalia; Odunsi, Kunle; Edwards, Robert P.; Kelley, Joseph L.; Modugno, Francesmary; Ness, Roberta B.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; Orsulic, Sandra; Fridley, Brooke L.; Vierkant, Robert A.; Cunningham, Julie M.; Wu, Xifeng; Lu, Karen; Liang, Dong; Hildebrandt, Michelle A.T.; Weber, Rachel Palmieri; Iversen, Edwin S.; Tworoger, Shelley S.; Poole, Elizabeth M.; Salvesen, Helga B.; Krakstad, Camilla; Bjorge, Line; Tangen, Ingvild L.; Pejovic, Tanja; Bean, Yukie; Kellar, Melissa; Wentzensen, Nicolas; Brinton, Louise A.; Lissowska, Jolanta; Garcia-Closas, Montserrat; Campbell, Ian G.; Eccles, Diana; Whittemore, Alice S.; Sieh, Weiva; Rothstein, Joseph H.; Anton-Culver, Hoda; Ziogas, Argyrios; Phelan, Catherine M.; Moysich, Kirsten B.; Goode, Ellen L.; Schildkraut, Joellen M.; Berchuck, Andrew; Pharoah, Paul D.P.; Sellers, Thomas A.; Brooks-Wilson, Angela; Cook, Linda S.; Le, Nhu D.

2014-01-01

Scope We re-evaluated previously reported associations between variants in pathways of one-carbon (folate) transfer genes and ovarian carcinoma (OC) risk, and in related pathways of purine and pyrimidine metabolism, and assessed interactions with folate intake. Methods and Results Odds ratios (OR) for 446 genetic variants were estimated among 13,410 OC cases and 22,635 controls and among 2,281 cases and 3,444 controls with folate information. Following multiple testing correction, the most significant main effect associations were for DPYD variants rs11587873 (OR=0.92, P=6x10−5) and rs828054 (OR=1.06, P=1x10−4). Thirteen variants in the pyrimidine metabolism genes, DPYD, DPYS, PPAT and TYMS, also interacted significantly with folate in a multi-variant analysis (corrected P=9.9x10−6) but collectively explained only 0.2% of OC risk. Although no other associations were significant after multiple testing correction, variants in SHMT1 in one-carbon transfer, previously reported with OC, suggested lower risk at higher folate (Pinteraction=0.03-0.006). Conclusions Variation in pyrimidine metabolism genes, particularly DPYD, which was previously reported to be associated with OC, may influence risk; however, stratification by folate intake is unlikely to modify disease risk appreciably in these women. SHMT1 SNP-byfolate interactions are plausible but require further validation. Polymorphisms in selected genes in purine metabolism were not associated with OC. PMID:25066213

A novel approach to select differential pathways associated with hypertrophic cardiomyopathy based on gene co‑expression analysis.

PubMed

Chen, Xiao-Min; Feng, Ming-Jun; Shen, Cai-Jie; He, Bin; Du, Xian-Feng; Yu, Yi-Bo; Liu, Jing; Chu, Hui-Min

2017-07-01

The present study was designed to develop a novel method for identifying significant pathways associated with human hypertrophic cardiomyopathy (HCM), based on gene co‑expression analysis. The microarray dataset associated with HCM (E‑GEOD‑36961) was obtained from the European Molecular Biology Laboratory‑European Bioinformatics Institute database. Informative pathways were selected based on the Reactome pathway database and screening treatments. An empirical Bayes method was utilized to construct co‑expression networks for informative pathways, and a weight value was assigned to each pathway. Differential pathways were extracted based on weight threshold, which was calculated using a random model. In order to assess whether the co‑expression method was feasible, it was compared with traditional pathway enrichment analysis of differentially expressed genes, which were identified using the significance analysis of microarrays package. A total of 1,074 informative pathways were screened out for subsequent investigations and their weight values were also obtained. According to the threshold of weight value of 0.01057, 447 differential pathways, including folding of actin by chaperonin containing T‑complex protein 1 (CCT)/T‑complex protein 1 ring complex (TRiC), purine ribonucleoside monophosphate biosynthesis and ubiquinol biosynthesis, were obtained. Compared with traditional pathway enrichment analysis, the number of pathways obtained from the co‑expression approach was increased. The results of the present study demonstrated that this method may be useful to predict marker pathways for HCM. The pathways of folding of actin by CCT/TRiC and purine ribonucleoside monophosphate biosynthesis may provide evidence of the underlying molecular mechanisms of HCM, and offer novel therapeutic directions for HCM.

[Scrining workers employed at the weapons and military technology recycling and disposal facilitie].

PubMed

2012-01-01

A screening study was performed to identify factors of the raised risk of cardiovascular and bronchopulmonary diseases among workers involved in weapons and military technology recycling and disposal. A high prevalence of risk factors such as smoking, excess body mass, adiposis, disorders of the carbohydrate, lipid and purine metabolism was found for workers aged 35 years and older. Circulatory, musculoskeletal, and eye diseases prevailed in the structure of first diagnosed diseases. Taking into account an unfavorable profile of risk factors, groups with the raised risk of cardiovascular and bronchopulmonary diseases were identified. An expansion of the standard scope of examination in the framework of regular medical monitoring in compliance with the applicable regulatory documents by inclusion of screening studies would provide additional opportunities for the early diagnosing and prevention of the above diseases.

Incorporation of Exogenous Purines and Pyrimidines by Methanococcus voltae and Isolation of Analog-Resistant Mutants

PubMed Central

Bowen, Timothy L.; Whitman, William B.

1987-01-01

Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 μg/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments. PMID:16347408

HCN - A plausible source of purines, pyrimidines and amino acids on the primitive earth

NASA Technical Reports Server (NTRS)

Ferris, J.-P.; Joshi, P. C.; Edelson, E. H.; Lawless, J. G.

1978-01-01

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 9.2, and hydrolysis of these oligomers yields 4,5-dihydroxypyrimidine, orotic acid, 5-hydroxyuracil, adenine, 4-aminoimidazole-5-carboxamide, and amino acids. It is suggested that the three main classes of nitrogen-containing biomolecules - purines, pyrimidines, and amino acids may have originated from HCN on the primitive earth. It is also suggested that the presence of orotic acid and 4-aminoimidazole-5-carboxamide might indicate that contemporary biosynthetic pathways for nucleotides evolved from the compounds released on hydrolysis of HCN oligomers.

Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors.

PubMed

Debaigt, Colin; Hirling, Harald; Steiner, Pascal; Vincent, Jean-Pierre; Mazella, Jean

2004-08-20

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.

Nicotinamide Riboside and Nicotinic Acid Riboside Salvage in Fungi and Mammals

PubMed Central

Belenky, Peter; Christensen, Kathryn C.; Gazzaniga, Francesca; Pletnev, Alexandre A.; Brenner, Charles

2009-01-01

NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

Facilitating Understanding of the Purine Nucleotide Cycle and the One-Carbon Pool: Part II--Metabolism of the One-Carbon Pool

ERIC Educational Resources Information Center

Arinze, Ifeanyi J.

2005-01-01

Some metabolic processes such as glycolysis, gluconeogenesis, and lipogenesis are readily understood because they are circumscribed in metabolic pathways that have clearly identifiable beginning points, end products, and other features. Other metabolic pathways that do not appear to be straightforward pose difficulties for students. In part I of…

The Purine-Utilizing Bacterium Clostridium acidurici 9a: A Genome-Guided Metabolic Reconsideration

PubMed Central

Hartwich, Katrin; Poehlein, Anja; Daniel, Rolf

2012-01-01

Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp) and one small circular plasmid (2,913 bp). The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed. PMID:23240052

The purine-utilizing bacterium Clostridium acidurici 9a: a genome-guided metabolic reconsideration.

PubMed

Hartwich, Katrin; Poehlein, Anja; Daniel, Rolf

2012-01-01

Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp) and one small circular plasmid (2,913 bp). The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed.

Adenosine metabolism in Toxoplasma gondii: potential targets for chemotherapy.

PubMed

el Kouni, Mahmoud H

2007-01-01

Toxoplasma gondii is an intracellular parasitic protozoan that infects approximately a billion people worldwide. Infection with T. gondii represents a major health problem for immunocompromised individuals, such as AIDS patients, organ transplant recipients, and the unborn children of infected mothers. Currently available drugs usually do not eradicate infection and as many as 50% of the patients do not respond to this therapy. Furthermore, they are ineffective against T. gondii tissue cysts. In addition, prolonged exposure to these drugs induces serious host toxicity forcing the discontinuation of the therapy. Finally, there is no effective vaccine currently available for the treatment of toxoplasmosis. Therefore, it is necessary to develop new and effective drugs for the treatment and management of toxoplasmosis. The rational design of a drug depends on the exploitation of fundamental biochemical or physiological differences between pathogens and their host. Some of the most striking differences between T. gondii and their mammalian host are found in purine metabolism. T. gondii, like most parasites studied, lack the ability to synthesize purines do novo and depend on the salvage of purines from their host to satisfy their requirements of purines. In this respect, the salvage of adenosine is the major source of purines in T. gondii. Therefore, interference with adenosine uptake and metabolism in T. gondii can be selectively detrimental to the parasite. The host cells, on the other hand, can still obtain their purine requirements by their de novo pathways. This review will focus on the broad aspects of the adenosine transport and the enzyme adenosine kinase (EC 2.7.1.20) which are the two primary routes for adenosine utilization in T. gondii, in an attempt to illustrate their potentials as targets for chemotherapy against this parasite.

Guanine- Formation During the Thermal Polymerization of Amino Acids

NASA Technical Reports Server (NTRS)

Mc Caw, B. K.; Munoz, E. F.; Ponnamperuma, C.; Young, R. S.

1964-01-01

The action of heat on a mixture of amino acids was studied as a possible abiological pathway for the synthesis of purines and pyrimidines. Guanine was detected. This result is significant in the context of chemical evolution.

Src regulates sequence-dependent beta-2 adrenergic receptor recycling via cortactin phosphorylation*

PubMed Central

Vistein, Rachel; Puthenveedu, Manojkumar A.

2014-01-01

The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains, and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin-stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways. PMID:25077552

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

PubMed Central

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases

PubMed Central

2017-01-01

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface. PMID:28768685

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication

PubMed Central

Wang, Yijin; Wang, Wenshi; Xu, Lei; Zhou, Xinying; Shokrollahi, Ehsan; Felczak, Krzysztof; van der Laan, Luc J. W.; Pankiewicz, Krzysztof W.; Sprengers, Dave; Raat, Nicolaas J. H.; Metselaar, Herold J.; Peppelenbosch, Maikel P.

2016-01-01

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication. PMID:26926637

Are scarce metals in cars functionally recycled?

PubMed

Andersson, Magnus; Ljunggren Söderman, Maria; Sandén, Björn A

2017-02-01

Improved recycling of end-of-life vehicles (ELVs) may serve as an important strategy to address resource security risks related to increased global demand for scarce metals. However, in-depth knowledge of the magnitude and fate of such metals entering ELV recycling is lacking. This paper quantifies input of 25 scarce metals to Swedish ELV recycling, and estimates the extent to which they are recycled to material streams where their metal properties are utilised, i.e. are functionally recycled. Methodologically, scarce metals are mapped to main types of applications within newly produced Swedish car models and subsequently, material flow analysis of ELV waste streams is used as basis for identifying pathways of these applications and assessing whether contained metals are functionally recycled. Results indicate that, of the scarce metals, only platinum may be functionally recycled in its main application. Cobalt, gold, manganese, molybdenum, palladium, rhodium and silver may be functionally recycled depending on application and pathways taken. For remaining 17 metals, functional recycling is absent. Consequently, despite high overall ELV recycling rates of materials in general, there is considerable risk of losing ELV scarce metals to carrier metals, construction materials, backfilling materials and landfills. Given differences in the application of metals and identified pathways, prospects for increasing functional recycling are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission

PubMed Central

Sabeva, Nadezhda; Cho, Richard W.; Vasin, Alexander; Gonzalez, Agustin; Littleton, J. Troy

2017-01-01

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1–43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1–43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1–43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion. SIGNIFICANCE STATEMENT Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process but can partially intermix during stimulation. PMID:28077717

Serial Metabolome Changes in a Prospective Cohort of Subjects with Influenza Viral Infection and Comparison with Dengue Fever.

PubMed

Cui, Liang; Fang, Jinling; Ooi, Eng Eong; Lee, Yie Hou

2017-07-07

Influenza virus infection (IVI) and dengue virus infection (DVI) are major public health threats. Between IVI and DVI, clinical symptoms can be overlapping yet infection-specific, but host metabolome changes are not well-described. Untargeted metabolomics and targeted oxylipinomic analyses were performed on sera serially collected at three phases of infection from a prospective cohort study of adult subjects with either H3N2 influenza infection or dengue fever. Untargeted metabolomics identified 26 differential metabolites, and major perturbed pathways included purine metabolism, fatty acid biosynthesis and β-oxidation, tryptophan metabolism, phospholipid catabolism, and steroid hormone pathway. Alterations in eight oxylipins were associated with the early symptomatic phase of H3N2 flu infection, were mostly arachidonic acid-derived, and were enriched in the lipoxygenase pathway. There was significant overlap in metabolome profiles in both infections. However, differences specific to IVI and DVI were observed. DVI specifically attenuated metabolites including serotonin, bile acids and biliverdin. Additionally, metabolome changes were more persistent in IVI in which metabolites such as hypoxanthine, inosine, and xanthine of the purine metabolism pathway remained significantly elevated at 21-27 days after fever onset. This study revealed the dynamic metabolome changes in IVI subjects and provided biochemical insights on host physiological similarities and differences between IVI and DVI.

Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism.

PubMed

Gevi, Federica; Zolla, Lello; Gabriele, Stefano; Persico, Antonio M

2016-01-01

Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of "control" status. ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2-7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway. Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B 6 , riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate. The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Metabolic Mitigation of Staphylococcus aureus Vancomycin Intermediate-Level Susceptibility.

PubMed

Gardner, Stewart G; Marshall, Darrell D; Daum, Robert S; Powers, Robert; Somerville, Greg A

2018-01-01

Staphylococcus aureus is a major human pathogen whose infections are increasingly difficult to treat due to increased antibiotic resistance, including resistance to vancomycin. Vancomycin-intermediate S. aureus (VISA) strains develop resistance to vancomycin through adaptive changes that are incompletely understood. Central to this adaptation are metabolic changes that permit growth in the presence of vancomycin. To define the metabolic changes associated with adaptive resistance to vancomycin in S. aureus , the metabolomes of a vancomycin-sensitive and VISA strain pair isolated from the same patient shortly after vancomycin therapy began and following vancomycin treatment failure were analyzed. The metabolic adaptations included increases in acetogenesis, carbon flow through the pentose phosphate pathway, wall teichoic acid and peptidoglycan precursor biosynthesis, purine biosynthesis, and decreased tricarboxylic acid (TCA) cycle activity. The significance of these metabolic pathways for vancomycin-intermediate susceptibility was determined by assessing the synergistic potential of human-use-approved inhibitors of these pathways in combination with vancomycin against VISA strains. Importantly, inhibitors of amino sugar and purine biosynthesis acted synergistically with vancomycin to kill a diverse set of VISA strains, suggesting that combinatorial therapy could augment the efficacy of vancomycin even in patients infected with VISA strains. Copyright © 2017 American Society for Microbiology.

The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase*

PubMed Central

Franzolin, Elisa; Salata, Cristiano; Bianchi, Vera; Rampazzo, Chiara

2015-01-01

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus. PMID:26342080

Elevated Airway Purines in COPD

PubMed Central

Lazaar, Aili L.; Bordonali, Elena; Qaqish, Bahjat; Boucher, Richard C.

2011-01-01

Background: Adenosine and related purines have established roles in inflammation, and elevated airway concentrations are predicted in patients with COPD. However, accurate airway surface purine measurements can be confounded by stimulation of purine release during collection of typical respiratory samples. Methods: Airway samples were collected noninvasively as exhaled breath condensate (EBC) from 36 healthy nonsmokers (NS group), 28 healthy smokers (S group), and 89 subjects with COPD (29 with GOLD [Global Initiative for Chronic Obstructive Lung Disease] stage II, 29 with GOLD stage III, and 31 with GOLD stage IV) and analyzed with mass spectrometry for adenosine, adenosine monophosphate (AMP), and phenylalanine, plus urea as a dilution marker. Variable dilution of airway secretions in EBC was controlled using ratios to urea, and airway surface concentrations were calculated using EBC to serum urea-based dilution factors. Results: EBC adenosine to urea ratios were similar in NS (0.20 ± 0.21) and S (0.22 ± 0.20) groups but elevated in those with COPD (0.32 ± 0.30, P < .01 vs NS). Adenosine to urea ratios were highest in the most severely affected cohort (GOLD IV, 0.35 ± 0.34, P < .01 vs NS) and negatively correlated with FEV1 (r = −0.27, P < .01). Elevated AMP to urea ratios were also observed in the COPD group (0.58 ± 0.97 COPD, 0.29 ± 0.35 NS, P < .02), but phenylalanine to urea ratios were similar in all groups. Airway surface adenosine concentrations calculated in a subset of subjects were 3.2 ± 2.7 μM in those with COPD (n = 28) relative to 1.7 ± 1.5 μM in the NS group (n = 16, P < .05). Conclusions: Airway purines are present on airway surfaces at physiologically significant concentrations, are elevated in COPD, and correlate with markers of COPD severity. Purinergic signaling pathways are potential therapeutic targets in COPD, and EBC purines are potential noninvasive biomarkers. PMID:21454402

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

PubMed

Nemkov, Travis; Sun, Kaiqi; Reisz, Julie A; Song, Anren; Yoshida, Tatsuro; Dunham, Andrew; Wither, Matthew J; Francis, Richard O; Roach, Robert C; Dzieciatkowska, Monika; Rogers, Stephen C; Doctor, Allan; Kriebardis, Anastasios; Antonelou, Marianna; Papassideri, Issidora; Young, Carolyn T; Thomas, Tiffany A; Hansen, Kirk C; Spitalnik, Steven L; Xia, Yang; Zimring, James C; Hod, Eldad A; D'Alessandro, Angelo

2018-02-01

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13 C 1 -aspartate or 13 C 5 -adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates. Copyright© 2018 Ferrata Storti Foundation.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage

PubMed Central

Nemkov, Travis; Sun, Kaiqi; Reisz, Julie A.; Song, Anren; Yoshida, Tatsuro; Dunham, Andrew; Wither, Matthew J.; Francis, Richard O.; Roach, Robert C.; Dzieciatkowska, Monika; Rogers, Stephen C.; Doctor, Allan; Kriebardis, Anastasios; Antonelou, Marianna; Papassideri, Issidora; Young, Carolyn T.; Thomas, Tiffany A.; Hansen, Kirk C.; Spitalnik, Steven L.; Xia, Yang; Zimring, James C.; Hod, Eldad A.; D’Alessandro, Angelo

2018-01-01

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates. PMID:29079593

Identification, Biochemical Characterization, and Subcellular Localization of Allantoate Amidohydrolases from Arabidopsis and Soybean1[W

PubMed Central

Werner, Andrea K.; Sparkes, Imogen A.; Romeis, Tina; Witte, Claus-Peter

2008-01-01

Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO2, and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 μm manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of l-asparagine and l-aspartate but not d-asparagine. l-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum. PMID:18065556

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.

Targeting the cell cycle and the PI3K pathway: a possible universal strategy to reactivate innate tumor suppressor programmes in cancer cells.

PubMed

David-Pfeuty, Thérèse; Legraverend, Michel; Ludwig, Odile; Grierson, David S

2010-04-01

Corruption of the Rb and p53 pathways occurs in virtually all human cancers. This could be because it lends oncogene-bearing cells a surfeit of Cdk activity and growth, enabling them to elaborate strategies to evade tumor-suppressive mechanisms and divide inappropriately. Targeting both Cdk activities and the PI3K pathway might be therefore a potentially universal means to palliate their deficiency in cancer cells. We showed that the killing efficacy of roscovitine and 16 other purines and potentiation of roscovitine-induced apoptosis by the PI3K inhibitor, LY294002, decreased with increasing corruption of the Rb and p53 pathways. Further, we showed that purines differing by a single substitution, which exerted little lethal effect on distant cell types in rich medium, could display widely-differing cytotoxicity profiles toward the same cell types in poor medium. Thus, closely-related compounds targeting similar Cdks may interact with different targets that could compete for their interaction with therapeutically-relevant Cdk targets. In the perspective of clinical development in association with the PI3K pathway inhibitors, it might thus be advisable to select tumor cell type-specific Cdk inhibitors on the basis of their toxicity in cell-culture-based assays performed at a limiting serum concentration sufficient to suppress their interaction with undesirable crossreacting targets whose range and concentration would depend on the cell genotype.

Trisubstituted purine inhibitors of PDGFRα and their antileukemic activity in the human eosinophilic cell line EOL-1.

PubMed

Malínková, Veronika; Řezníčková, Eva; Jorda, Radek; Gucký, Tomáš; Kryštof, Vladimír

2017-12-15

Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G 1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pathways of nitrogen assimilation in cowpea nodules studied using /sup 15/N/sub 2/ and allopurinol. [Vigna unguiculata L. Walp. cv Vita

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

Atkins, C.A.; Storer, P.J.; Pate, J.S.

1988-01-01

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo (3,4-d)pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (vigna unguiculata L. Walp. cv Vita 3) formed (/sup 15/N)xanthine from /sup 15/N/sub 2/ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.87.99.2) activity.more » Negligible /sup 15/N -labeling of asparagine from /sup 15/N/sub 2/ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.« less

The biochemical origins of the surface-enhanced Raman spectra of bacteria: a metabolomics profiling by SERS.

PubMed

Premasiri, W Ranjith; Lee, Jean C; Sauer-Budge, Alexis; Théberge, Roger; Costello, Catherine E; Ziegler, Lawrence D

2016-07-01

The dominant molecular species contributing to the surface-enhanced Raman spectroscopy (SERS) spectra of bacteria excited at 785 nm are the metabolites of purine degradation: adenine, hypoxanthine, xanthine, guanine, uric acid, and adenosine monophosphate. These molecules result from the starvation response of the bacterial cells in pure water washes following enrichment from nutrient-rich environments. Vibrational shifts due to isotopic labeling, bacterial SERS spectral fitting, SERS and mass spectrometry analysis of bacterial supernatant, SERS spectra of defined bacterial mutants, and the enzymatic substrate dependence of SERS spectra are used to identify these molecular components. The absence or presence of different degradation/salvage enzymes in the known purine metabolism pathways of these organisms plays a central role in determining the bacterial specificity of these purine-base SERS signatures. These results provide the biochemical basis for the development of SERS as a rapid bacterial diagnostic and illustrate how SERS can be applied more generally for metabolic profiling as a probe of cellular activity. Graphical Abstract Bacterial typing by metabolites released under stress.

Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

PubMed

Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi

2018-02-16

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

Electronic and Structural Elements That Regulate the Excited-State Dynamics in Purine Nucleobase Derivatives

PubMed Central

2015-01-01

The excited-state dynamics of the purine free base and 9-methylpurine are investigated using experimental and theoretical methods. Femtosecond broadband transient absorption experiments reveal that excitation of these purine derivatives in aqueous solution at 266 nm results primarily in ultrafast conversion of the S2(ππ*) state to the vibrationally excited 1nπ* state. Following vibrational and conformational relaxation, the 1nπ* state acts as a doorway state in the efficient population of the triplet manifold with an intersystem crossing lifetime of hundreds of picoseconds. Experiments show an almost 2-fold increase in the intersystem crossing rate on going from polar aprotic to nonpolar solvents, suggesting that a solvent-dependent energy barrier must be surmounted to access the singlet-to-triplet crossing region. Ab initio static and surface-hopping dynamics simulations lend strong support to the proposed relaxation mechanism. Collectively, the experimental and computational results demonstrate that the accessibility of the nπ* states and the topology of the potential energy surfaces in the vicinity of conical intersections are key elements in controlling the excited-state dynamics of the purine derivatives. From a structural perspective, it is shown that the purine chromophore is not responsible for the ultrafast internal conversion in the adenine and guanine monomers. Instead, C6 functionalization plays an important role in regulating the rates of radiative and nonradiative relaxation. C6 functionalization inhibits access to the 1nπ* state while simultaneously facilitating access to the 1ππ*(La)/S0 conical intersection, such that population of the 1nπ* state cannot compete with the relaxation pathways to the ground state involving ring puckering at the C2 position. PMID:25763596

Informal E-waste recycling in developing countries: review of metal(loid)s pollution, environmental impacts and transport pathways.

PubMed

Ackah, Michael

2017-11-01

Crude or primitive recycling practices are often adopted in material resource recovery from E-waste in developing nations. Significant human health and environmental impacts may occur because of such practices. Literature on metal(loid)s pollution during E-waste processing is fragmented. Here, I review the health and environmental impacts of E-waste recycling operations and transport pathways of metal(loid)s, dispersed during operations. This paper is organised into five sections. Section 1 relates to the background of global E-waste generation and legal/illegal trade, citing specific cases from Ghana and other developing nations. Section 2 provides a brief information on sources of metal(loid)s in E-waste. Section 3 describes characteristics of informal E-waste recycling operations in developing nations. Section 4 examines the health and environmental impacts in E-waste recycling while section 5 evaluates major transport pathways of metal(loid)s contaminants.

The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

PubMed Central

Wiederkehr, Andreas; Avaro, Sandrine; Prescianotto-Baschong, Cristina; Haguenauer-Tsapis, Rosine; Riezman, Howard

2000-01-01

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling. PMID:10769031

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism.

PubMed

Gooding, Jessica R; Jensen, Mette V; Dai, Xiaoqing; Wenner, Brett R; Lu, Danhong; Arumugam, Ramamani; Ferdaoussi, Mourad; MacDonald, Patrick E; Newgard, Christopher B

2015-10-06

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet β cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in β cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing β cell dysfunction in T2D. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Uric Acid Induces Hepatic Steatosis by Generation of Mitochondrial Oxidative Stress

PubMed Central

Lanaspa, Miguel A.; Sanchez-Lozada, Laura G.; Choi, Yea-Jin; Cicerchi, Christina; Kanbay, Mehmet; Roncal-Jimenez, Carlos A.; Ishimoto, Takuji; Li, Nanxing; Marek, George; Duranay, Murat; Schreiner, George; Rodriguez-Iturbe, Bernardo; Nakagawa, Takahiko; Kang, Duk-Hee; Sautin, Yuri Y.; Johnson, Richard J.

2012-01-01

Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states. PMID:23035112

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

PubMed Central

Vistein, Rachel; Puthenveedu, Manojkumar A.

2013-01-01

The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling. PMID:24003153

First Evidence on the Role of Heavy Ion Irradiation of Meteorites and Formamide in the Origin of Biomolecules

NASA Astrophysics Data System (ADS)

Saladino, Raffaele; Carota, Eleonora; Botta, Giorgia; Kapralov, Michail; Timoshenko, Gennady N.; Rozanov, Alexei; Krasavin, Eugene; Di Mauro, Ernesto

2016-11-01

Formamide (NH2CHO) has been irradiated in condensed phase at 273 K by 11B-boron beams in the presence of powdered meteorites of the chondrite and stony-iron types. Relative to the controls (no radiation or no catalysis), a variegate panel of compounds was observed, including purine and pyrimidine nucleobases (uracil, cytosine, adenine, and guanine), nucleobase analogues, heterocycles, and carboxylic acids involved in metabolic pathways. The presence of amino imidazole carbonitrile (AICN), 4,6-diamino purine (4,6-DAP) and 2,4-diamino pyrimidine (2,4-DAPy) among the observed products suggests the occurrence of an unified mechanism based on the generation of radical cyanide species (•CN). These observations contribute to outline plausible prebiotic scenarios involving 11B-boron as energy source.

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion

PubMed Central

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-01-01

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry. PMID:22328521

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion.

PubMed

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-04-15

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

Metabolic Profiles and Free Radical Scavenging Activity of Cordyceps bassiana Fruiting Bodies According to Developmental Stage

PubMed Central

Hyun, Sun-Hee; Lee, Seok-Young; Sung, Gi-Ho; Kim, Seong Hwan; Choi, Hyung-Kyoon

2013-01-01

The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana. PMID:24058459

An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

PubMed Central

Tugizov, Sharof; Maidji, Ekaterina; Xiao, Jianqiao; Pereira, Lenore

1999-01-01

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway. PMID:10482621

Mechanical and chemical recycling of solid plastic waste.

PubMed

Ragaert, Kim; Delva, Laurens; Van Geem, Kevin

2017-11-01

This review presents a comprehensive description of the current pathways for recycling of polymers, via both mechanical and chemical recycling. The principles of these recycling pathways are framed against current-day industrial reality, by discussing predominant industrial technologies, design strategies and recycling examples of specific waste streams. Starting with an overview on types of solid plastic waste (SPW) and their origins, the manuscript continues with a discussion on the different valorisation options for SPW. The section on mechanical recycling contains an overview of current sorting technologies, specific challenges for mechanical recycling such as thermo-mechanical or lifetime degradation and the immiscibility of polymer blends. It also includes some industrial examples such as polyethylene terephthalate (PET) recycling, and SPW from post-consumer packaging, end-of-life vehicles or electr(on)ic devices. A separate section is dedicated to the relationship between design and recycling, emphasizing the role of concepts such as Design from Recycling. The section on chemical recycling collects a state-of-the-art on techniques such as chemolysis, pyrolysis, fluid catalytic cracking, hydrogen techniques and gasification. Additionally, this review discusses the main challenges (and some potential remedies) to these recycling strategies and ground them in the relevant polymer science, thus providing an academic angle as well as an applied one. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.

PubMed

Wang, Feifei; Chen, Xiaoqing; Zhang, Xiaoqing; Ma, Lan

2008-08-01

Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.

Reversal of autism-like behaviors and metabolism in adult mice with single-dose antipurinergic therapy

PubMed Central

Naviaux, J C; Schuchbauer, M A; Li, K; Wang, L; Risbrough, V B; Powell, S B; Naviaux, R K

2014-01-01

Autism spectrum disorders (ASDs) now affect 1–2% of the children born in the United States. Hundreds of genetic, metabolic and environmental factors are known to increase the risk of ASD. Similar factors are known to influence the risk of schizophrenia and bipolar disorder; however, a unifying mechanistic explanation has remained elusive. Here we used the maternal immune activation (MIA) mouse model of neurodevelopmental and neuropsychiatric disorders to study the effects of a single dose of the antipurinergic drug suramin on the behavior and metabolism of adult animals. We found that disturbances in social behavior, novelty preference and metabolism are not permanent but are treatable with antipurinergic therapy (APT) in this model of ASD and schizophrenia. A single dose of suramin (20 mg kg−1 intraperitoneally (i.p.)) given to 6-month-old adults restored normal social behavior, novelty preference and metabolism. Comprehensive metabolomic analysis identified purine metabolism as the key regulatory pathway. Correction of purine metabolism normalized 17 of 18 metabolic pathways that were disturbed in the MIA model. Two days after treatment, the suramin concentration in the plasma and brainstem was 7.64 μM pmol μl−1 (±0.50) and 5.15 pmol mg−1 (±0.49), respectively. These data show good uptake of suramin into the central nervous system at the level of the brainstem. Most of the improvements associated with APT were lost after 5 weeks of drug washout, consistent with the 1-week plasma half-life of suramin in mice. Our results show that purine metabolism is a master regulator of behavior and metabolism in the MIA model, and that single-dose APT with suramin acutely reverses these abnormalities, even in adults. PMID:24937094

Aminoimidazole Carboxamide Ribotide Exerts Opposing Effects on Thiamine Synthesis in Salmonella enterica

PubMed Central

Bazurto, Jannell V.; Heitman, Nicholas J.

2015-01-01

ABSTRACT In Salmonella enterica, the thiamine biosynthetic intermediate 5-aminoimidazole ribotide (AIR) can be synthesized de novo independently of the early purine biosynthetic reactions. This secondary route to AIR synthesis is dependent on (i) 5-amino-4-imidazolecarboxamide ribotide (AICAR) accumulation, (ii) a functional phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase (PurC; EC 6.3.2.6), and (iii) methionine and lysine in the growth medium. Studies presented here show that AICAR is a direct precursor to AIR in vivo. PurC-dependent conversion of AICAR to AIR was recreated in vitro. Physiological studies showed that exogenous nutrients (e.g., methionine and lysine) antagonize the inhibitory effects of AICAR on the ThiC reaction and decreased the cellular thiamine requirement. Finally, genetic results identified multiple loci that impacted the effect of AICAR on thiamine synthesis and implicated cellular aspartate levels in AICAR-dependent AIR synthesis. Together, the data here clarify the mechanism that allows conditional growth of a strain lacking the first five biosynthetic enzymes, and they provide additional insights into the complexity of the metabolic network and its plasticity. IMPORTANCE In bacteria, the pyrimidine moiety of thiamine is derived from aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis. A previous study described conditions under which AIR synthesis is independent of purine biosynthesis. This work is an extension of that previous study and describes a new synthetic pathway to thiamine that depends on a novel thiamine precursor and a secondary activity of the biosynthetic enzyme PurC. These findings provide mechanistic details of redundancy in the synthesis of a metabolite that is essential for nucleotide and coenzyme biosynthesis. Metabolic modifications that allow the new pathway to function or enhance it are also described. PMID:26100042

In silico analysis of the amido phosphoribosyltransferase inhibition by PY873, PY899 and a derivative of isophthalic acid.

PubMed

Batool, Sidra; Nawaz, Muhammad Sulaman; Kamal, Mohammad A

2013-10-01

Selectively decreasing the availability of precursors for the de novo biosynthesis of purine nucleotides is a valid approach towards seeking a cure for leukaemia. Nucleotides and deoxynucleotides are required by living cells for syntheses of RNA, DNA, and cofactors such as NADP(+), FAD(+), coenzyme A and ATP. Nucleotides contain purine and pyrimidine bases, which can be synthesized through salvage pathway as well. Amido phosphoribosyltransferase (APRT), also known as glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), is an enzyme that in humans is encoded by the PPAT (phosphoribosyl pyrophosphate amidotransferase) gene. APRT catalyzes the first committed step of the de novo pathway using its substrate, phosphoribosyl pyrophosphate (PRPP). As APRT is inhibited by many folate analogues, therefore, in this study we focused on the inhibitory effects of three folate analogues on APRT activity. This is extension of our previous wet lab work to analyze and dissect molecular interaction and inhibition mechanism using molecular modeling and docking tools in the current study. Comparative molecular docking studies were carried out for three diamino folate derivatives employing a model of the human enzyme that was built using the 3D structure of Bacillus subtilis APRT (PDB ID; 1GPH) as the template. Binding orientation of interactome indicates that all compounds having nominal cluster RMSD in same active site's deep narrow polar fissure. On the basis of comparative conformational analysis, electrostatic interaction, binding free energy and binding orientation of interactome, we support the possibility that these molecules could behave as APRT inhibitors and therefore may block purine de novo biosynthesis. Consequently, we suggest that PY899 is the most active biological compound that would be a more potent inhibitor for APRT inhibition than PY873 and DIA, which also confirms previous wet lab report.

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines

PubMed Central

McKeague, Maureen; Wang, Yen-Hsiang; Cravens, Aaron; Win, Maung Nyan; Smolke, Christina D.

2016-01-01

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270 μg/L, 61 μg/L, and 3700 μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives. PMID:27519552

On the Origin of Heterotrophy.

PubMed

Schönheit, Peter; Buckel, Wolfgang; Martin, William F

2016-01-01

The theory of autotrophic origins of life posits that the first cells on Earth satisfied their carbon needs from CO2. At hydrothermal vents, spontaneous synthesis of methane via serpentinization links an energy metabolic reaction with a geochemical homologue. If the first cells were autotrophs, how did the first heterotrophs arise, and what was their substrate? We propose that cell mass roughly similar to the composition of Escherichia coli was the substrate for the first chemoorganoheterotrophs. Amino acid fermentations, pathways typical of anaerobic clostridia and common among anaerobic archaea, in addition to clostridial type purine fermentations, might have been the first forms of heterotrophic carbon and energy metabolism. Ribose was probably the first abundant sugar, and the archaeal type III RubisCO pathway of nucleoside monophosphate conversion to 3-phosphoglycerate might be a relic of ancient heterotrophy. Participation of chemiosmotic coupling and flavin-based electron bifurcation--a soluble energy coupling process--in clostridial amino acid and purine fermentations is consistent with an autotrophic origin of both metabolism and heterotrophy, as is the involvement of S(0) as an electron acceptor in the facilitated fermentations of anaerobic heterotrophic archaea. Copyright © 2015 Elsevier Ltd. All rights reserved.

GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

PubMed Central

Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

2018-01-01

The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

Isolation and Characterization of Anaerobic Bacteria for Symbiotic Recycling of Uric Acid Nitrogen in the Gut of Various Termites

PubMed Central

Thong-On, Arunee; Suzuki, Katsuyuki; Noda, Satoko; Inoue, Jun-ichi; Kajiwara, Susumu; Ohkuma, Moriya

2012-01-01

Recycling of the nitrogenous waste uric acid (UA) of wood-feeding termites by their gut bacteria is one of the significant aspects of symbiosis for the conservation of nitrogen sources. Diverse anaerobic UA-degrading bacteria comprising 16 species were isolated from the gut of eight termite species, and were assigned to Clostridia, Enterobacteriaceae, and low G+C Gram-positive cocci. UA-degrading Clostridia had never been isolated from termite guts. UA-degrading ability was sporadically distributed among phylogenetically various culturable anaerobic bacteria from termite guts. A strain of Clostridium sp., which was commonly isolated from three termite species and represented a probable new species in cluster XIVa of clostridia, utilized UA as a nitrogen source but not as a sole carbon and energy source. This feature is in clear contrast to that of well-studied purinolytic clostridia or previously isolated UA degraders from termite guts, which also utilize UA as a sole carbon and energy source. Ammonia is the major nitrogenous product of UA degradation. Various purines stimulated the growth of this strain when added to an otherwise growth-limiting, nitrogen poor medium. The bacterial species involved the recycling of UA nitrogen in the gut microbial community of termites are more diverse in terms of both taxonomy and nutritional physiology than previously recognized. PMID:22791052

Clathrin-independent internalization and recycling

PubMed Central

Gong, Qiang; Huntsman, Christopher; Ma, Dzwokai

2008-01-01

Abstract The functionality of receptor and channel proteins depends directly upon their expression level on the plasma membrane. Therefore, the ability to selectively adjust the surface level of a particular receptor or channel protein is pivotal to many cellular signalling events. The internalization and recycling pathway plays a major role in the regulation of protein surface level, and thus has been a focus of research for many years. Although several endocytic pathways have been identified, most of our knowledge has come from the clathrin-dependent pathway, while the other pathways remain much less well defined. Considering that clathrin-independent internalization may account for as much as 50% of the total endocytic activity in the cell, the lack of such knowledge constitutes a major gap in our efforts to understand how different internalization pathways are utilized and co-ordinated. Recent studies have provided valuable insights into this area, yet many more questions still remain. In this review, we will give a panoramic introduction to the current knowledge of various internalization and recycling pathways, with an emphasis on the latest findings that have broadened our view of the clathrin-independent pathways. We will also dedicate one section to the emerging studies of the clathrin-independent internalization pathways in neuronal cells. PMID:18039352

ARH directs megalin to the endocytic recycling compartment to regulate its proteolysis and gene expression

PubMed Central

Shah, Mehul; Baterina, Oscar Y.; Taupin, Vanessa

2013-01-01

Receptors internalized by endocytosis can return to the plasma membrane (PM) directly from early endosomes (EE; fast recycling) or they can traffic from EE to the endocytic recycling compartment (ERC) and recycle from there (slow recycling). How receptors are sorted for trafficking along these two pathways remains unclear. Here we show that autosomal recessive hypercholesterolemia (ARH) is required for trafficking of megalin, a member of the LDL receptor family, from EE to the ERC by coupling it to dynein; in the absence of ARH, megalin returns directly to the PM from EE via the connecdenn2/Rab35 fast recycling pathway. Binding of ARH to the endocytic adaptor AP-2 prevents fast recycling of megalin. ARH-mediated trafficking of megalin to the ERC is necessary for γ-secretase mediated cleavage of megalin and release of a tail fragment that mediates transcriptional repression. These results identify a novel mechanism for sorting receptors for trafficking to the ERC and link ERC trafficking to regulated intramembrane proteolysis (RIP) and expression of megalin. PMID:23836931

Expression for caffeine biosynthesis and related enzymes in Camellia sinensis.

PubMed

Kato, Misako; Kitao, Naoko; Ishida, Mariko; Morimoto, Hanayo; Irino, Fumi; Mizuno, Kouichi

2010-01-01

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid that is present in high concentrations in the tea plant Camellia sinensis. Caffeine synthase (CS, EC 2.1.1.160) catalyzes the S-adenosyl-L-methionine-dependent N-3- and N-1-methylation of the purine base to form caffeine, the last step in the purine alkaloid biosynthetic pathway. We studied the expression profile of the tea caffeine synthase (TCS) gene in developing leaves and flowers by means of northern blot analysis, and compared it with those of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), and S-adenosyl-L-methionine synthase (SAMS, EC 2.5.1.6). The amount of TCS transcripts was highest in young leaves and declined markedly during leaf development, whereas it remained constant throughout the development of the flower. Environmental stresses other than heavy metal stress and plant hormone treatments had no effect on the expression of TCS genes, unlike the other three genes. Drought stress suppressed TCS gene expression in leaves, and the expression pattern mirrored that of the dehydrin gene. The amounts of TCS transcripts increased slightly on supply of a nitrogen source. We discuss the regulation of TCS gene expression.

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

PubMed Central

Çağlayan, Melike; Horton, Julie K.; Dai, Da-Peng; Stefanick, Donna F.; Wilson, Samuel H.

2017-01-01

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides. PMID:28067232

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin.

PubMed

Lesteberg, Kelsey; Orange, Jordan; Makedonas, George

2017-10-01

Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein, we aimed to determine how new perforin transits to the synapse if not via lytic granules. We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response.

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

PubMed Central

Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

2018-01-01

Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

Protein recycling pathways in neurodegenerative diseases

PubMed Central

2014-01-01

Many progressive neurodegenerative diseases, including Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and frontotemporal lobe dementia, are associated with the formation of insoluble intracellular proteinaceous inclusions. It is therefore imperative to understand the factors that regulate normal, as well as abnormal, protein recycling in neurons. Dysfunction of the ubiquitin-proteasome or autophagy pathways might contribute to the pathology of various neurodegenerative diseases. Induction of these pathways may offer a rational therapeutic strategy for a number of these diseases. PMID:25031631

Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells

PubMed Central

Linford, Andrea; Yoshimura, Shin-ichiro; Bastos, Ricardo Nunes; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J.; Barr, Francis A.

2012-01-01

Summary Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. PMID:22595670

Effects of nucleotides adenosine monophosphate and adenosine triphosphate in combination with L-arginine on male rabbit corpus cavernosum tissue.

PubMed

Hupertan, V; Neuzillet, Y; Stücker, O; Pons, C; Leammel, E; Lebret, T

2012-12-01

Purines and more specifically adenosine monophosphate (AMP) and adenosine triphosphate (ATP) have a strong relaxant effect on smooth muscle cells of the dog, rabbit and human corpus cavernosum, to approximately the same degree as nitric oxide (NO). However, purines are considered as modulators of erectile function rather than key mediators. This suggests that the use of purines combined with NO donors could be effective to treat some specific erectile disorders. The relaxation induced by the combination of l-arginine (Arg), a natural substrate for NO synthase, was assessed with a purine-nucleotide (AMP, ATP) on a rabbit corpus cavernosum model, to determine if these substances could potentiate each other's effect. When a pre-contraction was induced by phenylephrine, AMP alone induced a 43% CC relaxation rate and ATP alone a 26% rate. The relaxation rate induced by Arg was lower in comparison (8% at 5.10(-4) m vs. 25% at AMP 5.10(-4) m and 15% at ATP 5.10(-4) m). NO synthase inhibitor n-nitro-l-arginine did not modify the relaxing effect provoked by AMP suggesting that the mechanism of action of this nucleotide does not involve the NO pathway. The combination of Arg at 5.10(-4) m with either AMP or ATP at different doses ranging from 5.10(-4) to 10(-3) m significantly enhanced the relaxing response reaching rates of 62 and 80% respectively, leading to a synergistic effect. The present data indicate that a 'NO donor' combined with an 'adenosine donor' could be an effective therapeutic approach. © 2012 The Authors. International Journal of Andrology © 2012 European Academy of Andrology.

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer.

PubMed

Singh, Himanshu Narayan; Rajeswari, Moganty R

2016-01-01

Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich's ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed "PuRepeatFinder.pl" algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein-protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.

Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular Parasites

PubMed Central

Heinz, Eva; Hacker, Christian; Dean, Paul; Mifsud, John; Goldberg, Alina V.; Williams, Tom A.; Nakjang, Sirintra; Gregory, Alison; Hirt, Robert P.; Lucocq, John M.; Kunji, Edmund R. S.; Embley, T. Martin

2014-01-01

Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis. PMID:25474405

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma.

PubMed

Wang, Xiuxing; Yang, Kailin; Xie, Qi; Wu, Qiulian; Mack, Stephen C; Shi, Yu; Kim, Leo J Y; Prager, Briana C; Flavahan, William A; Liu, Xiaojing; Singer, Meromit; Hubert, Christopher G; Miller, Tyler E; Zhou, Wenchao; Huang, Zhi; Fang, Xiaoguang; Regev, Aviv; Suvà, Mario L; Hwang, Tae Hyun; Locasale, Jason W; Bao, Shideng; Rich, Jeremy N

2017-05-01

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation.

PubMed

Baresova, Veronika; Krijt, Matyas; Skopova, Vaclava; Souckova, Olga; Kmoch, Stanislav; Zikanova, Marie

2016-11-01

Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolic pathways of lung inflammation revealed by high-resolution metabolomics (HRM) of H1N1 influenza virus infection in mice.

PubMed

Chandler, Joshua D; Hu, Xin; Ko, Eun-Ju; Park, Soojin; Lee, Young-Tae; Orr, Michael; Fernandes, Jolyn; Uppal, Karan; Kang, Sang-Moo; Jones, Dean P; Go, Young-Mi

2016-11-01

Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function, and severe lung inflammation with elevated cytokines [interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids, and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycosphingolipid, linoleate, and tryptophan metabolites with IFN-γ contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1β, TNF-α, and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D, purine, and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation. Copyright © 2016 the American Physiological Society.

Metabolic pathways of lung inflammation revealed by high-resolution metabolomics (HRM) of H1N1 influenza virus infection in mice

PubMed Central

Chandler, Joshua D.; Hu, Xin; Ko, Eun-Ju; Park, Soojin; Lee, Young-Tae; Orr, Michael; Fernandes, Jolyn; Uppal, Karan; Kang, Sang-Moo; Jones, Dean P.

2016-01-01

Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function, and severe lung inflammation with elevated cytokines [interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids, and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycosphingolipid, linoleate, and tryptophan metabolites with IFN-γ contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1β, TNF-α, and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D, purine, and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation. PMID:27558316

The Evolutionary Fate of the Genes Encoding the Purine Catabolic Enzymes in Hominoids, Birds, and Reptiles

PubMed Central

Keebaugh, Alaine C.; Thomas, James W.

2010-01-01

Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes. PMID:20106906

The evolutionary fate of the genes encoding the purine catabolic enzymes in hominoids, birds, and reptiles.

PubMed

Keebaugh, Alaine C; Thomas, James W

2010-06-01

Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes.

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers

PubMed Central

Delevoye, Cédric; Romao, Maryse; Owen, David J.; Raposo, Graça

2016-01-01

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1–dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3–dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky–Pudlak syndrome variants. PMID:27482051

Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

PubMed

Lenz, Stefan A P; Wetmore, Stacey D

2016-02-09

Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many different DNA-protein interactions used by AAG to facilitate BER, as well as the crucial role of the general base and water (nucleophile) positioning. The insights gained from our work will aid the understanding of the function of other enzymes that use flexible active sites to exhibit diverse substrate specificity.

Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya

PubMed Central

2012-01-01

Background The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. Results We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. Conclusions We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants. PMID:23216749

Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya.

PubMed

Aryal, Rishi; Yang, Xiaozeng; Yu, Qingyi; Sunkar, Ramanjulu; Li, Lei; Ming, Ray

2012-12-05

The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff's purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants.

Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland

PubMed Central

Sanderson, Julie; Dartt, Darlene A.; Trinkaus-Randall, Vickery; Pintor, Jesus; Civan, Mortimer M.; Delamere, Nicholas A.; Fletcher, Erica L.; Salt, Thomas E.; Grosche, Antje; Mitchell, Claire H.

2014-01-01

This review highlights recent findings that describe how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca2+ concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target may be a key issue in understanding how physiological signaling becomes pathological in ocular disease. PMID:25151301

MOEX: Solvent extraction approach for recycling enriched 98Mo/ 100Mo material

DOE PAGES

Tkac, Peter; Brown, M. Alex; Momen, Abdul; ...

2017-03-20

Several promising pathways exist for the production of 99Mo/ 99mTc using enriched 98Mo or 100Mo. Use of Mo targets require a major change in current generator technology, and the necessity for an efficient recycle pathway to recover valuable enriched Mo material. High recovery yields, purity, suitable chemical form and particle size are required. Results on the development of the MOEX– molybdenum solvent extraction – approach to recycle enriched Mo material are presented. Furthermore, the advantages of the MOEX process are very high decontamination factors from potassium and other elements, high throughput, easy scalability, automation, and minimal waste generation.

MOEX: Solvent extraction approach for recycling enriched 98Mo/ 100Mo material

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

Tkac, Peter; Brown, M. Alex; Momen, Abdul

Several promising pathways exist for the production of 99Mo/ 99mTc using enriched 98Mo or 100Mo. Use of Mo targets require a major change in current generator technology, and the necessity for an efficient recycle pathway to recover valuable enriched Mo material. High recovery yields, purity, suitable chemical form and particle size are required. Results on the development of the MOEX– molybdenum solvent extraction – approach to recycle enriched Mo material are presented. Furthermore, the advantages of the MOEX process are very high decontamination factors from potassium and other elements, high throughput, easy scalability, automation, and minimal waste generation.

Determination of four different purines and their content change in seafood by high-performance liquid chromatography.

PubMed

Qu, Xin; Sui, Jianxin; Mi, Nasha; Lin, Hong

2017-01-01

Seafood is regarded as a high-purine food that may induce gout, which has attracted extensive attention concerning its safety. Therefore, the aim of this study was to develop a simple and reliable method to determine the purine content in seafood and its change during storage to offer consumers healthy diet information. Chromatographic separation was carried out using Waters Atlantis dC 18 column, and potassium phosphate monobasic solution (0.02 mol L -1 , pH 3.6) as a mobile phase. The average recovery yields of four purines were 91.5-105.0%, and relative standard deviation values were around 1.8-6.5%. Shrimp and snail contained higher amounts of purine than fish and bivalves; the livers and skins of fish contained higher amounts of purine than muscles; and the main purine varied depending on the type of seafood. Also, purine content of seafood changed during storage. The purine content of seafood differed depending on species, body part and degree of freshness, which could recommend consumers a healthy diet, especially for people with hyperuricemia and gout. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Retriever, a multiprotein complex for retromer-independent endosomal cargo recycling

PubMed Central

McNally, Kerrie E.; Faulkner, Rebecca; Steinberg, Florian; Gallon, Matthew; Ghai, Rajesh; Pim, David; Langton, Paul; Pearson, Neil; Danson, Chris M.; Nägele, Heike; Morris, Lindsey M; Singla, Arnika; Overlee, Brittany L; Heesom, Kate J.; Sessions, Richard; Banks, Lawrence; Collins, Brett M; Berger, Imre; Billadeau, Daniel D.; Burstein, Ezra; Cullen, Peter J.

2018-01-01

Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway. PMID:28892079

Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration.

PubMed

Majeed, Sophia R; Vasudevan, Lavanya; Chen, Chih-Ying; Luo, Yi; Torres, Jorge A; Evans, Timothy M; Sharkey, Andrew; Foraker, Amy B; Wong, Nicole M L; Esk, Christopher; Freeman, Theresa A; Moffett, Ashley; Keen, James H; Brodsky, Frances M

2014-05-23

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of β1-integrin by interference with recycling following normal endocytosis of inactive β1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces β1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin-actin interactions needed for recycling by G-clathrin during migration.

COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal

PubMed Central

Xu, Peng; Hankins, Hannah M; MacDonald, Chris; Erlinger, Samuel J; Frazier, Meredith N; Diab, Nicholas S; Piper, Robert C; Jackson, Lauren P; MacGurn, Jason A

2017-01-01

The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway. PMID:29058666

Xanthine Oxidase Inhibition with Febuxostat Attenuates Systolic Overload-induced Left Ventricular Hypertrophy and Dysfunction in Mice

PubMed Central

Xu, Xin; Hu, Xinli; Lu, Zhongbing; Zhang, Ping; Zhao, Lin; Wessale, Jerry L.; Bache, Robert J.; Chen, Yingjie

2008-01-01

The purine analog xanthine oxidase (XO) inhibitors (XOIs), allopurinol and oxypurinol, have been reported to protect against heart failure secondary to myocardial infarction or rapid ventricular pacing. Since these agents might influence other aspects of purine metabolism that could influence their effect, this study examined the effect of the non-purine XOI, febuxostat, on pressure overload-induced left ventricular (LV) hypertrophy and dysfunction. Transverse aortic constriction (TAC) in mice caused LV hypertrophy and dysfunction as well as increased myocardial nitrotyrosine at 8 days. TAC also caused increased phosphorylated Akt (p-AktSer473), p42/44 extracellular signal-regulated kinase (p-ErkThr202/Tyr204) and mammalian target of rapamycin (mTOR) (p-mTORSer2488). XO inhibition with febuxostat (5mg/kg/day by gavage for 8 days) beginning ~60 minutes after TAC attenuated the TAC-induced LV hypertrophy and dysfunction. Febuxostat blunted the TAC-induced increases in nitrotyrosine (indicating reduced myocardial oxidative stress), p-ErkThr202/Tyr204 and p-mTORSer2488, with no effect on total Erk or total mTOR. Febuxostat had no effect on myocardial p-AktSer473 or total Akt. The results suggest that XO inhibition with febuxostat reduced oxidative stress in the pressure overloaded LV, thereby diminishing the activation of pathways that result in pathologic hypertrophy and contractile dysfunction. PMID:18995179

[Identification and quantitation of purine derivatives in urinary calculi as markers of abnormal purine metabolism by using high-performance liquid chromatography (HPLC)].

PubMed

Safranow, K

2000-01-01

The objective of this study was to develop a practical method for the analysis of purine derivatives in urinary calculi using high-performance liquid chromatography (HPLC). The method presented herein includes extraction of purine derivatives from urinary stones, followed by chromatography on a reversed-phase column with UV detection. A simpler isocratic method was applied to quantitate 6 purines known to be components of urinary stones, namely uric acid, xanthine, hypoxanthine, 2,8-dihydroxyadenine, oxypurinol and allopurinol. Gradient method separated 10 additional peaks representing methyl derivatives of uric acid or xanthine (1-, 3-, 7-, and 9-methyluric acid, 1,3-,1,7-, and 3,7-dimethyluric acid, and 1-, 3-, and 7-methylxanthine) (Fig. 1). Detection limits for individual compounds ranged from 25 to 140 micrograms purine per g stone weight and precision (RSD%) was 0.5-2.4%. Both methods were next used to analyze purine derivatives in urinary calculi from 48 residents of Western Pomerania. Uric acid was the main component of 9 stones. All of the uric acid stones showed admixtures of 9 other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methyl derivatives of uric acid (1-,3-, and 7-methyluric acid, 1,3-dimethyluric acid, 3-, and 7-methylxanthine) originating from the metabolism of exogenous methylxanthines (caffeine, theophylline and theobromine) (Tab. 1,2). Methyl derivatives of uric acid and xanthine, with a maximal content in stones of 1.7%, have hitherto not been considered constituents of urinary calculi. Statistical analysis of the results revealed strong positive correlations between the level of uric acid and of other purine derivatives in stones (Fig. 2). Correlations were also found between levels of some purines and inorganic compounds (Tab. 3). The sensitivity and specificity of HPLC with UV detection satisfy the requirements of a reference method for the analysis of purines in urinary stones. Isocratic separation is simpler in terms of technique and equipment, and therefore more suitable for hospital laboratories. Examination of purine derivatives in stones may be very helpful for the diagnosis of abnormal purine metabolism and urolithiasis, particularly in dihydroxyadeninuria, xanthinuria and during treatment with allopurinol. Gradient separation requiring more sophisticated instrument seems useful for research purposes when the content of methyl derivatives of purines must be known. The present results indicate that urinary purines at concentrations lower than saturation point may nevertheless coprecipitate with oversaturated uric acid and appear as admixtures in urinary stones. The content of a purine derivative in stone depends on its average urinary excretion in the general population, similarity to the chemical structure of uric acid, and content of the latter in stone. These findings suggest that purines in stones represent a solid solution with uric acid as solvent. It is also plausible that methylxanthines, ubiquitous components of the diet and drugs, are involved in the pathogenesis of urolithiasis. Interpretation of results and practical significance of the determination of purine derivatives in stones is discussed, and future studies to assess the clinical importance of endo- and exogenous purine derivatives in urinary calculi are suggested.

40 CFR 721.4685 - Substituted purine metal salt (generic name).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Substituted purine metal salt (generic... Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... as a substituted purine metal salt (PMN P-95-175) is subject to reporting under this section for the...

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism.

PubMed

Fukuuchi, T; Iyama, N; Yamaoka, N; Kaneko, K

2018-04-13

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

Purine derivate content and amino acid profile in larval stages of three edible insects.

PubMed

Bednářová, Martina; Borkovcová, Marie; Komprda, Tomáš

2014-01-15

Considering their high content of protein, insects are a valuable alternative protein source. However, no evaluation of their purine content has so far been done. High content of purine derivates may lead to the exclusion of such food from the diet of people with specific diseases. The aim of this study was to analyse the content of selected purine derivates and amino acid profile in the three insect species most often used for entomophagy in Europe and compare them with the purine content in egg white and chicken breast. The content of individual purine derivates and their total content were significantly dependent on insect species. The purine content in all three species was significantly higher (P < 0.05) than in egg white, but some values were significantly lower (P < 0.05) than in chicken breast. The total protein content was 548.9 g kg(-1) dry matter (DM) in mealworm (Tenebrio molitor), 551.6 g kg(-1) DM in superworm (Zophobas atratus) and 564.9 g kg(-1) DM in cricket (Gryllus assimilis). Larvae of mealworm and superworm are protein-rich and purine-low meat alternatives. In contrast, cricket nymphs are protein-rich and purine-rich and cannot be recommended for people with hyperuricemia or gout. © 2013 Society of Chemical Industry.

Purine Restriction Induces Pronounced Translational Upregulation of the NT1 Adenosine/Pyrimidine Nucleoside Transporter in Leishmania major

PubMed Central

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A.; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M.

2010-01-01

Summary Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild type promastigotes that were transferred to medium deficient in purines. Pulse labeling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. PMID:20735779

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major.

PubMed

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M

2010-10-01

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. © 2010 Blackwell Publishing Ltd.

Adherens junction turnover: regulating adhesion through cadherin endocytosis, degradation, and recycling

PubMed Central

Nanes, Benjamin A.; Kowalczyk, Andrew P.

2014-01-01

Adherens junctions are important mediators of intercellular adhesion, but they are not static structures. They are regularly formed, broken, and rearranged in a variety of situations, requiring changes in the amount of cadherins, the main adhesion molecule in adherens junctions, present at the cell surface. Thus, endocytosis, degradation, and recycling of cadherins are crucial for dynamic regulation of adherens junctions and control of intercellular adhesion. In this chapter, we review the involvement of cadherin endocytosis in development and disease. We discuss the various endocytic pathways available to cadherins, the adaptors involved, and the sorting of internalized cadherin for recycling or lysosomal degradation. In addition, we review the regulatory pathways controlling cadherin endocytosis and degradation, including regulation of cadherin endocytosis by catenins, cadherin ubiquitination, and growth factor receptor signaling pathways. Lastly, we discuss the proteolytic cleavage of cadherins at the plasma membrane. PMID:22674073

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

PubMed

Pisithkul, Tippapha; Jacobson, Tyler B; O'Brien, Thomas J; Stevenson, David M; Amador-Noguez, Daniel

2015-09-01

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. Copyright © 2015, Pisithkul et al.

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

PubMed Central

Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.; Stevenson, David M.

2015-01-01

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID:26070680

Phenolic amides are potent inhibitors of De Novo nucleotide biosynthesis

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

Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposuremore » leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [ 15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. Furthermore, the results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.« less

Phenolic amides are potent inhibitors of De Novo nucleotide biosynthesis

DOE PAGES

Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.; ...

2015-06-12

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposuremore » leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [ 15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. Furthermore, the results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.« less

Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure

PubMed Central

Nath, Anjali K.; Roberts, Lee D.; Liu, Yan; Mahon, Sari B.; Kim, Sonia; Ryu, Justine H.; Werdich, Andreas; Januzzi, James L.; Boss, Gerry R.; Rockwood, Gary A.; MacRae, Calum A.; Brenner, Matthew; Gerszten, Robert E.; Peterson, Randall T.

2013-01-01

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.—Nath, A. K., Roberts, L. D., Liu, Y., Mahon, S. B., Kim, S., Ryu, J. H., Werdich, A., Januzzi, J. L., Boss, G. R., Rockwood, G. A., MacRae, C. A., Brenner, M., Gerszten, R. E., Peterson, R. T. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure. PMID:23345455

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments.

PubMed

Stoops, Emily H; Hull, Michael; Caplan, Michael J

2016-12-01

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans-Golgi network is a well-established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post-endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post-endocytic pools of this protein are subjected to distinct sorting processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

PubMed Central

Jin, Lu; Bhuiya, Mohammad Wadud; Li, Mengmeng; Liu, XiangQi; Han, Jixiang; Deng, WeiWei; Wang, Min; Yu, Oliver; Zhang, Zhengzhu

2014-01-01

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed. PMID:25133732

The methionine salvage pathway in Bacillus subtilis

PubMed Central

Sekowska, Agnieszka; Danchin, Antoine

2002-01-01

Background Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis. Results Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling. Conclusions A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane). PMID:12022921

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes.

PubMed

Henriques, Cristina; Sanchez, Marco A; Tryon, Rob; Landfear, Scott M

2003-08-31

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

Effects of breeds, tissues and genders on purine contents in pork and the relationships between purine content and other meat quality traits.

PubMed

Zheng, Min; Huang, Yizhong; Ji, Jiuxiu; Xiao, Shijun; Ma, Junwu; Huang, Lusheng

2018-09-01

The purine contents of animal foods are becoming widely concerned because excess intake of purine increases the risk of hyperuricemia and gout. In this study, we investigated the impacts of breed, tissue and sex on pork purine content and its correlations with multiple meat quality traits. Among six pig breeds, the average value of total purine contents (TP) in longissimus lumborum muscle was lowest in Chinese Laiwu pigs (114.2 mg/100 g) while highest in Chinese Bamaxiang mini pigs (139.3 mg/100 g). Considerable variations in TP were observed within most breeds, as well as among twelve pork organs with the range from 7 to 245 mg/100 g. However, no significant differences in TP were found between barrows and gilts. Intriguingly, lower purine content in meat was significantly associated with higher ultimate pH, better meat color and more abundant intramuscular fat content and marbling. The results thus suggest that the selection of low-purine pig species is available, which may simultaneously improve other meat quality traits. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Purine in common plant food in China].

PubMed

Rong, Shengzhong; Zou, Lina; Wang, Zhaoxu; Pan, Hongzhi; Yang, Yuexin

2012-01-01

To determine the content of purine in plant food in China with HPLC. HPLC analysis was applied on Waters Atlantis T3 column (4.6mm x 250mm x 5 microm), using 10.0 mmol/L NH4COOH (pH 3.6) and CH3OH (99%/1%) as mobile phase and running at a flow rate of 1.0 ml/min. The column temperature was 30 degrees C, and the detection wavelength was at 254nm. The content of purine varied significantly in different kinds of plant food. The content of purine in dried fungi and dried legumes and legume products was higher than that in other food, the content of purine in vegetables and vegetable products and fruits and fruit products was low. As a whole, the content of purine was: dried fungi and algae > dried legumes and legume products > nuts and fresh > seeds fungi and algae > cereal and cereals products > vegetables and vegetable products > fruit and fruit products > tubers, starches and products. The content of purine of dried fungi and algae and dried legumes and legume products in plant food was high. The content of purine was varied significantly in different kinds of plant food.

Metabolic Pathways and Networks Associated with Tobacco Use in Military Personnel

PubMed Central

Jones, Dean P.; Walker, Douglas I.; Uppal, Karan; Rohrbeck, Patricia; Mallon, Timothy M.; Go, Young-Mi

2016-01-01

Objective Use high-resolution metabolomics (HRM) to identify metabolic pathways and networks associated with tobacco use in military personnel. Methods Four hundred de-identified samples obtained from the Department of Defense Serum Repository were classified as tobacco users or non-users according to cotinine content. HRM and bioinformatic methods were used to determine pathways and networks associated with classification. Results Eighty individuals were classified as tobacco users compared to 320 non-users based on cotinine levels ≥10 ng/mL. Alterations in lipid and xenobiotic metabolism, and diverse effects on amino acid, sialic acid and purine and pyrimidine metabolism were observed. Importantly, network analysis showed broad effects on metabolic associations not simply linked to well-defined pathways. Conclusions Tobacco use has complex metabolic effects which must be considered in evaluation of deployment-associated environmental exposures in military personnel. PMID:27501098

Metabolic Pathways and Networks Associated With Tobacco Use in Military Personnel.

PubMed

Jones, Dean P; Walker, Douglas I; Uppal, Karan; Rohrbeck, Patricia; Mallon, Col Timothy M; Go, Young-Mi

2016-08-01

The aim of this study is to use high-resolution metabolomics (HRM) to identify metabolic pathways and networks associated with tobacco use in military personnel. Four hundred deidentified samples obtained from the Department of Defense Serum Repository were classified as tobacco users or nonusers according to cotinine content. HRM and bioinformatic methods were used to determine pathways and networks associated with classification. Eighty individuals were classified as tobacco users compared with 320 nonusers on the basis of cotinine levels at least 10 ng/mL. Alterations in lipid and xenobiotic metabolism, and diverse effects on amino acid, sialic acid, and purine and pyrimidine metabolism were observed. Importantly, network analysis showed broad effects on metabolic associations not simply linked to well-defined pathways. Tobacco use has complex metabolic effects that must be considered in evaluation of deployment-associated environmental exposures in military personnel.

Divergent prebiotic synthesis of pyrimidine and 8-oxo-purine ribonucleotides

NASA Astrophysics Data System (ADS)

Stairs, Shaun; Nikmal, Arif; Bučar, Dejan-Krešimir; Zheng, Shao-Liang; Szostak, Jack W.; Powner, Matthew W.

2017-05-01

Understanding prebiotic nucleotide synthesis is a long standing challenge thought to be essential to elucidating the origins of life on Earth. Recently, remarkable progress has been made, but to date all proposed syntheses account separately for the pyrimidine and purine ribonucleotides; no divergent synthesis from common precursors has been proposed. Moreover, the prebiotic syntheses of pyrimidine and purine nucleotides that have been demonstrated operate under mutually incompatible conditions. Here, we tackle this mutual incompatibility by recognizing that the 8-oxo-purines share an underlying generational parity with the pyrimidine nucleotides. We present a divergent synthesis of pyrimidine and 8-oxo-purine nucleotides starting from a common prebiotic precursor that yields the β-ribo-stereochemistry found in the sugar phosphate backbone of biological nucleic acids. The generational relationship between pyrimidine and 8-oxo-purine nucleotides suggests that 8-oxo-purine ribonucleotides may have played a key role in primordial nucleic acids prior to the emergence of the canonical nucleotides of biology.

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

PubMed

Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

2007-08-01

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

Relaxation mechanisms of UV-photoexcited DNA and RNA nucleobases

PubMed Central

Barbatti, Mario; Aquino, Adélia J. A.; Szymczak, Jaroslaw J.; Nachtigallová, Dana; Hobza, Pavel; Lischka, Hans

2010-01-01

A comprehensive effort in photodynamical ab initio simulations of the ultrafast deactivation pathways for all five nucleobases adenine, guanine, cytosine, thymine, and uracil is reported. These simulations are based on a complete nonadiabatic surface-hopping approach using extended multiconfigurational wave functions. Even though all five nucleobases share the basic internal conversion mechanisms, the calculations show a distinct grouping into purine and pyrimidine bases as concerns the complexity of the photodynamics. The purine bases adenine and guanine represent the most simple photodeactivation mechanism with the dynamics leading along a diabatic ππ* path directly and without barrier to the conical intersection seam with the ground state. In the case of the pyrimidine bases, the dynamics starts off in much flatter regions of the ππ* energy surface due to coupling of several states. This fact prohibits a clear formation of a single reaction path. Thus, the photodynamics of the pyrimidine bases is much richer and includes also nπ* states with varying importance, depending on the actual nucleobase considered. Trapping in local minima may occur and, therefore, the deactivation time to the ground state is also much longer in these cases. Implications of these findings are discussed (i) for identifying structural possibilities where singlet/triplet transitions can occur because of sufficient retention time during the singlet dynamics and (ii) concerning the flexibility of finding other deactivation pathways in substituted pyrimidines serving as candidates for alternative nucleobases. PMID:21115845

Heterogeneity and dynamics of the ligand recognition mode in purine-sensing riboswitches.

PubMed

Jain, Niyati; Zhao, Liang; Liu, John D; Xia, Tianbing

2010-05-04

High-resolution crystal structures and biophysical analyses of purine-sensing riboswitches have revealed that a network of hydrogen bonding interactions appear to be largey responsible for discrimination of cognate ligands against structurally related compounds. Here we report that by using femtosecond time-resolved fluorescence spectroscopy to capture the ultrafast decay dynamics of the 2-aminopurine base as the ligand, we have detected the presence of multiple conformations of the ligand within the binding pockets of one guanine-sensing and two adenine-sensing riboswitches. All three riboswitches have similar conformational distributions of the ligand-bound state. The known crystal structures represent the global minimum that accounts for 50-60% of the population, where there is no significant stacking interaction between the ligand and bases of the binding pocket, but the hydrogen-bonding cage collectively provides an electronic environment that promotes an ultrafast ( approximately 1 ps) charge transfer pathway. The ligand also samples multiple conformations in which it significantly stacks with either the adenine or the uracil bases of the A21-U75 and A52-U22 base pairs that form the ceiling and floor of the binding pocket, respectively, but favors the larger adenine bases. These alternative conformations with well-defined base stacking interactions are approximately 1-1.5 kcal/mol higher in DeltaG degrees than the global minimum and have distinct charge transfer dynamics within the picosecond to nanosecond time regime. Inside the pocket, the purine ligand undergoes dynamic motion on the low nanosecond time scale, sampling the multiple conformations based on time-resolved anisotropy decay dynamics. These results allowed a description of the energy landscape of the bound ligand with intricate details and demonstrated the elastic nature of the ligand recognition mode by the purine-sensing riboswitches, where there is a dynamic balance between hydrogen bonding and base stacking interactions, yielding the high affinity and specificity by the aptamer domain.

Structure-activity relationships and molecular studies of novel arylpiperazinylalkyl purine-2,4-diones and purine-2,4,8-triones with antidepressant and anxiolytic-like activity.

PubMed

Zagórska, Agnieszka; Kołaczkowski, Marcin; Bucki, Adam; Siwek, Agata; Kazek, Grzegorz; Satała, Grzegorz; Bojarski, Andrzej J; Partyka, Anna; Wesołowska, Anna; Pawłowski, Maciej

2015-06-05

A novel series of arylpiperazinylalkyl purine-2,4-diones (4-27) and purine-2,4,8-triones (31-38) was synthesized and tested to evaluated their affinity for the serotoninergic (5-HT1A, 5-HT6, 5-HT7) and dopaminergic (D2) receptors. Compounds with purine-2,4-dione nucleus generally had affinity values higher than the corresponding purine-2,4,8-trione compounds. A spectrum of receptor activities was observed for compounds with a substituent at the 7-position of the imidazo[2,1-f]purine-2,4-dione system and some potent 5-HT1A (18, 25), 5-HT7 (14) and mixed 5-HT1A/5-HT7 (8, 9) receptor ligands with additional affinity for dopamine D2 receptors (15) has been identified. Moreover, docking studies proved that a substituent at the 7-position of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione could be essential for receptor affinity and selectivity, especially towards 5-HT1A and 5-HT7. The results of the preliminary pharmacological in vivo studies of selected derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione, including 9 as a potential anxiolytic, 8 and 15 as potential antidepressants, and 18 and 25 as potential antidepressant and anxiolytic agents. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Enhanced activity of the purine nucleotide cycle of the exercising muscle in patients with hyperthyroidism.

PubMed

Fukui, H; Taniguchi , S; Ueta, Y; Yoshida, A; Ohtahara, A; Hisatome, I; Shigemasa, C

2001-05-01

Myopathy frequently develops in patients with hyperthyroidism, but its precise mechanism is not clearly understood. In this study we focused on the purine nucleotide cycle, which contributes to ATP balance in skeletal muscles. To investigate purine metabolism in muscles, we measured metabolites related to the purine nucleotide cycle using the semiischemic forearm test. We examined the following four groups: patients with untreated thyrotoxic Graves' disease (untreated group), patients with Graves' disease treated with methimazole (treated group), patients in remission (remission group), and healthy volunteers (control group). To trace the glycolytic process, we measured glycolytic metabolites (lactate and pyruvate) as well as purine metabolites (ammonia and hypoxanthine). In the untreated group, the levels of lactate, pyruvate, and ammonia released were remarkably higher than those in the control group. Hypoxanthine release also increased in the untreated group, but the difference among the patient groups was not statistically significant. The accelerated purine catabolism did not improve after 3 months of treatment with methimazole, but it was completely normalized in the remission group. This indicated that long-term maintenance of thyroid function was necessary for purine catabolism to recover. We presume that an unbalanced ATP supply or conversion of muscle fiber type may account for the acceleration of the purine nucleotide cycle under thyrotoxicosis. Such acceleration of the purine nucleotide cycle is thought to be in part a protective mechanism against a rapid collapse of the ATP energy balance in exercising muscles of patients with hyperthyroidism.

Characterization of solution-phase and gas-phase reactions in on-line electrochemistry-thermospray tandem mass spectrometry.

PubMed

Volk, K J; Yost, R A; Brajter-Toth, A

1989-07-14

Electrochemistry was used on-line with high-performance liquid chromatography-thermospray tandem mass spectrometry to provide insight into the solution-phase decomposition reactions of electrochemically generated oxidation products. Products formed during electrooxidation were monitored as the electrode potential was varied. The solution reactions which follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe and the composition of the thermospray buffer. Either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur with pH 7 ammonium acetate buffer. Both the electrochemically generated and the synthesized disulfide of 6-thiopurine decompose under thermospray conditions to produce 6-thiopurine and purine-6-sulfinate. Solution-phase studies indicate that nucleophilic and electrophilic substitution reactions with purine-6-sulfinate result in the formation of purine, adenine, and hypoxanthine. Products were identified and characterized by tandem mass spectrometry. This work shows the first example of high-performance liquid chromatography used on-line with electrochemistry to separate stable oxidation products prior to analysis by thermospray tandem mass spectrometry. In addition, solution-phase and gas-phase studies with methylamine show that the site of the nucleophilic and electrophilic reactions is probably inside the thermospray probe. Most importantly, these results also show that the on-line combination of electrochemistry with thermospray tandem mass spectrometry provides valuable information about redox and associated chemical reactions of biological molecules such as the structures of intermediates or products as well as providing insight into reaction pathways.

Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2.

PubMed

An, Songon; Kyoung, Minjoung; Allen, Jasmina J; Shokat, Kevan M; Benkovic, Stephen J

2010-04-09

The reversible association and dissociation of a metabolic multi-enzyme complex participating in de novo purine biosynthesis, the purinosome, was demonstrated in live cells to respond to the levels of purine nucleotides in the culture media. We also took advantage of in vitro proteomic scale studies of cellular substrates of human protein kinases (e.g. casein kinase II (CK2) and Akt), that implicated several de novo purine biosynthetic enzymes as kinase substrates. Here, we successfully identified that purinosome formation in vivo was significantly promoted in HeLa cells by the addition of small-molecule CK2-specific inhibitors (i.e. 4,5,6,7-tetrabromo-1H-benzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, tetrabromocinammic acid, 4,4',5,5',6,6'-hexahydroxydiphenic acid 2,2',6,6'-dilactone (ellagic acid) as well as by silencing the endogenous human CK2alpha catalytic subunit with small interfering RNA. However, 4,5,6,7-tetrabromobenzotriazole, another CK2-specific inhibitor, triggered the dissociation of purinosome clusters in HeLa cells. Although the mechanism by which 4,5,6,7-tetrabromobenzotriazole affects purinosome clustering is not clear, we were capable of chemically reversing purinosome formation in cells by the sequential addition of two CK2 inhibitors. Collectively, we provide compelling cellular evidence that CK2-mediated pathways reversibly regulate purinosome assembly, and thus the purinosome may be one of the ultimate targets of kinase inhibitors.

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection.

PubMed

Safranow, Krzysztof; Machoy, Zygmunt

2005-05-25

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.

Twenty-seven Years of Cerebral Pyruvate Recycling.

PubMed

Cerdán, Sebastián

2017-06-01

Cerebral pyruvate recycling is a metabolic pathway deriving carbon skeletons and reducing equivalents from mitochondrial oxaloacetate and malate, to the synthesis of mitochondrial and cytosolic pyruvate, lactate and alanine. The pathway allows both, to provide the tricarboxylic acid cycle with pyruvate molecules produced from alternative substrates to glucose and, to generate reducing equivalents necessary for the operation of NADPH requiring processes. At the cellular level, pyruvate recycling involves the activity of malic enzyme, or the combined activities of phosphoenolpyruvate carboxykinase and pyruvate kinase, as well as of those transporters of the inner mitochondrial membrane exchanging the corresponding intermediates. Its cellular localization between the neuronal or astrocytic compartments of the in vivo brain has been controversial, with evidences favoring either a primarily neuronal or glial localizations, more recently accepted to occur in both environments. This review provides a brief history on the detection and characterization of the pathway, its relations with the early developments of cerebral high resolution 13 C NMR, and its potential neuroprotective functions under hypoglycemic conditions or ischemic redox stress.

UNC-108/Rab2 Regulates Postendocytic Trafficking in Caenorhabditis elegans

PubMed Central

Chun, Denise K.; McEwen, Jason M.; Burbea, Michelle

2008-01-01

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking. PMID:18434599

An unusual mode of DNA duplex association: Watson-Crick interaction of all-purine deoxyribonucleic acids.

PubMed

Battersby, Thomas R; Albalos, Maria; Friesenhahn, Michel J

2007-05-01

Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N(3)-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

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

Pan, Huiling; Hu, Qian; Wang, Jingyuan

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanningmore » fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.« less

Methylated purines in urinary stones.

PubMed

Safranow, Krzysztof; Machoy, Zygmunt

2005-08-01

The aim of the study was to measure the content of methylated purines that appear as admixtures in uric acid stones. We analyzed urinary calculi from 48 residents of Western Pomerania who underwent surgery at the urology ward in Szczecin. Stone samples were dissolved in 0.1 mol/L NaOH. Extracts were diluted in 50 mmol/L KH(2)PO(4) and analyzed by reversed-phase HPLC with ultraviolet detection and use of a gradient of methanol concentration and pH. Uric acid was the main component of 9 stones. All 9 showed admixtures of 9 other purine derivatives: endogenous purine breakdown products (xanthine, hypoxanthine, and 2,8-dihydroxyadenine) and exogenous methyl derivatives of uric acid and xanthine (1-, 3-, and 7-methyluric acid; 1,3-dimethyluric acid; and 3- and 7-methylxanthine). Amounts of these purine derivatives ranged from the limit of detection to 12 mg/g of stone weight and showed a strong positive correlation (Spearman rank correlation coefficients, 0.63-0.94) with the uric acid content of the samples. The main methylated purine in the stones was 1-methyluric acid. Urinary purines at concentrations below their saturation limits may coprecipitate in samples supersaturated with uric acid and appear as admixtures in urinary stones. The amount of each purine depends on its average urinary excretion, similarity to the chemical structure of uric acid, and concentration of the latter in the stone. These findings suggest that purines in stones represent a substitutional solid solution with uric acid as solvent. Methylxanthines, which are ubiquitous components of the diet, drugs, and uric acid calculi, may be involved in the pathogenesis of urolithiasis.

A tiered approach to evaluate an iodine recycling inhibition adverse outcome pathway (AOP) in amphibians

EPA Science Inventory

The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces thyroid hormone synthesis, which leads to insufficiency in tissues and subsequent ne...

Hot shot induction and reperfusion with a specific blocker of the es-ENT1 nucleoside transporter before and after hypothermic cardioplegia abolishes myocardial stunning in acutely ischemic hearts despite metabolic derangement: Hot shot drug delivery before hypothermic cardioplegia

PubMed Central

Abd-Elfattah, Anwar Saad; Tuchy, Gert E.; Jessen, Michael E.; Salter, David R.; Goldstein, Jacques P.; Brunsting, Louis A.; Wechsler, Andrew S.

2013-01-01

Objective Simultaneous inhibition of the cardiac equilibrative-p-nitrobenzylthioinosine (NBMPR)–sensitive (es) type of the equilibrative nucleoside transport 1 (ENT1) nucleoside transporter, with NBMPR, and adenosine deaminase, with erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), prevents release of myocardial purines and attenuates myocardial stunning and fibrillation in canine models of warm ischemia and reperfusion. It is not known whether prolonged administration of hypothermic cardioplegia influences purine release and EHNA/NBMPR-mediated cardioprotection in acutely ischemic hearts. Methods Anesthetized dogs (n = 46), which underwent normothermic aortic crossclamping for 20 minutes on-pump, were divided to determine (1) purine release with induction of intermittent antegrade or continuous retrograde hypothermic cardioplegia and reperfusion, (2) the effects of postischemic treatment with 100 µM EHNA and 25 µM NBMPR on purine release and global functional recovery, and (3) whether a hot shot and reperfusion with EHNA/NBMPR inhibits purine release and attenuates ventricular dysfunction of ischemic hearts. Myocardial biopsies and coronary sinus effluents were obtained and analyzed using high-performance liquid chromatography. Results Warm ischemia depleted myocardial adenosine triphosphate and elevated purines (ie, inosine > adenosine) as markers of ischemia. Induction of intermittent antegrade or continuous retrograde hypothermic (4°C) cardioplegia releases purines until the heart becomes cold (<20°C). During reperfusion, the levels of hypoxanthine and xanthine (free radical substrates) were >90% of purines in coronary sinus effluent. Reperfusion with EHNA/NBMPR abolished ventricular dysfunction in acutely ischemic hearts with and without a hot shot and hypothermic cardioplegic arrest. Conclusions Induction of hypothermic cardioplegia releases purines from ischemic hearts until they become cold, whereas reperfusion induces massive purine release and myocardial stunning. Inhibition of cardiac es-ENT1 nucleoside transporter abolishes postischemic reperfusion injury in warm and cold cardiac surgery. PMID:23422047

Comparisons of four categories of waste recycling in China's paper industry based on physical input-output life-cycle assessment model.

PubMed

Liang, Sai; Zhang, Tianzhu; Xu, Yijian

2012-03-01

Waste recycling for paper production is an important component of waste management. This study constructs a physical input-output life-cycle assessment (PIO-LCA) model. The PIO-LCA model is used to investigate environmental impacts of four categories of waste recycling in China's paper industry: crop straws, bagasse, textile wastes and scrap paper. Crop straw recycling and wood utilization for paper production have small total intensity of environmental impacts. Moreover, environmental impacts reduction of crop straw recycling and wood utilization benefits the most from technology development. Thus, using crop straws and wood (including wood wastes) for paper production should be promoted. Technology development has small effects on environmental impacts reduction of bagasse recycling, textile waste recycling and scrap paper recycling. In addition, bagasse recycling and textile waste recycling have big total intensity of environmental impacts. Thus, the development of bagasse recycling and textile waste recycling should be properly limited. Other pathways for reusing bagasse and textile wastes should be explored and evaluated. Moreover, imports of scrap paper should be encouraged to reduce large indirect impacts of scrap paper recycling on domestic environment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.

PubMed

Yudowski, Guillermo A; Puthenveedu, Manojkumar A; Henry, Anastasia G; von Zastrow, Mark

2009-06-01

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (approximately twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.

A transthyretin-related protein is functionally expressed in Herbaspirillum seropedicae.

PubMed

Matiollo, Camila; Vernal, Javier; Ecco, Gabriela; Bertoldo, Jean Borges; Razzera, Guilherme; de Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

2009-10-02

Transthyretin-related proteins (TRPs) constitute a family of proteins structurally related to transthyretin (TTR) and are found in a large range of bacterial, fungal, plant, invertebrate, and vertebrate species. However, it was recently recognized that both prokaryotic and eukaryotic members of this family are not functionally related to transthyretins. TRPs are in fact involved in the purine catabolic pathway and function as hydroxyisourate hydrolases. An open reading frame encoding a protein similar to the Escherichia coli TRP was identified in Herbaspirillum seropedicae genome (Hs_TRP). It was cloned, overexpressed in E. coli, and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein, and circular dichroism spectrum indicated a predominance of beta-sheet structure, as expected for a TRP. We have demonstrated that Hs_TRP is a 5-hydroxyisourate hydrolase and by site-directed mutagenesis the importance of three conserved catalytic residues for Hs_TRP activity was further confirmed. The production of large quantities of this recombinant protein opens up the possibility of obtaining its 3D-structure and will help further investigations into purine catabolism.

Metabolic intervention to affect myocardial recovery following ischemia.

PubMed Central

Pasque, M K; Wechsler, A S

1984-01-01

Myocardial recovery during reperfusion following ischemia is critical to patient survival in a broad spectrum of clinical settings. Myocardial functional recovery following ischemia correlates well with recovery of myocardial adenosine triphosphate (ATP). Adenosine triphosphate recovery is uniformly incomplete during reperfusion following moderate ischemic injury and is therefore subject to manipulation by metabolic intervention. By definition ATP recovery is limited either by (1) energy availability and application in the phosphorylation of adenosine monophosphate (AMP) to ATP or (2) availability of AMP for this conversion. Experimental data suggest that substrate energy and the mechanisms required for its application in the creation of high energy phosphate bonds (AMP conversion to ATP) are more than adequate during reperfusion following moderate ischemic injury. Adenosine monophosphate availability, however, is inadequate following ischemia due to loss of diffusable adenine nucleotide purine metabolites. These purine precursors are necessary to fuel adenine nucleotide salvage pathways. Metabolic interventions that enhance AMP recovery rather than those that improve substrate energy availability during reperfusion are therefore recommended. The mechanisms of various metabolic interventions are discussed in this framework along with the rationale for or against their clinical application. PMID:6428332

Inner-shell chemical shift of DNA/RNA bases and inheritance from their parent purine and pyrimidine.

PubMed

Wang, Feng; Zhu, Quan; Ivanova, Elena

2008-11-01

Inner-shell electronic structures, properties and ionization spectra of DNA/RNA bases are studied with respect to their parent pyrimidine and purine species. Density functional theory B3LYP/aug-cc-pVTZ has been employed to produce the geometries of the bases, whereas LB94/et-pVQZ//B3LYP/aug-cc-pVTZ is used to calculate site-related Hirshfeld charges and core (vertical) ionization energies, as well as inner-shell spectra of C1s, N1s and O1s for DNA/RNA bases and their parent pyrimidine and purine species. The site-dependent variations of properties indicate the changes and inheritance of chemical environment when pyrimidine and purine become substituted. In general, although the changes are site-dependent, they are also ring-dependent. Pyrimidine bases change less significantly with respect to their parent pyrimidine than the purine bases with respect to their parent purine. Pyrimidine bases such as uracil, thymine and cytosine inherit certain properties from their parent pyrimidine, such as the Hirshfeld charge distributions and the order of core ionization energy level etc. No particular sites in the pyrimidine derivatives are engaged with a dramatic chemical shift nor with energy crossings to other sites. For the core shell spectra, the purine bases inherit very little from their parent purine, and guanine exhibits the least similarities to the parent among all the DNA/RNA bases.

Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11.

PubMed

Leiva, Natalia; Pavarotti, Martín; Colombo, María I; Damiani, María T

2006-06-10

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.

Uric acid induces hepatic steatosis by generation of mitochondrial oxidative stress: potential role in fructose-dependent and -independent fatty liver.

PubMed

Lanaspa, Miguel A; Sanchez-Lozada, Laura G; Choi, Yea-Jin; Cicerchi, Christina; Kanbay, Mehmet; Roncal-Jimenez, Carlos A; Ishimoto, Takuji; Li, Nanxing; Marek, George; Duranay, Murat; Schreiner, George; Rodriguez-Iturbe, Bernardo; Nakagawa, Takahiko; Kang, Duk-Hee; Sautin, Yuri Y; Johnson, Richard J

2012-11-23

Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. Rather than a consequence, uric acid induces fatty liver Hyperuricemic people are more prone to develop fructose-induced fatty liver. Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.

Intrinsic Apoptosis Pathway in Fallopian Tube Epithelial Cells Induced by Cladribine

PubMed Central

Chylińska-Wrzos, Patrycja; Lis-Sochocka, Marta; Bulak, Kamila; Jodłowska-Jędrych, Barbara

2014-01-01

Cladribine is a purine nucleoside analog which initiates the apoptotic mechanism within cells. Moreover, the available data confirms that cladribine, with the participation of the p53 protein, as well as the proapoptotic proteins from the Bcl-2 family, also induces the activation of the intrinsic apoptosis pathway. However, while there has been a lot of research devoted to the effect of cladribine on lymphatic system cells, little is known about the impact of cladribine on the reproductive system. The aim of our study was to evaluate apoptosis in oviduct epithelial cells sourced from 15 different female rats. In so doing, the sections were stained with caspases 3, 9, and 8. Results suggest that cladribine also induces apoptosis in the oviduct epithelial cells by way of the intrinsic pathway. Indeed, the discontinuing of the administration of cladribine leads to a reduction in the amount of apoptotic cells in the oviduct epithelium. PMID:25431797

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2001-07-03

The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Purine derivatives with antituberculosis activity

NASA Astrophysics Data System (ADS)

Gruzdev, D. A.; Musiyak, V. V.; Levit, G. L.; Krasnov, V. P.; Charushin, V. N.

2018-06-01

The review summarizes the data published over the last 10–15 years concerning the key groups of purine derivatives with antituberculosis activity. The structures of purines containing heteroatoms (S, O, N), fragments of heterocycles, amino acids and peptides, in the 6-position, as well as of purine nucleosides are presented. The possible targets for the action of such compounds and structure–activity relationship are discussed. Particular attention is paid to the most active compounds, which are of considerable interest as a basis for the development of efficient antituberculosis drugs. The bibliography includes 99 references.

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

Meadows, J.; Smith, R.C.

Uric acid has been proposed to be an important antioxidant and free radical scavenger in humans. Of the purine and pyrimidine compounds examined in this study, uric acid showed the greatest susceptibility to ozone-induced degradation. The parent compounds, purine and pyrimidine, were more resistant to ozonation than were the nucleobases. When the degradation of OH-substituted purines was examined, it was found that the more OH groups on the purine ring, the more readily the purine was degraded. Urea and allantoin were identified as degradation products of uric acid. The relative rates of nucleobase degradation in the presence and absence ofmore » uric acid were compared. Uric acid protected thymine, guanine, and uracil from degradation by ozone. In this system uric acid was found to protect the nucleobases as effectively as reduced glutathione.« less

Dietary purines in vegetarian meat analogues.

PubMed

Havlik, Jaroslav; Plachy, Vladimir; Fernandez, Javier; Rada, Vojtech

2010-11-01

The meat alternatives market offers a wide range of products resembling meat in taste, flavour or texture but based on vegetable protein sources. These high protein-low purine foods may find application in a low purine or purine-free diet, which is sometimes suggested for subjects with increased serum urate levels, i.e. hyperuricaemia. We determined purine content (uric acid, adenine, guanine, hypoxanthine, xanthine) in 39 commercially available meat substitutes and evaluated them in relation to their protein content. Some of the products contained a comparable sum of adenine and hypoxanthine per protein as meat. Analysis of variance showed an influence of protein source used. Mycoprotein-based products had significantly higher contents (2264 mg kg(-1)) of adenine and hypoxanthine per kg of 100% protein than soybean-based products (1648 mg kg(-1)) or mixtures consisting of soybean protein and wheat protein (1239 mg kg(-1)). Protein-rich vegetable-based meat substitutes might be generally accepted as meat alternatives for individuals on special diets. The type of protein used to manufacture these products determines the total content of purines, which is relatively higher in the case of mycoprotein or soybean protein, while appearing lower in wheat protein and egg white-based products. These are therefore more suitable for dietary considerations in a low-purine diet for hyperuricaemic subjects. 2010 Society of Chemical Industry

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

PubMed

Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Peptidoglycan turnover and recycling in Gram-positive bacteria.

PubMed

Reith, Jan; Mayer, Christoph

2011-10-01

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

Gene modification of the acetate biosynthesis pathway in Escherichia coli and implementation of the cell recycling technology to increase L-tryptophan production

PubMed Central

Bai, Fang; Chen, Ning; Bai, Gang

2017-01-01

The implementation of a novel cell recycling technology based on a special disk centrifuge during microbial fermentation process can continuously separate the product and harmful intermediates, while maintaining the cell viability owing to the installed cooling system. Acetate accumulation is an often encountered problem in L-tryptophan fermentation by Escherichia coli. To extend our previous studies, the current study deleted the key genes underlying acetate biosynthesis to improve l-tryptophan production. The deletion of the phosphotransacetylase (pta)–acetate kinase (ackA) pathway in a gltB (encoding glutamate synthase) mutant of E. coli TRTHB, led to the highest production of l-tryptophan (47.18 g/L) and glucose conversion rate (17.83%), with a marked reduction in acetate accumulation (1.22 g/L). This strain, TRTHBPA, was then used to investigate the effects of the cell recycling process on L-tryptophan fermentation. Four different strategies were developed concerning two issues, the volume ratio of the concentrated cell solution and clear solution and the cell recycling period. With strategy I (concentrated cell solution: clear solution, 1: 1; cell recycling within 24–30 h), L-tryptophan production and the glucose conversion rate increased to 55.12 g/L and 19.75%, respectively, 17.55% and 10.77% higher than those without the cell recycling. In addition, the biomass increased by 13.52% and the fermentation period was shortened from 40 h to 32 h. These results indicated that the cell recycling technology significantly improved L-tryptophan production by E. coli. PMID:28622378

Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling CompartmentD⃞

PubMed Central

Strick, David J.; Elferink, Lisa A.

2005-01-01

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways. PMID:16195351

Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

Synaptic vesicle recycling: steps and principles.

PubMed

Rizzoli, Silvio O

2014-04-16

Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Abdul-Zani, Izma; Wheatcroft, Stephen B.; Tomlinson, Darren C.; Harrison, Michael A.

2017-01-01

ABSTRACT Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. PMID:28798148

Determination of purine contents in different parts of pork and beef by high performance liquid chromatography.

PubMed

Rong, Shengzhong; Zou, Lina; Zhang, Yannan; Zhang, Guangteng; Li, Xiaoxia; Li, Miaojing; Yang, Fenghua; Li, Chunmei; He, Yingjuan; Guan, Hongjun; Guo, Yupeng; Wang, Dong; Cui, Xinyu; Ye, Hongting; Liu, Fenghai; Pan, Hongzhi; Yang, Yuexin

2015-03-01

Determination of adenine, hypoxanthine, guanine and xanthine in different parts of pork and beef using high performance liquid chromatography was described. Chromatographic separation was carried out on Waters Atlantis T3 column (4.6 mm × 250 mm × 5 μm) with column temperature at 30 °C. The mobile phase contained 99% 10.0 mmol/L ammonium formate solution at pH 3.6 and 1.0% methanol. Chromatography was achieved at a flow rate of 1.0 mL/min and detection wavelength at 254 nm. The results indicated that total purine amounts in pork rump and beef sirloin were higher than those in other parts (P<0.05). The principal purine bases were hypoxanthine and adenine, and hypoxanthine content was the most highest in all samples (P<0.05). As pork rump and beef sirloin contain considerable amounts of total purine and uricogenic purine base, we suggest that excess consumption of them be avoid, whereas pork loin chop and beef rib eye are more suitable for a low-purine diet. Copyright © 2014 Elsevier Ltd. All rights reserved.

GGA3 Functions as a Switch to Promote Met Receptor Recycling, Essential for Sustained ERK and Cell Migration

PubMed Central

Parachoniak, Christine Anna; Luo, Yi; Abella, Jasmine Vanessa; Keen, James H.; Park, Morag

2011-01-01

Summary Cells are dependent on correct sorting of activated receptor tyrosine kinases (RTKs) for the outcome of growth factor signaling. Upon activation, RTKs are coupled through the endocytic machinery for degradation, or recycled to the cell surface. However, the molecular mechanisms governing RTK recycling are poorly understood. Here, we show that Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3) interacts selectively with the Met/Hepatocyte Growth Factor RTK when stimulated, to sort it for recycling in association with “gyrating”-clathrin. GGA3 loss abrogates Met recycling from a Rab4 endosomal subdomain, resulting in pronounced trafficking of Met towards degradation. Decreased Met recycling attenuates ERK activation and cell migration. Met recycling, sustained ERK activation and migration require interaction of GGA3 with Arf6 and an unexpected association with the Crk adaptor. The data show that GGA3 defines an active recycling pathway and support a broader role for GGA3-mediated cargo selection in targeting receptors destined for recycling. PMID:21664574

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

PubMed Central

Simm, Roger; Torgersen, Maria Lyngaas; Sandvig, Kirsten

2015-01-01

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus. PMID:26017782

(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.

PubMed

Gebril, Hoda M; Avula, Bharathi; Wang, Yan-Hong; Khan, Ikhlas A; Jekabsons, Mika B

2016-02-01

Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

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

Taylor,E.; Rinaldo-Matthis, A.; Li, L.

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast formore » AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.« less

Study on the activity of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and molecular docking

NASA Astrophysics Data System (ADS)

Li, Peizhen; Tian, Yueli; Zhai, Honglin; Deng, Fangfang; Xie, Meihong; Zhang, Xiaoyun

2013-11-01

Non-purine derivatives have been shown to be promising novel drug candidates as xanthine oxidase inhibitors. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) methods including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), two 3D-QSAR models for a series of non-purine xanthine oxidase (XO) inhibitors were established, and their reliability was supported by statistical parameters. Combined 3D-QSAR modeling and the results of molecular docking between non-purine xanthine oxidase inhibitors and XO, the main factors that influenced activity of inhibitors were investigated, and the obtained results could explain known experimental facts. Furthermore, several new potential inhibitors with higher activity predicted were designed, which based on our analyses, and were supported by the simulation of molecular docking. This study provided some useful information for the development of non-purine xanthine oxidase inhibitors with novel structures.

Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

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

Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.

We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPsmore » are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.« less

Ligand-activated epidermal growth factor receptor (EGFR) signaling governs endocytic trafficking of unliganded receptor monomers by non-canonical phosphorylation.

PubMed

Tanaka, Tomohiro; Zhou, Yue; Ozawa, Tatsuhiko; Okizono, Ryuya; Banba, Ayako; Yamamura, Tomohiro; Oga, Eiji; Muraguchi, Atsushi; Sakurai, Hiroaki

2018-02-16

The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand lead to a higher proportion of receptor that is recycled and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no connections have been made between these pathways; i.e. it has yet to be established what happens to unbound receptors following stimulation with ligand. Here we demonstrate that a minimal level of activation of epidermal growth factor receptor (EGFR) tyrosine kinase by low levels of ligand is sufficient to fully activate downstream mitogen-activated protein kinase (MAPK) pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated mitogen-activated protein kinase. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser-1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

EARP, a multisubunit tethering complex involved in endocytic recycling

PubMed Central

Schindler, Christina; Chen, Yu; Pu, Jing; Guo, Xiaoli; Bonifacino, Juan S.

2015-01-01

Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named Endosome-Associated Recycling Protein (EARP) that is structurally related to the previously described Golgi-Associated Retrograde Protein (GARP) complex. Both complexes share the Ang2, Vps52 and Vps53 subunits, but EARP comprises an uncharacterized protein, Syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target-SNARE Syntaxin 6 and various cognate SNAREs. Depletion of Syndetin or Syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling. PMID:25799061

Regulation of the sphingosine-recycling pathway for ceramide generation by oxidative stress, and its role in controlling c-Myc/Max function

PubMed Central

Sultan, Iyad; Senkal, Can E.; Ponnusamy, Suriyan; Bielawski, Jacek; Szulc, Zdzislaw; Bielawska, Alicja; Hannun, Yusuf A.; Ogretmen, Besim

2005-01-01

In the present study, the regulation of the sphingosine-recycling pathway in A549 human lung adenocarcinoma cells by oxidative stress was investigated. The generation of endogenous long-chain ceramide in response to exogenous C6-cer (C6-ceramide), which is FB1 (fumonisin B1)-sensitive, was employed to probe the sphingosine-recycling pathway. The data showed that ceramide formation via this pathway was significantly blocked by GSH and NAC (N-acetylcysteine) whereas it was enhanced by H2O2, as detected by both palmitate labelling and HPLC/MS. Similar data were also obtained using a novel approach that measures the incorporation of 17Sph (sphingosine containing 17 carbons) of 17C6-cer (C6-cer containing a 17Sph backbone) into long-chain 17C16-cer in cells by HPLC/MS, which was significantly decreased and increased in response to GSH and H2O2 respectively. TNF (tumour necrosis factor)-α, which decreases the levels of endogenous GSH, increased the generation of C16-cer in response to C6-cer, and this was blocked by exogenous GSH or NAC, or by the overexpression of TPx I (thioredoxin peroxidase I), an enzyme that reduces the generation of intracellular ROS (reactive oxygen species). Additional data showed that ROS regulated both the deacylation and reacylation steps of C6-cer. At a functional level, C6-cer inhibited the DNA-binding function of the c-Myc/Max oncogene. Inhibition of the generation of longchain ceramide in response to C6-cer by FB1 or NAC significantly blocked the modulation of the c-Myc/Max function. These data demonstrate that the sphingosine-recycling pathway for the generation of endogenous long-chain ceramide in response to exogenous C6-cer is regulated by ROS, and plays an important biological role in controlling c-Myc function. PMID:16201965

Exploring metabolic pathway disruption in the subchronic phencyclidine model of schizophrenia with the Generalized Singular Value Decomposition

PubMed Central

2011-01-01

Background The quantification of experimentally-induced alterations in biological pathways remains a major challenge in systems biology. One example of this is the quantitative characterization of alterations in defined, established metabolic pathways from complex metabolomic data. At present, the disruption of a given metabolic pathway is inferred from metabolomic data by observing an alteration in the level of one or more individual metabolites present within that pathway. Not only is this approach open to subjectivity, as metabolites participate in multiple pathways, but it also ignores useful information available through the pairwise correlations between metabolites. This extra information may be incorporated using a higher-level approach that looks for alterations between a pair of correlation networks. In this way experimentally-induced alterations in metabolic pathways can be quantitatively defined by characterizing group differences in metabolite clustering. Taking this approach increases the objectivity of interpreting alterations in metabolic pathways from metabolomic data. Results We present and justify a new technique for comparing pairs of networks--in our case these networks are based on the same set of nodes and there are two distinct types of weighted edges. The algorithm is based on the Generalized Singular Value Decomposition (GSVD), which may be regarded as an extension of Principle Components Analysis to the case of two data sets. We show how the GSVD can be interpreted as a technique for reordering the two networks in order to reveal clusters that are exclusive to only one. Here we apply this algorithm to a new set of metabolomic data from the prefrontal cortex (PFC) of a translational model relevant to schizophrenia, rats treated subchronically with the N-methyl-D-Aspartic acid (NMDA) receptor antagonist phencyclidine (PCP). This provides us with a means to quantify which predefined metabolic pathways (Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolite pathway database) were altered in the PFC of PCP-treated rats. Several significant changes were discovered, notably: 1) neuroactive ligands active at glutamate and GABA receptors are disrupted in the PFC of PCP-treated animals, 2) glutamate dysfunction in these animals was not limited to compromised glutamatergic neurotransmission but also involves the disruption of metabolic pathways linked to glutamate; and 3) a specific series of purine reactions Xanthine ← Hypoxyanthine ↔ Inosine ← IMP → adenylosuccinate is also disrupted in the PFC of PCP-treated animals. Conclusions Network reordering via the GSVD provides a means to discover statistically validated differences in clustering between a pair of networks. In practice this analytical approach, when applied to metabolomic data, allows us to quantify the alterations in metabolic pathways between two experimental groups. With this new computational technique we identified metabolic pathway alterations that are consistent with known results. Furthermore, we discovered disruption in a novel series of purine reactions that may contribute to the PFC dysfunction and cognitive deficits seen in schizophrenia. PMID:21575198

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)

Raman Spectroscopy of the Interferon-Induced 2’,5’-Oligoadenylates

DTIC Science & Technology

1987-06-25

generation of the Raman spectrum of triethyl ammonium ion ••••••••••••••••••••••••••••••• 41 12. structures of purine, adenine, purine riboside , adenosine...ribose 5 1-phosphate, AMP, and ATP........ 48 13. Raman spectra of adenine and purine •••••••.••••••••• 49 14. Raman spectra of purine riboside and... nicotinamide adenine dinucleotide; TFAB, triethyl anunonium bicarbonate; TFA, triethyl amm::mium. ion; CD circular _dichroism; NMR, nuclear magnetic

Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

PubMed

Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

2013-05-01

P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

A novel requirement for C. elegans Alix/ALX-1 in RME-1 mediated membrane transport

PubMed Central

Shi, Anbing; Pant, Saumya; Balklava, Zita; Chen, Carlos Chih-Hsiung; Figueroa, Vanesa; Grant, Barth D.

2007-01-01

Summary Background Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition Alix is associated with the actin cytoskeleton and may regulate cytoskeletal dynamics. Results Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane called RME-1. Analysis of alx-1 mutants indicates that ALX-1 is required for endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by analysis of rme-1 mutants. Expression of truncated human Alix in HeLa cells disrupts recycling of MHCI, a known Ehd1/RME-1 dependent transport step, suggesting phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears dispensable for ALX-1 function in MVEs/late endosomes. Conclusions This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1. PMID:17997305

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.

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

PubMed Central

Cadet, Jean; Wagner, J. Richard; Shafirovich, Vladimir; Geacintov, Nicholas E.

2014-01-01

Purpose The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. Conclusion There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation. PMID:24369822

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA.

PubMed

Cadet, Jean; Wagner, J Richard; Shafirovich, Vladimir; Geacintov, Nicholas E

2014-06-01

The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

The Last Enzyme of the De Novo Purine Synthesis Pathway 5-aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC) Plays a Central Role in Insulin Signaling and the Golgi/Endosomes Protein Network*

PubMed Central

Boutchueng-Djidjou, Martial; Collard-Simard, Gabriel; Fortier, Suzanne; Hébert, Sébastien S.; Kelly, Isabelle; Landry, Christian R.; Faure, Robert L.

2015-01-01

Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome hepatic protein fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with insulin receptor (IR) internalization. The IR interactome (IRGEN) connects ATIC to AMPK within the Golgi/endosome protein network (GEN). Forty-five percent of the IR Golgi/endosome protein network have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalized within the plasma membrane (PM) and Golgi/endosome fractions after insulin stimulation and that ATIC later accumulates in the Golgi/endosome fraction. Using an in vitro reconstitution system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPK-Thr172 phosphorylation in IR complexes. We observed that IR internalization was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signaling mechanism that senses adenylate synthesis, ATP levels, and IR activation states and that acts in regulating IR autophosphorylation and endocytosis. PMID:25687571

Metabolites Associated With Malnutrition in the Intensive Care Unit Are Also Associated With 28-Day Mortality.

PubMed

Mogensen, Kris M; Lasky-Su, Jessica; Rogers, Angela J; Baron, Rebecca M; Fredenburgh, Laura E; Rawn, James; Robinson, Malcolm K; Massarro, Anthony; Choi, Augustine M K; Christopher, Kenneth B

2017-02-01

We hypothesized that metabolic profiles would differ in critically ill patients with malnutrition relative to those without. We performed a prospective cohort study on 85 adult patients with systemic inflammatory response syndrome or sepsis admitted to a 20-bed medical intensive care unit (ICU) in Boston. We generated metabolomic profiles using gas and liquid chromatography and mass spectroscopy. We followed this by logistic regression and partial least squares discriminant analysis to identify individual metabolites that were significant. We then interrogated the entire metabolomics profile using metabolite set enrichment analysis and network model construction of chemical-protein target interactions to identify groups of metabolites and pathways that were differentiates in patients with and without malnutrition. Of the cohort, 38% were malnourished at admission to the ICU. Metabolomic profiles differed in critically ill patients with malnutrition relative to those without. Ten metabolites were significantly associated with malnutrition ( P < .05). A parsimonious model of 5 metabolites effectively differentiated patients with malnutrition (AUC = 0.76), including pyroglutamine and hypoxanthine. Using pathway enrichment analysis, we identified a critical role of glutathione and purine metabolism in predicting nutrition. Nutrition status was associated with 28-day mortality, even after adjustment for known phenotypic variables associated with ICU mortality. Importantly, 7 metabolites associated with nutrition status were also associated with 28-day mortality. Malnutrition is associated with differential metabolic profiles early in critical illness. Common to all of our metabolome analyses, glutathione and purine metabolism, which play principal roles in cellular redox regulation and accelerated tissue adenosine triphosphate degradation, respectively, were significantly altered with malnutrition.

The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling

PubMed Central

Cruse, Glenn; Beaven, Michael A.; Music, Stephen C.; Bradding, Peter; Gilfillan, Alasdair M.; Metcalfe, Dean D.

2015-01-01

MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1–enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases. PMID:25717186

Comparisons of four categories of waste recycling in China's paper industry based on physical input-output life-cycle assessment model

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

Liang Sai; Zhang, Tianzhu, E-mail: zhangtz@mail.tsinghua.edu.cn; Xu Yijian

Highlights: Black-Right-Pointing-Pointer Using crop straws and wood wastes for paper production should be promoted. Black-Right-Pointing-Pointer Bagasse and textile waste recycling should be properly limited. Black-Right-Pointing-Pointer Imports of scrap paper should be encouraged. Black-Right-Pointing-Pointer Sensitivity analysis, uncertainties and policy implications are discussed. - Abstract: Waste recycling for paper production is an important component of waste management. This study constructs a physical input-output life-cycle assessment (PIO-LCA) model. The PIO-LCA model is used to investigate environmental impacts of four categories of waste recycling in China's paper industry: crop straws, bagasse, textile wastes and scrap paper. Crop straw recycling and wood utilization for papermore » production have small total intensity of environmental impacts. Moreover, environmental impacts reduction of crop straw recycling and wood utilization benefits the most from technology development. Thus, using crop straws and wood (including wood wastes) for paper production should be promoted. Technology development has small effects on environmental impacts reduction of bagasse recycling, textile waste recycling and scrap paper recycling. In addition, bagasse recycling and textile waste recycling have big total intensity of environmental impacts. Thus, the development of bagasse recycling and textile waste recycling should be properly limited. Other pathways for reusing bagasse and textile wastes should be explored and evaluated. Moreover, imports of scrap paper should be encouraged to reduce large indirect impacts of scrap paper recycling on domestic environment.« less

Synthesis of Purine Nucleoside and Nucleotide Analogs as Antiparasitic Agents.

DTIC Science & Technology

1979-09-01

was to conduct studies on the synthesis of purine nucleoside and nucleotide analogs as anti- parasitic agents. The primary target compounds were 5...antiparasitic agents. - Jaffe has proposed that the susceptibility of pathogenic helminths and protozoa to fraudulent purine, in contrast to pyrimidine...8217-substituted derivatives are thus designed to inhibit nucleoside and nucleotide kinases as well as other parasitic enzymes. Mammalian cells, onthe

Metabonomics revealed xanthine oxidase-induced oxidative stress and inflammation in the pathogenesis of diabetic nephropathy.

PubMed

Liu, Jingping; Wang, Chengshi; Liu, Fang; Lu, Yanrong; Cheng, Jingqiu

2015-03-01

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM), which is a major public health problem in the world. To reveal the metabolic changes associated with DN, we analyzed the serum, urine, and renal extracts obtained from control and streptozotocin (STZ)-induced DN rats by (1)H NMR-based metabonomics and multivariate data analysis. A significant difference between control and DN rats was revealed in metabolic profiles, and we identified several important DN-related metabolites including increased levels of allantoin and uric acid (UA) in the DN rats, suggesting that disturbed purine metabolism may be involved in the DN. Combined with conventional histological and biological methods, we further demonstrated that xanthine oxidase (XO), a key enzyme for purine catabolism, was abnormally activated in the kidney of diabetic rats by hyperglycemia. The highly activated XO increased the level of intracellular ROS, which caused renal injury by direct oxidative damage to renal cells, and indirect inducing inflammatory responses via activating NF-κB signaling pathway. Our study highlighted that metabonomics is a promising tool to reveal the metabolic changes and the underlying mechanism involved in the pathogenesis of DN.

Metabolomics Analysis of Human Vitreous in Diabetic Retinopathy and Rhegmatogenous Retinal Detachment.

PubMed

Haines, Nathan R; Manoharan, Niranjan; Olson, Jeffrey L; D'Alessandro, Angelo; Reisz, Julie A

2018-06-19

The vitreous humor is a highly aqueous eye fluid interfacing with the retina and lens and providing shape. Its molecular composition provides a readout for the eye's physiological status. Changes in cellular metabolism underlie vitreoretinal pathologies, but despite routine surgical collection of vitreous, only limited reports of metabolism in the vitreous of human patients have been described. Vitreous samples from patients with rhegmatogenous retinal detachment ( n = 25) and proliferative diabetic retinopathy ( n = 9) were profiled along with control human vitreous samples ( n = 8) by untargeted mass-spectrometry-based metabolomics. Profound changes were observed in diabetic retinopathy vitreous, including altered glucose metabolism and activation of the pentose phosphate pathway, which provides reducing equivalents to counter oxidative stress. In addition, purine metabolism was altered in diabetic retinopathy, with decreased xanthine and elevated levels of related purines (inosine, hypoxanthine, urate, allantoate) generated in oxidant-producing reactions. In contrast, the vitreous metabolite profiles of retinal detachment patients were similar to controls. In total, our results suggest a rewiring of vitreous metabolism in diabetic retinopathy that underlies disease features such as oxidative stress and furthermore illustrates how the vitreous metabolic profile may be impacted by disease.

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling.

PubMed

Chen, Christine Y; Balch, William E

2006-08-01

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

Synaptic vesicle recycling: steps and principles

PubMed Central

Rizzoli, Silvio O

2014-01-01

Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle. PMID:24596248

Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

PubMed Central

Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

2015-01-01

Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

PubMed

Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

2015-01-01

Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

PubMed

Frank, K B; Cheng, Y C

1986-02-05

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

The electrochemical properties of the purine bases : at the interface between biological conjugates to inorganic surfaces

NASA Technical Reports Server (NTRS)

Hays, Charles C.

2003-01-01

The study of the charge transfer and interfacial reactions of the purine bases in physiological solutions provides valuable knowledge, as these processes are relevant to the origins of life. It has been proposed that the adsorption of the adsorption of the purine bases on an inorganic surface could serve as a template for specifying the arrangement of amino acids in peptides.

Isolation of Purines and Pyrimidines from the Murchison Meteorite

NASA Technical Reports Server (NTRS)

Glavin, D. P.; Bada, J. K.

2003-01-01

The origin of life on Earth, and possibly on other planets such as Mars, would have required the presence of liquid water and a continuous supply of prebiotic organic compounds. The delivery of organic matter by asteroids, comets, and carbonaceous meteorites could have contributed to the early Earth's prebiotic inventory by seeding the planet with biologically important organic compounds. A wide variety of prebiotic organic compounds have previously been detected in the Murchison CM type carbonaceous chondrite including amino acids, purines and pyrimidines'. These compounds play a major role in terrestrial biochemistry and are integral components of proteins, DNA and RNA. In this study we developed a new extraction technique using sublimation in order to isolate purines and pyrimidines from Murchison2, which is cleaner and more time efficient that traditional methods3. Several purines including adenine, guanine, hypoxanthine and xanthine were positively identified by high performance liquid chromatography and ultraviolet absorption detection in our Murchison extracts. The purines detected in Murchison do not correlate with the distribution of nucleobases found in geological environments on Earth4. Moreover, the abundance of extraterrestrial amino acids and the low level of terrestrial amino acid contaminants found in Murchison', support the idea that the purines in t h s meteorite are extraterrestrial in origin.

Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway

PubMed Central

Das, Utpal; Scott, David; Ganguly, Archan; Koo, Edward H.; Tang, Yong; Roy, Subhojit

2013-01-01

The convergence of APP (substrate) and BACE-1 (enzyme) is a rate-limiting, obligatory event triggering the amyloidogenic pathway – a key step in Alzheimer’s disease (AD) pathology. However, as both APP/BACE-1 are highly expressed in brain, mechanisms precluding their unabated convergence are unclear. Exploring dynamic localization of APP/BACE-1 in cultured hippocampal neurons, we found that after synthesis via the secretory-pathway, dendritic APP/BACE-1-containing vesicles are largely segregated in physiologic states. While BACE-1 is largely sorted into acidic recycling endosomes, APP is conveyed in Golgi-derived vesicles. However upon activity-induction – a known trigger of the amyloidogenic pathway – APP is routed into BACE-1-positive recycling endosomes via a clathrin-dependent mechanism. A partitioning/convergence of APP/BACE-1 vesicles is also apparent in control/AD brains respectively. Considering BACE-1 is optimally active in an acidic environment, our experiments suggest that neurons have evolved trafficking strategies that normally limit APP/BACE-1 proximity; and also uncover a pathway routing APP into BACE-1-containing organelles – triggering amyloidogenesis. PMID:23931995

Responses of Adenine Nucleotides in Germinating Soybean Embryonic Axes to Exogenously Applied Adenine and Adenosine

PubMed Central

Anderson, James D.

1977-01-01

The ATP content of soybean (Glycine max [L.] Merr. cv. Kent) axes incubated for 3 hours in 1 mm solutions of adenine and adenosine increased over 100% and 75%, respectively, over axes incubated in water. The increase in ATP was primarily due to the conversion of these purines to nucleotides via the nucleotide salvage pathway. The ATP formed was in a metabolically active pool because label from adenine was incorporated into acid-insoluble material. Adenine also increased the levels of GTP, UTP, and CTP, but not to the extent of the ATP level. PMID:16660165

Inhibition of Lysosome Membrane Recycling Causes Accumulation of Gangliosides that Contribute to Neurodegeneration.

PubMed

Boutry, Maxime; Branchu, Julien; Lustremant, Céline; Pujol, Claire; Pernelle, Julie; Matusiak, Raphaël; Seyer, Alexandre; Poirel, Marion; Chu-Van, Emeline; Pierga, Alexandre; Dobrenis, Kostantin; Puech, Jean-Philippe; Caillaud, Catherine; Durr, Alexandra; Brice, Alexis; Colsch, Benoit; Mochel, Fanny; El Hachimi, Khalid Hamid; Stevanin, Giovanni; Darios, Frédéric

2018-06-26

Lysosome membrane recycling occurs at the end of the autophagic pathway and requires proteins that are mostly encoded by genes mutated in neurodegenerative diseases. However, its implication in neuronal death is still unclear. Here, we show that spatacsin, which is required for lysosome recycling and whose loss of function leads to hereditary spastic paraplegia 11 (SPG11), promotes clearance of gangliosides from lysosomes in mouse and human SPG11 models. We demonstrate that spatacsin acts downstream of clathrin and recruits dynamin to allow lysosome membrane recycling and clearance of gangliosides from lysosomes. Gangliosides contributed to the accumulation of autophagy markers in lysosomes and to neuronal death. In contrast, decreasing ganglioside synthesis prevented neurodegeneration and improved motor phenotype in a SPG11 zebrafish model. Our work reveals how inhibition of lysosome membrane recycling leads to the deleterious accumulation of gangliosides, linking lysosome recycling to neurodegeneration. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Cell-autonomous regulation of Mu-opioid receptor recycling by substance P.

PubMed

Bowman, Shanna L; Soohoo, Amanda L; Shiwarski, Daniel J; Schulz, Stefan; Pradhan, Amynah A; Puthenveedu, Manojkumar A

2015-03-24

How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropeptide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

Cell-Autonomous Regulation of Mu-Opioid Receptor Recycling by Substance P

PubMed Central

Bowman, Shanna L.; Soohoo, Amanda L.; Shiwarski, Daniel J.; Schulz, Stefan; Pradhan, Amynah A.; Puthenveedu, Manojkumar A.

2015-01-01

SUMMARY How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropep-tide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the muopioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeo-static interaction between the pain and analgesic systems. PMID:25801029

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

PubMed

Strachan, Lauren R; Condic, Maureen L

2004-11-08

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Recycling potential of neodymium: the case of computer hard disk drives.

PubMed

Sprecher, Benjamin; Kleijn, Rene; Kramer, Gert Jan

2014-08-19

Neodymium, one of the more critically scarce rare earth metals, is often used in sustainable technologies. In this study, we investigate the potential contribution of neodymium recycling to reducing scarcity in supply, with a case study on computer hard disk drives (HDDs). We first review the literature on neodymium production and recycling potential. From this review, we find that recycling of computer HDDs is currently the most feasible pathway toward large-scale recycling of neodymium, even though HDDs do not represent the largest application of neodymium. We then use a combination of dynamic modeling and empirical experiments to conclude that within the application of NdFeB magnets for HDDs, the potential for loop-closing is significant: up to 57% in 2017. However, compared to the total NdFeB production capacity, the recovery potential from HDDs is relatively small (in the 1-3% range). The distributed nature of neodymium poses a significant challenge for recycling of neodymium.

Qishen Yiqi Drop Pill improves cardiac function after myocardial ischemia.

PubMed

JianXin, Chen; Xue, Xu; ZhongFeng, Li; Kuo, Gao; FeiLong, Zhang; ZhiHong, Li; Xian, Wang; HongCai, Shang

2016-04-14

Myocardial ischemia (MI) is one of the leading causes of death, while Qishen Yiqi Drop Pill (QYDP) is a representative traditional Chinese medicine to treat this disease. Unveiling the pharmacological mechanism of QYDP will provide a great opportunity to promote the development of novel drugs to treat MI. 64 male Sprague-Dawley (SD) rats were divided into four groups: MI model group, sham operation group, QYDP treatment group and Fosinopril treatment group. Echocardiography results showed that QYDP exhibited significantly larger LV end-diastolic dimension (LVEDd) and LV end-systolic dimension (LVEDs), compared with the MI model group, indicating the improved cardiac function by QYDP. (1)H-NMR based metabonomics further identify 9 significantly changed metabolites in the QYDP treatment group, and the QYDP-related proteins based on the protein-metabolite interaction networks and the corresponding pathways were explored, involving the pyruvate metabolism pathway, the retinol metabolism pathway, the tyrosine metabolism pathway and the purine metabolism pathway, suggesting that QYDP was closely associated with blood circulation. ELISA tests were further employed to identify NO synthase (iNOS) and cathepsin K (CTSK) in the networks. For the first time, our work combined experimental and computational methods to study the mechanism of the formula of traditional Chinese medicine.

One-Carbon Metabolism in Health and Disease

PubMed Central

Ducker, Gregory S.; Rabinowitz, Joshua D.

2017-01-01

One-carbon (1C) metabolism, mediated by the folate cofactor, supports multiple physiological processes. These include biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), epigenetic maintenance, and redox defense. Both within eukaryotic cells and across organs, 1C metabolic reactions are compartmentalized. Here we review the fundamentals of mammalian 1C metabolism, including the pathways active in different compartments, cell types, and biological states. Emphasis is given to recent discoveries enabled by modern genetics, analytical chemistry, and isotope tracing. An emerging theme is the biological importance of mitochondrial 1C reactions, both for producing 1C units that are exported to the cytosol and for making additional products, including glycine and NADPH. Increased clarity regarding differential folate pathway usage in cancer, stem cells, development, and adult physiology is reviewed and highlights new opportunities for selective therapeutic intervention. PMID:27641100

Visualizing the Search for Radiation-damaged DNA Bases in Real Time.

PubMed

Lee, Andrea J; Wallace, Susan S

2016-11-01

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

Visualizing the search for radiation-damaged DNA bases in real time

NASA Astrophysics Data System (ADS)

Lee, Andrea J.; Wallace, Susan S.

2016-11-01

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

PubMed Central

Sharma, Vimal Kumar; Jelen, Frantisek; Trnkova, Libuse

2015-01-01

Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications. PMID:25594595

Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

PubMed Central

Bowen, Aaron B; Bourke, Ashley M; Hiester, Brian G; Hanus, Cyril

2017-01-01

Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network. PMID:28875935

[Pharmacodynamic evaluation and molecular mechanism research of Huanshao capsule on irregular menstruation].

PubMed

Sun, Jian-Hui; Huo, Hai-Ru; Li, Xiao-Qin; Li, Hong-Mei; Qin, De-Huai; Wu, Chun

2018-04-01

Huanshao capsule is widely used in irregular menstruation and has achieved a good effect. Huanshao capsule can promote gonad development in mice, significantly improve the ovarian index in mice, increase estrogen level and reduce FSH level in rats, inhibit the pain response induced by oxytocin and estrogen, inhibit writhing reaction induced by acetic acid pain in mice. Due to the complexity of traditional Chinese medical formula, the pharmacological mechanism of the treatment on the irregular menstruation of the Huanshao capsule is unclear. In this study, the internet-based computation platform (www.tcmip.cn）was used to explore the molecular mechanism of Huanshao capsule on the menstrual. The aim of this study was to find the molecular mechanism of Huanshao capsule in treating menstrual. In the study of the molecular mechanism of Huanshao capsule in the treatment of menstrual by using the internet-based computation platform, Huanshao capsule maybe treat the menstrual by the pathway of endocrine system, GnRH signal transduction pathway, estrogen signal transduction pathway, oxytocin signaling pathway, thyroid hormone signaling pathway, VEGF signaling pathway, FCεRI signaling pathway and purine metabolism and nucleotide metabolism. The early pharmacological study confirmed Huanshao capsule could increase the serum estradiol level and decrease follicle stimulating hormone level and the traditional Chinese medicine pharmacology coincide with the prediction result of internet-based computation platform which roles as the pathway of GnRH signaling pathway and estrogen signal transduction pathway. Other pathway needs further experimental verification. Copyright© by the Chinese Pharmaceutical Association.

COPI selectively drives maturation of the early Golgi

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

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

COPI selectively drives maturation of the early Golgi

DOE PAGES

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham; ...

2015-12-28

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

Radical-induced purine lesion formation is dependent on DNA helical topology.

PubMed

Terzidis, Michael A; Prisecaru, Andreea; Molphy, Zara; Barron, Niall; Randazzo, Antonio; Dumont, Elise; Krokidis, Marios G; Kellett, Andrew; Chatgilialoglu, Chryssostomos

2016-11-01

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO • ) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5',8-cyclo-2'-deoxyguanosine (5',8-cdG) and 5',8-cyclo-2'-deoxyadenosine (5',8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT] 4 (TG4T), d[AGGG(TTAGGG) 3 ] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG) 3 A] (mutTel24) were exposed to HO • radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ≫ SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (∼50% solution folded) and mutTel24 (∼90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33 Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.

Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae.

PubMed

Xia, Peng-Fei; Zhang, Guo-Chang; Walker, Berkley; Seo, Seung-Oh; Kwak, Suryang; Liu, Jing-Jing; Kim, Heejin; Ort, Donald R; Wang, Shu-Guang; Jin, Yong-Su

2017-02-17

Global climate change caused by the emission of anthropogenic greenhouse gases (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and phosphoribulokinase from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with the synthetic rPPP was able to exhibit a higher yield of ethanol and lower yields of byproducts (xylitol and glycerol) than a control strain. In addition, the reduced release of carbon dioxide by the engineered strain was observed during xylose fermentation, suggesting that the carbon dioxide generated by pyruvate decarboxylase was partially reassimilated through the synthetic rPPP. These results demonstrated that recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative metabolic pathway. This strategy has a great potential to alleviate GHG emissions during the production of second-generation ethanol.

Reactions of Trimethylsilyl Fluorosulfonyldifluoroacetate with Purine and Pyrimidine Nucleosides

PubMed Central

Rapp, Magdalena; Cai, Xiaohong; Xu, Wei; Dolbier, William R.; Wnuk, Stanislaw F.

2008-01-01

Difluorocarbene, generated from trimethylsilyl fluorosulfonyldifluoroacetate (TFDA), reacts with the uridine and adenosine substrates preferentially at the enolizable amide moiety of the uracil ring and the 6-amino group of the purine ring. 2',3'-Di-O-acetyl-3'-deoxy-3'-methyleneuridine reacts with TFDA to produce 4-O-difluoromethyl product derived from an insertion of difluorocarbene into the 4-hydroxyl group of the enolizable uracil ring. Reaction of the difluorocarbene with the adenosine substrates having the unprotected 6-amino group in the purine ring produced the 6-N-difluoromethyl derivative, while reaction with 6-N-benzoyl protected adenosine analogues gave the difluoromethyl ether product derived from the insertion of difluorocarbene into the enol form of the 6-benzamido group. Treatment of the 6-N-phthaloyl protected adenosine analogues with TFDA resulted in the unexpected one-pot conversion of the imidazole ring of the purine into the corresponding N-difluoromethylthiourea derivatives. Treatment of the suitably protected pyrimidine and purine nucleosides bearing an exomethylene group at carbons 2', 3' or 4' of the sugar rings with TFDA afforded the corresponding spirodifluorocyclopropyl analogues but in low yields. PMID:20160856

Structure and Electronic Spectra of Purine-Methyl Viologen Charge Transfer Complexes

PubMed Central

Jalilov, Almaz S.; Patwardhan, Sameer; Singh, Arunoday; Simeon, Tomekia; Sarjeant, Amy A.; Schatz, George C.; Lewis, Frederick D.

2014-01-01

The structure and properties of the electron donor-acceptor complexes formed between methyl viologen (MV) and purine nucleosides and nucleotides in water and the solid state have been investigated using a combination of experimental and theoretical methods. Solution studies were performed using UV-vis and 1H NMR spectroscopy. Theoretical calculations were performed within the framework of density functional theory (DFT). Energy decomposition analysis indicates that dispersion and induction (charge-transfer) interactions dominate the total binding energy, whereas electrostatic interactions are largely repulsive. The appearance of charge transfer bands in the absorption spectra of the complexes are well described by time-dependent (TD) DFT and are further explained in terms of the redox properties of purine monomers and solvation effects. Crystal structures are reported for complexes of methyl viologen with the purines 2′-deoxyguanosine 3′-monophosphate GMP (DAD′DAD′ type) and 7-deazaguanosine zG (DAD′ADAD′ type). Comparison of the structures determined in the solid state and by theoretical methods in solution provides valuable insights into the nature of charge-transfer interactions involving purine bases as electron donors. PMID:24294996

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

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

Meadows, J.R.

The ozone-induced degradation rates of various purine bases, hydroxylated purine compounds, pyrimidine bases, and uric acid were compared. Of the compounds examined, uric acid was the one most readily degraded while the parent compounds, purine and pyrimidine, were the ones most resistant to ozonation. When the breakdown of hydroxylated purines was studied, it was determined that the more OH substituents on the purine, the more readily it was degraded. Because of the preferential attack by ozone on uric acid in solutions containing a nucleic acid base plus uric acid, the presence of the uric acid had a sparing effect onmore » the base. This effect was readily apparent for guanine, thymine, and uracil which were the bases more labile to ozone. Two of the ozonation products of uric acid were identified as allantoin and urea. Ozonation of bovine and swine erythrocyte suspensions resulted in oxidation of oxyhemoglobin to methemoglobin, formation of thiobarbituric acid-reactive materials-a measure of lipid oxidation- and lysis of the red cells. Each of these changes was inhibited by the presence of uric acid in the solution during ozonation.« less

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling*

PubMed Central

Tringali, Cristina; Lupo, Barbara; Silvestri, Ilaria; Papini, Nadia; Anastasia, Luigi; Tettamanti, Guido; Venerando, Bruno

2012-01-01

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy. PMID:23139422

Prebiotic syntheses of purines and pyrimidines

NASA Technical Reports Server (NTRS)

Basile, B.; Oro, J.; Lazcano, A.

1984-01-01

The results of experimental and theoretical investigations of the prebiotic synthesis of purines and pyramidines are surveyed. Topics examined include the synthesis of purines from HCN via 4,5-disubstituted imidazole derivatives in aqueous solutions or liquid NH3, simultaneous formation of amino acids and purines by electron irradiation of CH4-NH3-H2O mixtures, synthesis of pyrimadines from cynoacetylene, energetics, formation of bases under anhydrous or concentrated conditions, formation of bases under dilute conditions, Fischer-Tropsch-type reactions, and the role of activated intermediates. It is pointed out that the precursor compounds have been detected in the interstellar medium, on Titan, and in other solar-system bodies, and that solar-nebula HCN concentrations of the order of 1-10 mM have been estimated on the basis of meteorite measurements.

Automated Selection of Regions of Interest for Intensity-based FRET Analysis of Transferrin Endocytic Trafficking in Normal vs. Cancer Cells

PubMed Central

Talati, Ronak; Vanderpoel, Andrew; Eladdadi, Amina; Anderson, Kate; Abe, Ken; Barroso, Margarida

2013-01-01

The overexpression of certain membrane-bound receptors is a hallmark of cancer progression and it has been suggested to affect the organization, activation, recycling and down-regulation of receptor-ligand complexes in human cancer cells. Thus, comparing receptor trafficking pathways in normal vs. cancer cells requires the ability to image cells expressing dramatically different receptor expression levels. Here, we have presented a significant technical advance to the analysis and processing of images collected using intensity based Förster resonance energy transfer (FRET) confocal microscopy. An automated Image J macro was developed to select region of interests (ROI) based on intensity and statistical-based thresholds within cellular images with reduced FRET signal. Furthermore, SSMD (strictly standardized mean differences), a statistical signal-to-noise ratio (SNR) evaluation parameter, was used to validate the quality of FRET analysis, in particular of ROI database selection. The Image J ROI selection macro together with SSMD as an evaluation parameter of SNR levels, were used to investigate the endocytic recycling of Tfn-TFR complexes at nanometer range resolution in human normal vs. breast cancer cells expressing significantly different levels of endogenous TFR. Here, the FRET-based assay demonstrates that Tfn-TFR complexes in normal epithelial vs. breast cancer cells show a significantly different E% behavior during their endocytic recycling pathway. Since E% is a relative measure of distance, we propose that these changes in E% levels represent conformational changes in Tfn-TFR complexes during endocytic pathway. Thus, our results indicate that Tfn-TFR complexes undergo different conformational changes in normal vs. cancer cells, indicating that the organization of Tfn-TFR complexes at the nanometer range is significantly altered during the endocytic recycling pathway in cancer cells. In summary, improvements in the automated selection of FRET ROI datasets allowed us to detect significant changes in E% with potential biological significance in human normal vs. cancer cells. PMID:23994873

Uridine Affects Liver Protein Glycosylation, Insulin Signaling, and Heme Biosynthesis

PubMed Central

Urasaki, Yasuyo; Pizzorno, Giuseppe; Le, Thuc T.

2014-01-01

Purines and pyrimidines are complementary bases of the genetic code. The roles of purines and their derivatives in cellular signal transduction and energy metabolism are well-known. In contrast, the roles of pyrimidines and their derivatives in cellular function remain poorly understood. In this study, the roles of uridine, a pyrimidine nucleoside, in liver metabolism are examined in mice. We report that short-term uridine administration in C57BL/6J mice increases liver protein glycosylation profiles, reduces phosphorylation level of insulin signaling proteins, and activates the HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. Short-term uridine administration is also associated with reduced liver hemin level and reduced ability for insulin-stimulated blood glucose removal during an insulin tolerance test. Some of the short-term effects of exogenous uridine in C57BL/6J mice are conserved in transgenic UPase1 −/− mice with long-term elevation of endogenous uridine level. UPase1 −/− mice exhibit activation of the liver HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. UPase1 −/− mice also exhibit impaired ability for insulin-stimulated blood glucose removal. However, other short-term effects of exogenous uridine in C57BL/6J mice are not conserved in UPase1 −/− mice. UPase1 −/− mice exhibit normal phosphorylation level of liver insulin signaling proteins and increased liver hemin concentration compared to untreated control C57BL/6J mice. Contrasting short-term and long-term consequences of uridine on liver metabolism suggest that uridine exerts transient effects and elicits adaptive responses. Taken together, our data support potential roles of pyrimidines and their derivatives in the regulation of liver metabolism. PMID:24918436

Metabolic modeling helps interpret transcriptomic changes during malaria.

PubMed

Tang, Yan; Gupta, Anuj; Garimalla, Swetha; Galinski, Mary R; Styczynski, Mark P; Fonseca, Luis L; Voit, Eberhard O

2018-06-01

Disease represents a specific case of malfunctioning within a complex system. Whereas it is often feasible to observe and possibly treat the symptoms of a disease, it is much more challenging to identify and characterize its molecular root causes. Even in infectious diseases that are caused by a known parasite, it is often impossible to pinpoint exactly which molecular profiles of components or processes are directly or indirectly altered. However, a deep understanding of such profiles is a prerequisite for rational, efficacious treatments. Modern omics methodologies are permitting large-scale scans of some molecular profiles, but these scans often yield results that are not intuitive and difficult to interpret. For instance, the comparison of healthy and diseased transcriptome profiles may point to certain sets of involved genes, but a host of post-transcriptional processes and regulatory mechanisms renders predictions regarding metabolic or physiological consequences of the observed changes in gene expression unreliable. Here we present proof of concept that dynamic models of metabolic pathway systems may offer a tool for interpreting transcriptomic profiles measured during disease. We illustrate this strategy with the interpretation of expression data of genes coding for enzymes associated with purine metabolism. These data were obtained during infections of rhesus macaques (Macaca mulatta) with the malaria parasite Plasmodium cynomolgi or P. coatneyi. The model-based interpretation reveals clear patterns of flux redistribution within the purine pathway that are consistent between the two malaria pathogens and are even reflected in data from humans infected with P. falciparum. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang. Copyright © 2017 Elsevier B.V. All rights reserved.

Developmental and metabolic responses of zebrafish (Danio rerio) embryos and larvae to short-chain chlorinated paraffins (SCCPs) exposure.

PubMed

Ren, Xiaoqian; Zhang, Haijun; Geng, Ningbo; Xing, Liguo; Zhao, Yu; Wang, Feidi; Chen, Jiping

2018-05-01

Short-chain chlorinated paraffins (SCCPs) are highly toxic to aquatic organisms, but their toxicity is yet not well characterized. In this study, the developmental toxicity of SCCPs to zebrafish embryos/larvae was evaluated, and a metabolomics approach was adopted to explore the impact of SCCPs exposure on the metabolism in zebrafish embryos. Exposure to SCCPs at concentrations of 1-200μg/L did not produce an observable effect on the hatching rate and morphological deformities of zebrafish embryos/larvae. However, the survival rate of zebrafish larvae in SCCPs exposure groups decreased in a concentration-dependent manner. The 13-day 50% lethal concentration (LC 50 ) value of SCCPs was calculated to be 34.4μg/L. Exposure to SCCPs induced a significant change of overall metabolism, even at environmentally relevant concentrations (1-5μg/L). The most relevant pathways affected by SCCPs exposure were glycerophospholipid metabolism, fatty acid metabolism and purine metabolism. Exposure to SCCPs at concentrations of 1-5μg/L had begun to accelerate the β-oxidation of unsaturated fatty acids and very long chain fatty acids, and affect the transformation of guanine to xanthine in the pathway of purine metabolism. Furthermore, when the exposure concentrations of SCCPs were increased to 50-200μg/L, the levels of phospholipids and amino acids were significantly raised; whereas the levels of fatty acids, carnitines and inosine were significantly decreased. In view of the significant effect on metabolism, the sub-chronic and chronic toxicity of SCCPs to fish should be concerned. Copyright © 2017 Elsevier B.V. All rights reserved.

Prominent Steatosis with Hypermetabolism of the Cell Line Permissive for Years of Infection with Hepatitis C Virus

PubMed Central

Sugiyama, Kazuo; Ebinuma, Hirotoshi; Nakamoto, Nobuhiro; Sakasegawa, Noriko; Murakami, Yuko; Chu, Po-sung; Usui, Shingo; Ishibashi, Yuka; Wakayama, Yuko; Taniki, Nobuhito; Murata, Hiroko; Saito, Yoshimasa; Fukasawa, Masayoshi; Saito, Kyoko; Yamagishi, Yoshiyuki; Wakita, Takaji; Takaku, Hiroshi; Hibi, Toshifumi; Saito, Hidetsugu; Kanai, Takanori

2014-01-01

Most of experiments for HCV infection have been done using lytic infection systems, in which HCV-infected cells inevitably die. Here, to elucidate metabolic alteration in HCV-infected cells in a more stable condition, we established an HCV-persistently-infected cell line, designated as HPI cells. This cell line has displayed prominent steatosis and supported HCV infection for more than 2 years, which is the longest ever reported. It enabled us to analyze metabolism in the HCV-infected cells integrally combining metabolomics and expression arrays. It revealed that rate-limiting enzymes for biosynthesis of cholesterol and fatty acids were up-regulated with actual increase in cholesterol, desmosterol (cholesterol precursor) and pool of fatty acids. Notably, the pentose phosphate pathway was facilitated with marked up-regulation of glucose-6-phosphate dehydrogenase, a rete-limiting enzyme, with actual increase in NADPH. In its downstream, enzymes for purine synthesis were also up-regulated resulting in increase of purine. Contrary to common cancers, the TCA cycle was preferentially facilitated comparing to glycolysis pathway with a marked increase of most of amino acids. Interestingly, some genes controlled by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of antioxidation and metabolism, were constitutively up-regulated in HPI cells. Knockdown of Nrf2 markedly reduced steatosis and HCV infection, indicating that Nrf2 and its target genes play important roles in metabolic alteration and HCV infection. In conclusion, HPI cell is a bona fide HCV-persistently-infected cell line supporting HCV infection for years. This cell line sustained prominent steatosis in a hypermetabolic status producing various metabolites. Therefore, HPI cell is a potent research tool not only for persistent HCV infection but also for liver metabolism, overcoming drawbacks of the lytic infection systems. PMID:24718268

Stock dynamics and emission pathways of the global aluminum cycle

NASA Astrophysics Data System (ADS)

Müller, Daniel B.; Liu, Gang; Bangs, Colton

Climate change mitigation in the materials sector faces a twin challenge: satisfying rapidly rising global demand for materials while significantly curbing greenhouse-gas emissions. Process efficiency improvement and recycling can contribute to reducing emissions per material output; however, long-term material demand and scrap availability for recycling depend fundamentally on the dynamics of societies' stocks of products in use, an issue that has been largely neglected in climate science. Here, we show that aluminium in-use stock patterns set essential boundary conditions for future emission pathways, which has significant implications for mitigation priority setting. If developing countries follow industrialized countries in their aluminium stock patterns, a 50% emission reduction by 2050 below 2000 levels cannot be reached even under very optimistic recycling and technology assumptions. The target can be reached only if future global per-capita aluminium stocks saturate at a level much lower than that in present major industrialized countries. As long as global in-use stocks are growing rapidly, radical new technologies in primary production (for example, inert anode and carbon capture and storage) have the greatest impact in emission reduction; however, their window of opportunity is closing once the stocks begin to saturate and the largest reduction potential shifts to post-consumer scrap recycling.

Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway*

PubMed Central

Madsen, Kenneth L.; Thorsen, Thor S.; Rahbek-Clemmensen, Troels; Eriksen, Jacob; Gether, Ulrik

2012-01-01

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β2-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway. PMID:22303009

Ethenoguanines Undergo Glycosylation by Nucleoside 2′-Deoxyribosyltransferases at Non-Natural Sites

PubMed Central

Ye, Wenjie; Paul, Debamita; Gao, Lina; Seckute, Jolita; Jayaraj, Karupiah; Zhang, Zhenfa; Kaminski, P. Alexandre

2014-01-01

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N 2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2′-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O 9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route. PMID:25521390

Multivesicular bodies: co-ordinated progression to maturity

PubMed Central

Woodman, Philip G; Futter, Clare E

2008-01-01

Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB. PMID:18502633

Childhood Lead Exposure from Battery Recycling in Vietnam

PubMed Central

Van Tung, Lo; Wallace, Ryan M.; Havens, Deborah J.; Karr, Catherine J.; Bich Diep, Nguyen; Croteau, Gerry A.; Beaudet, Nancy J.; Duy Bao, Nguyen

2015-01-01

Background. Battery recycling facilities in developing countries can cause community lead exposure. Objective. To evaluate child lead exposure in a Vietnam battery recycling craft village after efforts to shift home-based recycling outside the village. Methods. This cross-sectional study evaluated 109 children in Dong Mai village, using blood lead level (BLL) measurement, parent interview, and household observation. Blood samples were analyzed with a LeadCare II field instrument; highest BLLs (≥45 μg/dL) were retested by laboratory analysis. Surface and soil lead were measured at 11 households and a school with X-ray fluorescence analyzer. Results. All children had high BLLs; 28% had BLL ≥45 μg/dL. Younger age, family recycling, and outside brick surfaces were associated with higher BLL. Surface and soil lead levels were high at all tested homes, even with no recycling history. Laboratory BLLs were lower than LeadCare BLLs, in 24 retested children. Discussion. In spite of improvements, lead exposure was still substantial and probably associated with continued home-based recycling, legacy contamination, and workplace take-home exposure pathways. There is a need for effective strategies to manage lead exposure from battery recycling in craft villages. These reported BLL values should be interpreted cautiously, although the observed field-laboratory discordance may reflect bias in laboratory results. PMID:26587532

Childhood Lead Exposure from Battery Recycling in Vietnam.

PubMed

Daniell, William E; Van Tung, Lo; Wallace, Ryan M; Havens, Deborah J; Karr, Catherine J; Bich Diep, Nguyen; Croteau, Gerry A; Beaudet, Nancy J; Duy Bao, Nguyen

2015-01-01

Battery recycling facilities in developing countries can cause community lead exposure. To evaluate child lead exposure in a Vietnam battery recycling craft village after efforts to shift home-based recycling outside the village. This cross-sectional study evaluated 109 children in Dong Mai village, using blood lead level (BLL) measurement, parent interview, and household observation. Blood samples were analyzed with a LeadCare II field instrument; highest BLLs (≥45 μg/dL) were retested by laboratory analysis. Surface and soil lead were measured at 11 households and a school with X-ray fluorescence analyzer. All children had high BLLs; 28% had BLL ≥45 μg/dL. Younger age, family recycling, and outside brick surfaces were associated with higher BLL. Surface and soil lead levels were high at all tested homes, even with no recycling history. Laboratory BLLs were lower than LeadCare BLLs, in 24 retested children. In spite of improvements, lead exposure was still substantial and probably associated with continued home-based recycling, legacy contamination, and workplace take-home exposure pathways. There is a need for effective strategies to manage lead exposure from battery recycling in craft villages. These reported BLL values should be interpreted cautiously, although the observed field-laboratory discordance may reflect bias in laboratory results.

Urinary and plasma purine derivatives in fed and fasted llamas (Lama glama and L. guanacoe).

PubMed

Bakker, M L; Chen, X B; Kyle, D J; Orskov, E R; Bourke, D A

1996-02-01

The changes in urinary and plasma purine derivatives in response to fasting and level of feeding in llamas were examines. In one experiment, four llamas were gradually deprived of feed within 3 days and then fasted for 6 days. Daily urinary excretion of purine derivatives decreased with feed intake and leveled on the last 3 days of fasting at 177 +/- 26 mumol/kg W0.75. Allantoin and uric acid comprised 71% and 15% of total purine derivatives, respectively, in both fed and fasted states, but hypoxanthine plus xanthine increased from 9% to 36%. Plasma concentration of allantoin declined with feed intake reduction, but those of uric acid (217 mumol/l) and hypoxanthine plus xanthine (27 mumol/l) remained relatively unchanged. Concentration of uric acid was higher than that of allantoin, probably due to a high reabsorption of uric acid in renal tubules, which was measured as over 90%. In a second experiment, the four llamas were fed at 860 and 1740 g dry matter/d in a crossover design. Urinary total purine derivatives excretion responded to feed intake (10.4 vs 14.4 mmol/d), although the observed differences did not reach significance. Compared with some ruminant species, it appears that the llama resembles sheep regarding the magnitude of urinary purine derivatives excretion but is unique in maintaining a high concentration of uric acid in plasma, which could be part of the llama's adaptation to their environment.

Rituximab with pentostatin or cladribine: an effective combination treatment for hairy cell leukemia after disease recurrence.

PubMed

Else, Monica; Dearden, Claire E; Matutes, Estella; Forconi, Francesco; Lauria, Francesco; Ahmad, Humayun; Kelly, Susan; Liyanage, Anandika; Ratnayake, Vijitha; Shankari, Jagadeesan; Whalley, Ioana; Catovsky, Daniel

2011-06-01

The purine analogs pentostatin and cladribine are effective treatments for hairy cell leukemia (HCL). However, alternative treatments are needed for patients with recurrent disease. We reviewed retrospectively data from 18 patients who were retreated with either pentostatin (n = 12) or cladribine (n = 6) in combination with rituximab, after 1-6 (median 2) previous treatments with either purine analog as a single agent. All 18 patients responded to therapy, with a complete response (CR) rate of 89%. This compared favorably with CR rates of 68% after second-line therapy and 47% after third-line therapy in 88 patients retreated one or more times with a purine analog alone. Toxicity with the combination treatment was minimal. At a median follow-up of 36 months (range 5-83 months) all 16 complete responders remained in CR, while one partial responder developed recurrent disease at 10 months. The estimated recurrence rate at 3 years was 7%. This compares with 21% after second-line therapy and 42% after third-line therapy in the 88 patients retreated with a purine analog alone. Furthermore, it was a marked improvement on the 55% recurrence at 3 years previously seen in these same 18 patients after their own first-line treatment with single-agent pentostatin or cladribine (p = 0.006). The combination of a purine analog with rituximab was safe and effective for patients with recurrent HCL. The results suggest an added benefit compared with single-agent purine analog therapy.

Crystal structure of yeast allantoicase reveals a repeated jelly roll motif.

PubMed

Leulliot, Nicolas; Quevillon-Cheruel, Sophie; Sorel, Isabelle; Graille, Marc; Meyer, Philippe; Liger, Dominique; Blondeau, Karine; Janin, Joël; van Tilbeurgh, Herman

2004-05-28

Allantoicase (EC 3.5.3.4) catalyzes the conversion of allantoate into ureidoglycolate and urea, one of the final steps in the degradation of purines to urea. The mechanism of most enzymes involved in this pathway, which has been known for a long time, is unknown. In this paper we describe the three-dimensional crystal structure of the yeast allantoicase determined at a resolution of 2.6 A by single anomalous diffraction. This constitutes the first structure for an enzyme of this pathway. The structure reveals a repeated jelly roll beta-sheet motif, also present in proteins of unrelated biochemical function. Allantoicase has a hexameric arrangement in the crystal (dimer of trimers). Analysis of the protein sequence against the structural data reveals the presence of two totally conserved surface patches, one on each jelly roll motif. The hexameric packing concentrates these patches into conserved pockets that probably constitute the active site.

Specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system

PubMed Central

Finetti, Francesca; Patrussi, Laura; Masi, Giulia; Onnis, Anna; Galgano, Donatella; Lucherini, Orso Maria; Pazour, Gregory J.; Baldari, Cosima T.

2014-01-01

ABSTRACT T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5+ endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis. PMID:24554435

Recycled wind turbine blades as a feedstock for second generation composites.

PubMed

Mamanpush, Seyed Hossein; Li, Hui; Englund, Karl; Tabatabaei, Azadeh Tavousi

2018-06-01

With an increase in renewable wind energy via turbines, an underlying problem of the turbine blade disposal is looming in many areas of the world. These wind turbine blades are predominately a mixture of glass fiber composites (GFCs) and wood and currently have not found an economically viable recycling pathway. This work investigates a series of second generation composites fabricated using recycled wind turbine material and a polyurethane adhesive. The recycled material was first comminuted via a hammer-mill through a range of varying screen sizes, resinated and compressed to a final thickness. The refined particle size, moisture content and resin content were assessed for their influence on the properties of recycled composites. Static bending, internal bond and water sorption properties were obtained for all composites panels. Overall improvement of mechanical properties correlated with increase in resin content, moisture content, and particle size. The current investigation demonstrates that it is feasible and promising to recycle the wind turbine blade to fabricate value-added high-performance composite. Copyright © 2018 Elsevier Ltd. All rights reserved.

Xanthine urolithiasis in a cat: a case report and evaluation of a candidate gene for xanthine dehydrogenase.

PubMed

Tsuchida, Shuichi; Kagi, Akiko; Koyama, Hidekazu; Tagawa, Masahiro

2007-12-01

Xanthine urolithiasis was found in a 4-year-old spayed female Himalayan cat with a 10-month history of intermittent haematuria and dysuria. Ultrasonographs indicated the existence of several calculi in the bladder that were undetectable by survey radiographic examination. Four bladder stones were removed by cystotomy. The stones were spherical brownish-yellow and their surface was smooth and glossy. Quantitative mineral analysis showed a representative urolith to be composed of more than 95% xanthine. Ultrasonographic examination of the bladder 4.5 months postoperatively indicated the recurrence of urolithiasis. Analysis of purine concentration in urine and blood showed that the cat excreted excessive amounts of xanthine. In order to test the hypothesis that xanthinuria was caused by a homozygote of the inherited mutant allele of a gene responsible for deficiency of enzyme activity in purine degradation pathway, the allele composition of xanthine dehydrogenase (XDH) gene (one of the candidate genes for hereditary xanthinuria) was evaluated. The cat with xanthinuria was a heterozygote of the polymorphism. A single nucleotide polymorphism analysis of the cat XDH gene strongly indicated that the XDH gene of the patient cat was composed of two kinds of alleles and ruled out the hypothesis that the cat inherited the same recessive XDH allele suggesting no activity from a single ancestor.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway.

PubMed

Jacobsen, Stine Engesgaard; Ammendrup-Johnsen, Ina; Jansen, Anna Mai; Gether, Ulrik; Madsen, Kenneth Lindegaard; Bräuner-Osborne, Hans

2017-04-28

The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

PubMed Central

Jacobsen, Stine Engesgaard; Ammendrup-Johnsen, Ina; Jansen, Anna Mai; Gether, Ulrik; Madsen, Kenneth Lindegaard; Bräuner-Osborne, Hans

2017-01-01

The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure. PMID:28280242

Potassium recycling pathways in the human cochlea.

PubMed

Weber, P C; Cunningham, C D; Schulte, B A

2001-07-01

Potential pathways for recycling potassium (K+) used in the maintenance of inner ear electrochemical gradients have been elucidated in animal models. However, little is known about K+ transport in the human cochlea. This study was designed to characterize putative K+ recycling pathways in the human ear and to determine whether observations from animal models can be extrapolated to humans. A prospective laboratory study using an immunohistochemical approach to analyze the distribution of key ion transport mediators in the human cochlea. Human temporal bones were fixed in situ within 1 to 6 hours of death and subsequently harvested at autopsy. Decalcification was accomplished with the aid of microwaving. Immunohistochemical staining was then performed to define the presence and cell type-specific distribution of Na,K-ATPase, sodium-potassium-chloride cotransporter (NKCC), and carbonic anhydrase (CA) in the inner ear. Staining patterns visualized in the human cochlea closely paralleled those seen in other species. Anti-Na,K-ATPase stained strongly the basolateral plasma membrane of strial marginal cells and nerve endings underlying hair cells. This antibody also localized Na,K-ATPase to type II, type IV, and type V fibrocytes in the spiral ligament and in limbal fibrocytes. NKCC was present in the basolateral membrane of strial marginal cells as well as in type II, type V, and limbal fibrocytes. Immunoreactive carbonic anhydrase was present in type I and type III fibrocytes and in epithelial cells lining Reissner's membrane and the spiral prominence. The distribution of several major ion transport proteins in the human cochlea is similar but not identical to that described in various rodent models. These results support the presence of a complex system for recycling and regulating K+ homeostasis in the human cochlea, similar to that described in other mammalian species.

[Direct determination of purine bases in tea by reversed-phase high performance liquid chromatography].

PubMed

Ding, M; Yang, H; Xiao, S; Chen, P

1999-09-01

A reversed-phase high performance liquid chromatographic(RP-HPLC) method for the direct determination of three purine bases(theobromin, theophyllin and caffeine) in tea was developed. An ODS column with Zorbax SB-C18(4.6 mm i.d. x 250 mm, 5 microns) was employed. The aqueous solution of methanol containing 0.05% of acetic acid and 0.25% of N,N-dimethylformamide(DMF) was used as eluent with a flow rate of 0.8 mL/min. In this method, the aqueous extract of tea can be injected into HPLC directly, but in current HPLC methods for purine bases the coexisted tea polyphenols must be pre-separated. The three purine bases in tea were separated without any interference from the coexisted tea polyphenols. This method is simple (without any special sample pretreatment) and sensitive with detection limits (S/N = 3) of 0.7, 0.9 and 1.8 mg/L for theobromin, theophyllin and caffeine respectively. The linear range of the calibration curve of peak area for the three purine bases were from 6 mg/L to 1,000 mg/L with a correlation coefficient (r) of 0.998-0.999.

The effects of fasting in Ramadan. 1. Serum uric acid and lipid concentrations.

PubMed

Gumaa, K A; Mustafa, K Y; Mahmoud, N A; Gader, A M

1978-11-01

1. The changes in serum levels of uric acid and lipids during 1 month of starvation-refeeding were measured in sixteen male volunteers. 2. Uric acid levels increased linearly with the duration of the experiment. The increase was positively correlated with the increase in serum triglycerides but not with cholesterol or phospholipids. 3. Triglycerides increased at a faster rate than uric acid implying that the increase in uric acid was secondary to that of the lipid. 4. It was concluded that the purine and lipid synthetic pathways are linked through a common small-molecular-weight effector rather than through the sharing of a common enzyme.

Partition coefficients of some purine derivatives and its application to pharmacokinetics.

PubMed

Chrzanowska, M; Sobiak, J; Kuehn, M; Dorawa, E; Hermann, T

2009-12-01

Metazathioprine (MAZA), a methylated derivative of azathioprine (AZA), demonstrated the greatest values of apparent and specific partition coefficients in n-octanol/phosphate buffer at pH 5.7 and pH 7.4 among purine derivatives such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and AZA. Introduction of a methyl group into the imidazole ring of AZA increases lipophilic properties of MAZA compared to AZA. Mass balance of purine derivatives in n-octanol and in phosphate buffer indicated their chemical stability in those media.

Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

PubMed Central

Clinch, Keith; Crump, Douglas R.; Evans, Gary B.; Hazleton, Keith Z.; Mason, Jennifer M.; Schramm, Vern L.

2013-01-01

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme. PMID:23810424

Integrating Candida albicans metabolism with biofilm heterogeneity by transcriptome mapping

NASA Astrophysics Data System (ADS)

Rajendran, Ranjith; May, Ali; Sherry, Leighann; Kean, Ryan; Williams, Craig; Jones, Brian L.; Burgess, Karl V.; Heringa, Jaap; Abeln, Sanne; Brandt, Bernd W.; Munro, Carol A.; Ramage, Gordon

2016-10-01

Candida albicans biofilm formation is an important virulence factor in the pathogenesis of disease, a characteristic which has been shown to be heterogeneous in clinical isolates. Using an unbiased computational approach we investigated the central metabolic pathways driving biofilm heterogeneity. Transcripts from high (HBF) and low (LBF) biofilm forming isolates were analysed by RNA sequencing, with 6312 genes identified to be expressed in these two phenotypes. With a dedicated computational approach we identified and validated a significantly differentially expressed subnetwork of genes associated with these biofilm phenotypes. Our analysis revealed amino acid metabolism, such as arginine, proline, aspartate and glutamate metabolism, were predominantly upregulated in the HBF phenotype. On the contrary, purine, starch and sucrose metabolism was generally upregulated in the LBF phenotype. The aspartate aminotransferase gene AAT1 was found to be a common member of these amino acid pathways and significantly upregulated in the HBF phenotype. Pharmacological inhibition of AAT1 enzyme activity significantly reduced biofilm formation in a dose-dependent manner. Collectively, these findings provide evidence that biofilm phenotype is associated with differential regulation of metabolic pathways. Understanding and targeting such pathways, such as amino acid metabolism, is potentially useful for developing diagnostics and new antifungals to treat biofilm-based infections.

Transcriptomic analysis of genes involved in the biosynthesis, recycling and degradation of L-ascorbic acid in pepper fruits (Capsicum annuum L.).

PubMed

Alós, Enriqueta; Rodrigo, María J; Zacarías, Lorenzo

2013-06-01

Sweet pepper (Capsicum annuum L.) is widely recognized among the vegetables with high content of ascorbic acid (AsA). However, the metabolic pathways involved in the biosynthesis, recycling and degradation of AsA and their relative contribution to the concentration of AsA have not been established yet. In the present work, the expression levels of selected genes involved in the AsA biosynthesis, degradation and recycling pathways were analyzed during development and ripening of pepper fruit cv. Palermo and in mature fruit of four cultivars (Lipari, C-116, Surrentino and Italverde) with different AsA concentrations. An inverse correlation was found between the expression of the biosynthetic genes and AsA concentrations, which could indicate that a feedback mechanism regulates AsA homeostasis in pepper fruits. Interestingly, analysis of mRNA levels of ascorbate oxidase, involved in the degradation of AsA, suggests that this enzyme plays a critical role in the regulation of the AsA pool during fruit development and ripening. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

PubMed

Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

2016-09-01

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. Copyright © 2016. Published by Elsevier Ltd.

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

PubMed Central

Dennis, Megan K.; Mantegazza, Adriana R.; Snir, Olivia L.; Tenza, Danièle; Acosta-Ruiz, Amanda; Delevoye, Cédric; Zorger, Richard; Sitaram, Anand; de Jesus-Rojas, Wilfredo; Ravichandran, Keerthana; Rux, John; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Setty, Subba Rao Gangi

2015-01-01

Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation. PMID:26008744

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

PubMed Central

Hoogenraad, Casper C.; Popa, Ioana; Futai, Kensuke; Sanchez-Martinez, Emma; Wulf, Phebe S.; van Vlijmen, Thijs; Dortland, Bjorn R.; Oorschot, Viola; Govers, Roland; Monti, Maria; Heck, Albert J. R.; Sheng, Morgan; Klumperman, Judith; Rehmann, Holger; Jaarsma, Dick; Kapitein, Lukas C.; van der Sluijs, Peter

2010-01-01

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking. PMID:20098723

GGA3 mediates TrkA endocytic recycling to promote sustained Akt phosphorylation and cell survival

PubMed Central

Li, Xuezhi; Lavigne, Pierre; Lavoie, Christine

2015-01-01

Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood. Here we demonstrate that Golgi-localized, γ adaptin-ear–containing ADP ribosylation factor-binding protein 3 (GGA3) interacts directly with the TrkA cytoplasmic tail through an internal DXXLL motif and mediates the functional recycling of TrkA to the plasma membrane. We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival. We also show that GGA3’s effect on TrkA recycling is dependent on the activation of Arf6. This work identifies GGA3 as a key player in a novel DXXLL-mediated endosomal sorting machinery that targets TrkA to the plasma membrane, where it prolongs the activation of Akt signaling and survival responses. PMID:26446845

Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

PubMed

Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

1998-12-08

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Effects of pH, temperature, and chemical structure on the stability of S-(purin-6-yl)-L-cysteine: evidence for a novel molecular rearrangement mechanism to yield N-(purin-6-yl)-L-cysteine.

PubMed

Elfarra, A A; Hwang, I Y

1996-01-01

The stability of S-(purin-6-yl)-L-cysteine (SPC), a kidney-selective prodrug of 6-mercaptopurine and a putative metabolite of 6-chloropurine, was investigated under various pH and temperature conditions. At room temperature, the half-life (t 1/2) of SPC at either highly acidic (pH 3.6) or basic conditions (pH 9.6) was longer than at neutral or slightly acidic or basic conditions (pH 5.7-8.75). The primary degradation product, N-(purin-6-yl)-L-cysteine (NPC), was isolated using Sephadex LH-20 chromatography and characterized by 1H NMR and FAB/MS after derivatization with 2-iodoacetic acid. These results reveal novel stability requirements and implicate the cysteinyl amino group and the purinyl N-1 nitrogen in the mechanism of SPC rearrangement to NPC. Further evidence for this hypothesis was provided by the findings that the stability of SPC in phosphate buffer (pH 7.4) at 37 degrees C was similar to that of S-(guanin-6-yl)-L-cysteine, whereas S-(purin-6-yl)-N-acetyl-L-cysteine and S-(purin-6-yl)glutathione which have their cysteine amino groups blocked were much more stable than SPC. S-(Purin-6-yl)-L-homocysteine (SPHC) was also more stable than SPC, possibly because the formation of a 6-membered ring transition state as would be expected with SPHC is kinetically less favored than the formation of a 5-membered ring transition state as would be expected with SPC. These results may explain previous in vivo metabolism results of SPC and its analogs and may contribute to a better understanding of stability of structurally related cysteine S-conjugates.

Metabolic Biomarkers and Neurodegeneration: A Pathway Enrichment Analysis of Alzheimer's Disease, Parkinson's Disease, and Amyotrophic Lateral Sclerosis.

PubMed

Kori, Medi; Aydın, Busra; Unal, Semra; Arga, Kazim Yalcin; Kazan, Dilek

2016-11-01

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) lack robust diagnostics and prognostic biomarkers. Metabolomics is a postgenomics field that offers fresh insights for biomarkers of common complex as well as rare diseases. Using data on metabolite-disease associations published in the previous decade (2006-2016) in PubMed, ScienceDirect, Scopus, and Web of Science, we identified 101 metabolites as putative biomarkers for these three neurodegenerative diseases. Notably, uric acid, choline, creatine, L-glutamine, alanine, creatinine, and N-acetyl-L-aspartate were the shared metabolite signatures among the three diseases. The disease-metabolite-pathway associations pointed out the importance of membrane transport (through ATP binding cassette transporters), particularly of arginine and proline amino acids in all three neurodegenerative diseases. When disease-specific and common metabolic pathways were queried by using the pathway enrichment analyses, we found that alanine, aspartate, glutamate, and purine metabolism might act as alternative pathways to overcome inadequate glucose supply and energy crisis in neurodegeneration. These observations underscore the importance of metabolite-based biomarker research in deciphering the elusive pathophysiology of neurodegenerative diseases. Future research investments in metabolomics of complex diseases might provide new insights on AD, PD, and ALS that continue to place a significant burden on global health.

AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes.

PubMed

Navone, Laura; Macagno, Juan Pablo; Licona-Cassani, Cuauhtémoc; Marcellin, Esteban; Nielsen, Lars K; Gramajo, Hugo; Rodriguez, Eduardo

2015-10-01

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes

PubMed Central

Navone, Laura; Macagno, Juan Pablo; Licona-Cassani, Cuauhtémoc; Marcellin, Esteban; Nielsen, Lars K.; Gramajo, Hugo

2015-01-01

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production. PMID:26187964

Synthesis and pH-dependent stability of purine-6-sulfenic acid, a putative reactive metabolite of 6-thiopurine.

PubMed

Abraham, R T; Benson, L M; Jardine, I

1983-10-01

Previous studies have shown that 6-thiopurine is metabolically activated by hepatic cytochrome P-450 to an intermediate capable of binding to proteins by a mixed disulfide linkage. The identity of the active metabolite was postulated to be purine-6-sulfenic acid. In the present report, we describe the synthesis of the sulfenic acid derivatives of 6-thiopurine and two structurally similar compounds, 9-methyl-6-thiopurine and 4-mercapto-1H-pyrazolo[3,4-d]-pyrimidine. The unusual pH-dependent stability profiles of these compounds in buffered aqueous media are presented and explained on the basis of a disproportionation mechanism of sulfenic acid decomposition. Studies with radiolabeled purine-6-sulfenic acid demonstrate that this species binds directly to hepatic microsomal protein. These results support the proposed involvement of purine-6-sulfenic acid in the metabolic activation and tissue binding of 6-thiopurine.

AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

PubMed Central

Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

2013-01-01

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

PubMed

Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K; Van Vleet, Jeremy; Fenstermaker, Ali G; Silhavy, Jennifer L; Scheliga, Judith S; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma M; Celep, Figen; Oraby, Azza; Zaki, Maha S; Al-Baradie, Raidah; Faqeih, Eissa A; Saleh, Mohammed A M; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W; Gleeson, Joseph G

2013-08-01

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Specific features of L-histidine production by Escherichia coli concerned with feedback control of AICAR formation and inorganic phosphate/metal transport.

PubMed

Malykh, Evgeniya A; Butov, Ivan A; Ravcheeva, Anna B; Krylov, Alexander A; Mashko, Sergey V; Stoynova, Nataliya V

2018-03-15

In the L-histidine (His) biosynthetic pathway of Escherichia coli, the first key enzyme, ATP-phosphoribosyltransferase (ATP-PRT, HisG), is subject to different types of inhibition. Eliminating the feedback inhibition of HisG by the His end product is an important step that enables the oversynthesis of His in breeding strains. However, the previously reported feedback inhibition-resistant mutant enzyme from E. coli, HisG E271K , is inhibited by purine nucleotides, particularly ADP and AMP, via competitive inhibition with its ATP substrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is formed not only during His biosynthesis but also during de novo purine biosynthesis, acts as a natural analog of AMP and substitutes for it in some enzymatic reactions. We hypothesized that AICAR could control its own formation, particularly through the His biosynthetic pathway, by negatively influencing HisG enzymatic activity, which would make preventing ATP-PRT transferase inhibition by AICAR crucial for His overproduction. For the first time, both the native E. coli HisG and the previously described feedback-resistant mutant HisG E271K enzymes were shown to be sensitive to inhibition by AICAR, a structural analog of AMP. To circumvent the negative effect that AICAR has on His synthesis, we constructed the new His-producing strain EA83 and demonstrated its improved histidine production. This increased production was particularly associated with the improved conversion of AICAR to ATP due to purH and purA gene overexpression; additionally, the PitA-dependent phosphate/metal (Me 2+ -P i ) transport system was modified by a pitA gene deletion. This His-producing strain unexpectedly exhibited decreased alkaline phosphatase activity at low P i concentrations. AICAR was consequently hypothesized inhibit the two-component PhoBR system, which controls Pho regulon gene expression. Inhibition of a key enzyme in the His biosynthetic pathway, HisG, by AICAR, which is formed in this pathway, generates a serious bottleneck during His production. The constructed His-producing strain demonstrated the enhanced expression of genes that encode enzymes involved in the metabolism of AICAR to ATP, which is a substrate of HisG, and thus led to improved His accumulation.

Urinary excretion of purine derivatives as an index of microbial protein synthesis in the camel (Camelus dromedarius).

PubMed

Guerouali, Abdelhai; El Gass, Youssef; Balcells, Joaquim; Belenguer, Alvaro; Nolan, John

2004-08-01

Five experiments were carried out to extend knowledge of purine metabolism in the camel (Camelus dromedarius) and to establish a model to enable microbial protein outflow from the forestomachs to be estimated from the urinary excretion of purine derivatives (PD; i.e. xanthine, hypoxanthine, uric acid, allantoin). In experiment 1, four camels were fasted for five consecutive days to enable endogenous PD excretion in urine to be determined. Total PD excretion decreased during the fasting period to 267 (SE 41.5) micromol/kg body weight (W)0.75 per d. Allantoin and xanthine + hypoxanthine were consistently 86 and 6.1 % of total urinary PD during this period but uric acid increased from 3.6 % to 7.4 %. Xanthine oxidase activity in tissues (experiment 2) was (micromol/min per g fresh tissue) 0.038 in liver and 0.005 in gut mucosa but was not detected in plasma. In experiment 3, the duodenal supply of yeast containing exogenous purines produced a linear increase in urinary PD excretion rate with the slope indicating that 0.63 was excreted in urine. After taking account of endogenous PD excretion, the relationship can be used to predict purine outflow from the rumen. From the latter prediction, and also the purine:protein ratio in bacteria determined in experiment 5, we predicted the net microbial outflow from the rumen. In experiment 4, with increasing food intake, the rate of PD excretion in the urine increased linearly by about 11.1 mmol PD/kg digestible organic matter intake (DOMI), equivalent to 95 g microbial protein/kg DOMI.

Evaluation of dogs with genetic hyperuricosuria and urate urolithiasis consuming a purine restricted diet: a pilot study.

PubMed

Westropp, Jodi L; Larsen, Jennifer A; Johnson, Eric G; Bannasch, Dannika; Fascetti, Andrea J; Biourge, Vincent; Queau, Yann

2017-02-08

Urate urolithiasis is a common problem in breed homozygous for the mutation that results in hyperuricosuria. Low purine diets have been recommended to reduce purine intake in these dogs. A higher protein, purine restricted diet with water added was evaluated in dogs with genetic hyperuricosuria and a history of clinical urate urolithiasis over a one year time period. Dogs were evaluated at baseline and 2, 6, and 12 months after initiating the test diet. Bloodwork, urinalysis, abdominal ultrasound, body composition, and 24-h urinary purine metabolite analyses were performed. Transient, mild, self-limited lower urinary tract signs were noted in only one dog on a single day, despite variable but usually mild and occasionally moderate amounts of echogenic bladder stones (<2-3 mm in size) in almost every dog at each visit. No significant differences were noted in urine specific gravity, urine pH, lean body condition score or body composition. Urinary uric acid concentration was lower on the test diet (p = 0.008), but 24-h uric acid excretions were similar (p = 0.220) compared to baseline. Significant differences between least squares mean plasma amino acid concentrations measured at the 0 and 12-month visits were found only for valine (p = 0.0119) and leucine (p = 0.0017). This study suggests the use of a low purine, higher protein diet with added water may be beneficial as part of the management of dogs with genetic hyperuricosuria and history of clinical urate urolithiasis.

Mechanisms of the Formation of Adenine, Guanine, and Their Analogues in UV-Irradiated Mixed NH3:H2O Molecular Ices Containing Purine

NASA Astrophysics Data System (ADS)

Bera, Partha P.; Stein, Tamar; Head-Gordon, Martin; Lee, Timothy J.

2017-08-01

We investigated the formation mechanisms of the nucleobases adenine and guanine and the nucleobase analogues hypoxanthine, xanthine, isoguanine, and 2,6-diaminopurine in a UV-irradiated mixed 10:1 H2O:NH3 ice seeded with precursor purine by using ab initio and density functional theory computations. Our quantum chemical investigations suggest that a multistep reaction mechanism involving purine cation, hydroxyl and amino radicals, together with water and ammonia, explains the experimentally obtained products in an independent study. The relative abundances of these products appear to largely follow from relative thermodynamic stabilities. The key role of the purine cation is likely to be the reason why purine is not functionalized in pure ammonia ice, where cations are promptly neutralized by free electrons from NH3 ionization. Amine group addition to purine is slightly favored over hydroxyl group attachment based on energetics, but hydroxyl is much more abundant due to higher abundance of H2O. The amino group is preferentially attached to the 6 position, giving 6-aminopurine, that is, adenine, while the hydroxyl group is preferentially attached to the 2 position, leading to 2-hydroxypurine. A second substitution by hydroxyl or amino group occurs at either the 6 or the 2 position depending on the first substitution. Given that H2O is far more abundant than NH3 in the experimentally studied ices (as well as based on interstellar abundances), xanthine and isoguanine are expected to be the most abundant bi-substituted photoproducts.

Rab11 in Recycling Endosomes Regulates the Sorting and Basolateral Transport of E-CadherinV⃞

PubMed Central

Lock, John G.; Stow, Jennifer L.

2005-01-01

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion. PMID:15689490

Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission

PubMed Central

Chung, ChiHye; Barlyko, Barbara; Leitz, Jeremy; Liu, Xinran; Kavalali, Ege T.

2010-01-01

Synapses maintain synchronous, asynchronous and spontaneous forms of neurotransmission that are distinguished by their Ca2+-dependence and time course. Despite recent advances in our understanding of the mechanisms that underlie these three forms of release, it remains unclear whether they originate from the same vesicle population or arise from distinct vesicle pools with diverse propensities for release. Here, we used a reversible inhibitor of dynamin, dynasore, to dissect the vesicle pool dynamics underlying the three forms of neurotransmitter release in hippocampal GABAergic inhibitory synapses. In dynasore, evoked synchronous release and asynchronous neurotransmission detected after activity showed marked and unrecoverable depression within seconds. In contrast, spontaneous release remained intact after intense stimulation in dynasore or during prolonged (~1 hour) application of dynasore at rest, suggesting that separate recycling pathways maintain evoked and spontaneous synaptic vesicle trafficking. In addition, simultaneous imaging of spectrally separable styryl dyes revealed that in a given synapse vesicles that recycle spontaneously and in response to activity do not mix. These findings suggest that evoked synchronous and asynchronous release originate from the same vesicle pool that recycles rapidly in a dynamin-dependent manner, while a distinct vesicle pool sustains spontaneous release independent of dynamin activation. This result lends further support to the notion that synapses harbor distinct vesicle populations with divergent release properties that maintain independent forms of neurotransmission. PMID:20107062

COPI selectively drives maturation of the early Golgi

PubMed Central

Papanikou, Effrosyni; Day, Kasey J; Austin, Jotham; Glick, Benjamin S

2015-01-01

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins. DOI: http://dx.doi.org/10.7554/eLife.13232.001 PMID:26709839

Serum Metabolomics Investigation of Humanized Mouse Model of Dengue Virus Infection.

PubMed

Cui, Liang; Hou, Jue; Fang, Jinling; Lee, Yie Hou; Costa, Vivian Vasconcelos; Wong, Lan Hiong; Chen, Qingfeng; Ooi, Eng Eong; Tannenbaum, Steven R; Chen, Jianzhu; Ong, Choon Nam

2017-07-15

Dengue is an acute febrile illness caused by dengue virus (DENV) and a major cause of morbidity and mortality in tropical and subtropical regions of the world. The lack of an appropriate small-animal model of dengue infection has greatly hindered the study of dengue pathogenesis and the development of therapeutics. In this study, we conducted mass spectrometry-based serum metabolic profiling from a model using humanized mice (humice) with DENV serotype 2 infection at 0, 3, 7, 14, and 28 days postinfection (dpi). Forty-eight differential metabolites were identified, including fatty acids, purines and pyrimidines, acylcarnitines, acylglycines, phospholipids, sphingolipids, amino acids and derivatives, free fatty acids, and bile acid. These metabolites showed a reversible-change trend-most were significantly perturbed at 3 or 7 dpi and returned to control levels at 14 or 28 dpi, indicating that the metabolites might serve as prognostic markers of the disease in humice. The major perturbed metabolic pathways included purine and pyrimidine metabolism, fatty acid β-oxidation, phospholipid catabolism, arachidonic acid and linoleic acid metabolism, sphingolipid metabolism, tryptophan metabolism, phenylalanine metabolism, lysine biosynthesis and degradation, and bile acid biosynthesis. Most of these disturbed pathways are similar to our previous metabolomics findings in a longitudinal cohort of adult human dengue patients across different infection stages. Our analyses revealed the commonalities of host responses to DENV infection between humice and humans and suggested that humice could be a useful small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics. IMPORTANCE Dengue virus is the most widespread arbovirus, causing an estimated 390 million dengue infections worldwide every year. There is currently no effective treatment for the disease, and the lack of an appropriate small-animal model of dengue infection has greatly increased the challenges in the study of dengue pathogenesis and the development of therapeutics. Metabolomics provides global views of small-molecule metabolites and is a useful tool for finding metabolic pathways related to disease processes. Here, we conducted a serum metabolomics study on a model using humanized mice with dengue infection that had significant levels of human platelets, monocytes/macrophages, and hepatocytes. Forty-eight differential metabolites were identified, and the underlying perturbed metabolic pathways are quite similar to the pathways found to be altered in dengue patients in previous metabolomics studies, indicating that humanized mice could be a highly relevant small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics. Copyright © 2017 Cui et al.

Development of a bacterial screen for novel hypoxanthine-guanine phosphoribosyltransferase substrates.

PubMed

Shivashankar, K; Subbayya, I N; Balaram, H

2001-10-01

The lack of de novo purine biosynthesis in many parasitic protozoans makes the enzymes in the salvage of purines attractive chemotherapeutic targets. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme for purine salvage and bacterial complementation screens for HGPRT inhibitors are known. The low KMS for purine bases makes purine analogs unattractive as competitive inhibitors for this enzyme. Despite the availability of many crystal structures of HGPRTs, it is only recently that selective inhibitors of the enzyme have been developed. Therefore, novel purine analogs which act as substrates for the HGPRT reaction and thereby inhibit downstream enzymes or get incorporated into the nucleotide pool are an attractive altenative for drug design. We have used a combination of two E. coli strains Sphi606 (ara, deltapro-gpt-lac, thi, hpt) and Sphi609 (ara, deltapro-gpt-lac, thi, hpt, pup, purH,J, strA) to identify inhibitors and substrates of HGPRT. E. coli Sphi609 is deficient in both de novo synthesis as well as salvage enzymes of purine nucleotide synthesis, while E. coli Sphi606 is deficient in salvage enzymes only. Hence, expression of functional HGPRTs in E. coli Sphi606 grown in minimal medium makes it susceptible to HGPRT substrates, which inhibit downstream processes. Growth of E. coli Sphi609 in minimal medium can be made conditional for the expression of a functional HGPRT and this growth would be susceptible to both HGPRT substrate analogs and inhibitors. A substance that strictly acts as an inhibitor will affect growth of transformed E. coli Sphi609 only. For this purpose, we compared the human and P. falciparum enzymes with known HGPRT substrate analogs. Our data with 6-mercaptopurine, 6-thioguanine and allopurinol show that these compounds act by being substrates for HGPRT. Our results with allopurinol suggest that it is a better substrate for P. falciparum HGXPRT than the human enzyme. Therefore, species-specific substrates can be tested out successfully in E. coli Sphi606. The formation of products from substrates like allopurinol lacking a labile proton at N7 raises the possibility that the deprotonation of substrates might occur at N9 rather than at N7 or a purine anion might be the true substrate for the reaction.

Changes in the natural history of progressive multifocal leukoencephalopathy in HIV-negative lymphoproliferative disorders: impact of novel therapies.

PubMed

García-Suárez, Julio; de Miguel, Dunia; Krsnik, Isabel; Bañas, Helena; Arribas, Ignacio; Burgaleta, Carmen

2005-12-01

The aims of this study were to evaluate the clinical characteristics of HIV-negative patients affected by lymphoproliferative disorders (LPD) who developed progressive multifocal leukoencephalopathy (PML), to delineate the risk factors, and to analyze whether the new antineoplastic therapies are changing the natural history of this infectious disease. We retrospectively analyzed 46 cases with confirmed LPD-associated PML published from 1958 to 2004. Patients were stratified according to two different time periods: group A included patients diagnosed before 1989, and group B included patients diagnosed since 1990, after introduction of purine analogues. Group A patients (n = 22) had received alkylating agents and/or radiotherapy, and the majority (63.6%) had advanced Hodgkin disease. At univariate analysis, uncontrolled Hodgkin disease was the only risk factor for PML. In group B patients (n = 24), the most frequent treatments received were purine analogues (58.3%) and high-dose therapy with hematopoietic stem cell transplantation (33.3%; HDT/HSCT). B-cell chronic lymphocytic leukemia (45.8%) and aggressive non-Hodgkin lymphoma (24.9%) were the most frequent underlying LPDs. Patients treated with purine analogues were more likely to have active LPD, lower CD4 cell counts, and to be older and male than were HSCT recipients. The median interval from purine analogues or HDT/HSCT to PML was 11 months. In HDT/HSCT recipients, this interval was delayed for 10 months when peri-transplantation rituximab was used. Univariate analysis identified age >55 years, male sex, and CD4 cell counts

Attempts to increase inosinic acid in broiler meat by using feed additives.

PubMed

Wang, X F; Liu, G H; Cai, H Y; Chang, W H; Ma, J S; Zheng, A J; Zhang, S

2014-11-01

To explore regulation of inosinic acid content in chicken meat as a result of feed additives, 576 one-day-old male Arbor Acres broilers were randomly allotted into 8 dietary treatments including control, purine nucleotide (P), betaine (B), soybean isoflavone (S), purine nucleotide + betaine (PB), purine nucleotide + soybean isoflavone (PS), betaine +soybean isoflavone (BS), and purine nucleotide + betaine + soybean isoflavone (PBS) by a 2 × 2 × 2 factorial arrangement. At d 42 of age, broilers were slaughtered, and growth performance, carcass characteristics, inosinic acid content, and activities of enzyme closely related to inosinic acid metabolism of broilers were measured. The results revealed that these feed additives did not affect ADG and ADFI of the broilers (P > 0.05). However, supplementing purine nucleotides lowered feed/gain of broilers in PS and PBS groups (P < 0.05). There was a significant interaction on feed/gain of broilers between purine nucleotides and soybean isoflavone (P < 0.05). The abdominal fat percentages in groups B, S, BS, and PBS were lower than the control group, respectively (P < 0.05). The thigh muscle percentages of groups P and B were higher than that of group PB (P < 0.05). There were certain interactions on the percentage of thigh muscle (P = 0.05) and abdominal fat (P < 0.05) between P, B, and S groups. Compared with the control group, inosinic acid content in broiler breast meat was improved by using feed additives (P < 0.05). Supplementing purine nucleotides, betaine, soybean isoflavone, and their combinations increased alkaline phosphatase activity in breast meat of broilers (P < 0.05). Purine nucleotides improved the activity of adenosine deaminase, but decreased the activity of 5'-nucleotidase. Soybean isoflavone lowered the activity of alkaline phosphatase. There were no significant interactions on activities of creatine kinase, adenosine deaminase, alkaline phosphatase, and 5'-nucleotidase between these additives (P > 0.05). The umami rating of broiler breast meat increased in conjunction with supplementing these additives. In conclusion, supplementing standard feed with the additives investigated in this study could improve inosinic acid content in chicken meat by increasing synthase activity or inhibiting degradation enzyme activity without inferior growth performance and carcass quality. ©2014 Poultry Science Association Inc.

Green Chemistry Challenge: 2017 Greener Synthetic Pathways Award

EPA Pesticide Factsheets

Green Chemistry Challenge 2017 award winners, Merck, developed a novel asymmetric aza-Michael cyclization, employing a chemically stable and fully recyclable organocatalyst to make Letermovir, an antiviral drug

Genomic and transcriptome analyses reveal that MAPK- and phosphatidylinositol-signaling pathways mediate tolerance to 5-hydroxymethyl-2-furaldehyde for industrial yeast Saccharomyces cerevisiae

PubMed Central

Zhou, Qian; Liu, Z. Lewis; Ning, Kang; Wang, Anhui; Zeng, Xiaowei; Xu, Jian

2014-01-01

The industrial yeast Saccharomyces cerevisiae is a traditional ethanologenic agent and a promising biocatalyst for advanced biofuels production using lignocellulose mateials. Here we present the genomic background of type strain NRRL Y-12632 and its transcriptomic response to 5-hydroxymethyl-2-furaldehyde (HMF), a commonly encountered toxic compound liberated from lignocellulosic-biomass pretreatment, in dissecting the genomic mechanisms of yeast tolerance. Compared with the genome of laboratory model strain S288C, we identified more than 32,000 SNPs in Y-12632 with 23,000 missense and nonsense SNPs. Enriched sequence mutations occurred for genes involved in MAPK- and phosphatidylinositol (PI)- signaling pathways in strain Y-12632, with 41 and 13 genes containing non-synonymous SNPs, respectively. Many of these mutated genes displayed consistent up-regulated signature expressions in response to challenges of 30 mM HMF. Analogous single-gene deletion mutations of these genes showed significantly sensitive growth response on a synthetic medium containing 20 mM HMF. Our results suggest at least three MAPK-signaling pathways, especially for the cell-wall integrity pathway, and PI-signaling pathways to be involved in mediation of yeast tolerance against HMF in industrial yeast Saccharomyces cerevisiae. Higher levels of sequence variations were also observed for genes involved in purine and pyrimidine metabolism pathways. PMID:25296911

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

PubMed Central

McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

2012-01-01

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

Airway purinergic responses in healthy, atopic nonasthmatic, and atopic asthmatic subjects exposed to ozone**

EPA Science Inventory

Context: Ozone exposure triggers airway inflammatory responses that maybe influenced bybiologically active purine metabolites. Objective:To examinethe relationships between airway purine metabolites and established inflammatory markers of ozone exposure, and to determine if thes...

Four new 6-oxy purine alkaloids from the South China Sea sponge, Haliclona cymaeformis

NASA Astrophysics Data System (ADS)

Chen, Min; Wu, Xudong; Shen, Nanxing; Wang, Changyun

2017-12-01

In this study, the chemical analysis of the marine sponge spieces, Haliclona cymaeformis, collected from the South China Sea was carried out, Two pairs of regioisomers of alkyl substitutional 6-oxy purine alkaloids ( 1a/ 1b and 2a/ 2b) were isolated. All of them possess two structural moieties, a 6-oxy purine nucleus and a pentan-2-one or hexan-2-one alkyl chain. Among them, 1a and 2a are the major N-9-substitutional regioisomers, and 1b and 2b are the minor N-7-substitutional regioisomers.

Changes in Purines Concentration in the Cerebrospinal Fluid of Pregnant Women Experiencing Pain During Active Labor.

PubMed

Schmidt, André P; Böhmer, Ana E; Hansel, Gisele; Soares, Félix A; Oses, Jean P; Giordani, Alex T; Posso, Irimar P; Auler, José Otávio C; Mendes, Florentino F; Félix, Elaine A; Portela, Luís V; Souza, Diogo O

2015-11-01

Labor pain has been reported as a severe pain and can be considered as a model of acute visceral pain. It is well known that extracellular purines have an important role in pain signaling in the central nervous system. This study analyzes the relationship between extracellular purines and pain perception during active labor. A prospective observational study was performed. Cerebrospinal fluid (CSF) levels of the purines and their metabolites were compared between women at term pregnancy with labor pain (n = 49) and without labor pain (Caesarian section; n = 47). Control groups (healthy men and women without chronic or acute pain-n = 40 and 32, respectively) were also investigated. The CSF levels of adenosine were significantly lower in the labor pain group (P = 0.026) and negatively correlated with pain intensity measured by a visual analogue scale (r = -0.48, P = 0.0005). Interestingly, CSF levels of uric acid were significantly higher in healthy men as compared to women. Additionally, pregnant women showed increased CSF levels of ADP, GDP, adenosine and guanosine and reduced CSF levels of AMP, GTP, and uric acid as compared to non-pregnant women (P < 0.05). These findings suggest that purines, in special the nucleoside adenosine, are associated with pregnancy and labor pain.

TD-DFT investigation of the magnetic circular dichroism spectra of some purine and pyrimidine bases of nucleic acids.

PubMed

Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Santoro, Fabrizio; Improta, Roberto; Coriani, Sonia

2015-05-28

We present a computational study of the magnetic circular dichroism (MCD) spectra in the 200-300 nm wavelength region of purine and its derivative hypoxanthine, as well as of the pyrimidine bases of nucleic acids uracil, thymine, and cytosine, using the B3LYP and CAM-B3LYP functionals. Solvent effects are investigated within the polarizable continuum model and by inclusion of explicit water molecules. In general, the computed spectra are found to be in good agreement with the experimental ones, apart from some overall blue shifts. Both the pseudo-A term shape of the MCD spectra of the purines and the B term shape of the spectra of pyrimidine bases are reproduced. Our calculations also correctly reproduce the reversed phase of the MCD bands in purine compared to that of its derivatives present in nucleic acids. Solvent effects are sizable and system specific, but they do not in general alter the qualitative shape of the spectra. The bands are dominated by the bright π → π* transitions, and our calculations in solution nicely reproduce their energy differences, improving the estimates obtained in the gas phase. Shoulders are predicted for purine and uracil due to n → π* excitations, but they are too weak to be observed in the experiment.

Distinct Distribution of Purines in CM and CR Carbonaceous Chondrites

NASA Technical Reports Server (NTRS)

Callahan, Michael P.; Stern, Jennifer C.; Glavin, Daniel P.; Smith, Karen E.; Martin, Mildred G.; Dworkin, Jason P.

2010-01-01

Carbonaceous meteorites contain a diverse suite of organic molecules and delivered pre biotic organic compounds, including purines and pyrimidines, to the early Earth (and other planetary bodies), seeding it with the ingredients likely required for the first genetic material. We have investigated the distribution of nucleobases in six different CM and CR type carbonaceous chondrites, including fivc Antarctic meteorites never before analyzed for nucleobases. We employed a traditional formic acid extraction protocol and a recently developed solid phase extraction method to isolate nucleobases. We analyzed these extracts by high performance liquid chromatography with UV absorbance detection and tandem mass spectrometry (HPLC-UV -MS/MS) targeting the five canonical RNAIDNA bases and hypoxanthine and xanthine. We detected parts-per-billion levels of nucleobases in both CM and CR meteorites. The relative abundances of the purines found in Antarctic CM and CR meteorites were clearly distinct from each other suggesting that these compounds are not terrestrial contaminants. One likely source of these purines is formation by HCN oligomerization (with other small molecules) during aqueous alteration inside the meteorite parent body. The detection of the purines adenine (A), guanine (0), hypoxanthine (HX), and xanthine (X) in carbonaceous meteorites indicates that these compounds should have been available on the early Earth prior to the origin of the first genetic material.

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

PubMed

Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

2009-06-17

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

Herbivorous turtle ants obtain essential nutrients from a conserved nitrogen-recycling gut microbiome.

PubMed

Hu, Yi; Sanders, Jon G; Łukasik, Piotr; D'Amelio, Catherine L; Millar, John S; Vann, David R; Lan, Yemin; Newton, Justin A; Schotanus, Mark; Kronauer, Daniel J C; Pierce, Naomi E; Moreau, Corrie S; Wertz, John T; Engel, Philipp; Russell, Jacob A

2018-03-06

Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants.

Physiological Roles of Plant Post-Golgi Transport Pathways in Membrane Trafficking.

PubMed

Uemura, Tomohiro

2016-10-01

Membrane trafficking is the fundamental system through which proteins are sorted to their correct destinations in eukaryotic cells. Key regulators of this system include RAB GTPases and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Interestingly, the numbers of RAB GTPases and SNAREs involved in post-Golgi transport pathways in plant cells are larger than those in animal and yeast cells, suggesting that plants have evolved unique and complex post-Golgi transport pathways. The trans-Golgi network (TGN) is an important organelle that acts as a sorting station in the post-Golgi transport pathways of plant cells. The TGN also functions as the early endosome, which is the first compartment to receive endocytosed proteins. Several endocytosed proteins on the plasma membrane (PM) are initially targeted to the TGN/EE, then recycled back to the PM or transported to the vacuole for degradation. The recycling and degradation of the PM localized proteins is essential for the development and environmental responses in plant. The present review describes the post-Golgi transport pathways that show unique physiological functions in plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

CED-10/Rac1 Regulates Endocytic Recycling through the RAB-5 GAP TBC-2

PubMed Central

Sun, Lin; Liu, Ou; Desai, Jigar; Karbassi, Farhad; Sylvain, Marc-André; Shi, Anbing; Zhou, Zheng; Rocheleau, Christian E.; Grant, Barth D.

2012-01-01

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane. PMID:22807685

Sorting of β1-Adrenergic Receptors Is Mediated by Pathways That Are Either Dependent on or Independent of Type I PDZ, Protein Kinase A (PKA), and SAP97*

PubMed Central

Nooh, Mohammed M.; Chumpia, Maryanne M.; Hamilton, Thomas B.; Bahouth, Suleiman W.

2014-01-01

The β1-adrenergic receptor (β1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β1-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β1-AR and on protein kinase A (PKA) activity (Gardner, L. A., Naren, A. P., and Bahouth, S. W. (2007) J. Biol. Chem. 282, 5085–5099). We explored the effects of point mutations in the PDZ and in the activity of PKA on recycling of the β1-AR and its binding to the PDZ-binding protein SAP97. These studies indicated that β1-AR recycling was inhibited by PKA inhibitors and by mutations in the PDZ that interfered with SAP97 binding. The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant β1-AR. β1-AR chimera containing the type I PDZ of the β2-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. β1-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and PKA-dependent manner. Non-PDZ β1-AR chimera derived from μ-opioid, dopamine 1, or GluR2 receptors promoted rapid recycling of chimeric β1-AR in a SAP97- and PKA-independent manner. Moreover, the nature of the residue at position −3 in the PDZ regulated whether the β1-AR was internalized alone or in complex with SAP97. These results indicate that divergent pathways were involved in trafficking the β1-AR and provide a roadmap for its trafficking via type I PDZs versus non-PDZs. PMID:24324269

Proteomic analysis of the signaling pathway mediated by the heterotrimeric Gα protein Pga1 of Penicillium chrysogenum.

PubMed

Carrasco-Navarro, Ulises; Vera-Estrella, Rosario; Barkla, Bronwyn J; Zúñiga-León, Eduardo; Reyes-Vivas, Horacio; Fernández, Francisco J; Fierro, Francisco

2016-10-06

The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. Penicillium chrysogenum mutants with different levels of activity of the Pga1-mediated signaling pathway were used to perform comparative proteomic analyses by 2D-DIGE and LC-MS/MS. Thirty proteins were identified which showed differences in abundance dependent on Pga1 activity level. By modifying the intracellular levels of cAMP we could establish cAMP-dependent and cAMP-independent pathways in Pga1-mediated signaling. Pga1 was shown to regulate abundance of enzymes in primary metabolic pathways involved in ATP, NADPH and cysteine biosynthesis, compounds that are needed for high levels of penicillin production. An in vivo phosphorylated protein containing a pleckstrin homology domain was identified; this protein is a candidate for signal transduction activity. Proteins with possible roles in purine metabolism, protein folding, stress response and morphogenesis were also identified whose abundance was regulated by Pga1 signaling. Thirty proteins whose abundance was regulated by the Pga1-mediated signaling pathway were identified. These proteins are involved in primary metabolism, stress response, development and signal transduction. A model describing the pathways through which Pga1 signaling regulates different cellular processes is proposed.

Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

PubMed Central

Connor, Michael G.; Pulsifer, Amanda R.; Ceresa, Brian K.

2018-01-01

ABSTRACT Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV. PMID:29463656

Growth in rice cells requires de novo purine biosynthesis by the blast fungus Magnaporthe oryzae

PubMed Central

Fernandez, Jessie; Yang, Kuan Ting; Cornwell, Kathryn M.; Wright, Janet D.; Wilson, Richard A.

2013-01-01

Increasing incidences of human disease, crop destruction and ecosystem perturbations are attributable to fungi and threaten socioeconomic progress and food security on a global scale. The blast fungus Magnaporthe oryzae is the most devastating pathogen of cultivated rice, but its metabolic requirements in the host are unclear. Here we report that a purine-requiring mutant of M. oryzae could develop functional appressoria, penetrate host cells and undergo the morphogenetic transition to elaborate bulbous invasive hyphae from primary hyphae, but further in planta growth was aborted. Invasive hyphal growth following rice cell ingress is thus dependent on de novo purine biosynthesis by the pathogen and, moreover, plant sources of purines are neither available to the mutant nor required by the wild type during the early biotrophic phase of infection. This work provides new knowledge about the metabolic interface between fungus and host that might be applicable to other important intracellular fungal pathogens. PMID:23928947

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides.

PubMed

Nakabeppu, Yusaku; Kajitani, Kosuke; Sakamoto, Katsumi; Yamaguchi, Hiroo; Tsuchimoto, Daisuke

2006-07-13

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.

Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

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

Timofeev, V. I., E-mail: tostars@mail.ru; Abramchik, Yu. A., E-mail: ugama@yandex.ru; Zhukhlistova, N. E., E-mail: inna@ns.crys.ras.ru

2016-03-15

Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment ofmore » the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB-ID: 4RJ2).« less

Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility.

PubMed

Lindsay, Andrew J; McCaffrey, Mary W

2017-12-01

Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of α5β1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells.

Patterns of purine nucleotides in fish erythrocytes.

PubMed

Leray, C

1979-01-01

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

Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

PubMed

Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

2003-12-05

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

A new route for the prebiotic synthesis of nucleobases and hydantoins in water/ice solutions involving the photochemistry of acetylene.

PubMed

Menor-Salván, César; Marín-Yaseli, Margarita R

2013-05-10

The origin of nucleobases and other heterocycles is a classic question in the chemistry of the origins of life. The construction of laboratory models for the abiotic synthesis of nitrogen heterocycles in plausible natural conditions also aids the understanding and prediction of chemical species in the Solar System. Here, we report a new explanation for the origin of hydantoins, purines, and pyrimidines in eutectic water/ice/urea solutions driven by ultraviolet irradiation (in the 185-254 nm range, UVC) of acetylene under anoxic conditions. An analysis of the products indicates the synthesis of hydantoin and 5-hydroxyhydantoin, the purines uric acid, xanthine, and guanine, and the pyrimidines uracil and cytosine. The synthesis occurred together with the photo-oxidation of bases in a complex process for which possible pathways are proposed. In conclusion, an acetylene-containing atmosphere could contribute to the origin of nucleobases in the presence of a urea/water system by an HCN-independent mechanism. The presence of ice has a dual role as a favorable medium for the synthesis of nucleobases and protection against degradation and as a source of free radicals for the synthesis of highly oxidized heterocycles. A mechanism for the origin of hydantoins and uracil from urea in plausible conditions for prebiotic chemistry is also proposed. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evidence of extensive plasma glucose recycling following a glucose load in seabass.

PubMed

Rito, João; Viegas, Ivan; Pardal, Miguel A; Jones, John G

2017-09-01

Seabass and other carnivorous fish are highly dependent on gluconeogenesis from dietary amino acids to maintain glycemia. Glucose recycling (glucose→C3-intermediate→glucose) may potentiate the effects of glucose administration in sparing amino acid gluconeogenesis. To date, very few measurements of glucose recycling have been reported in fish. Thus, to determine the extent of glucose recycling following a glycemic challenge, juvenile seabass were given an intraperitoneal glucose load (2gkg -1 ) enriched with [U- 13 C]glucose. 13 C NMR analysis of plasma glucose 13 C-isotopomers was used to determine the fractional contributions of glucose derived directly from the load versus that from glucose recycling at 48h after the load. Both fed and 21-day fasted fish (20 per condition) were studied. In fasted fish, 18±4% of plasma glucose was directly derived from the load while 13±2% was derived from glucose recycling. In fed fish, the load accounted for 6±1% of plasma glucose levels while glucose recycling contributed 16±4%. 13 C NMR analysis of plasma lactate revealed 13 C-isotopomers corresponding to the expected C3-intermediates of peripheral [U- 13 C]glucose catabolism indicating that circulating lactate was a key intermediate in glucose carbon recycling under these conditions. In conclusion, glucose recycling was shown to contribute a significant portion of plasma glucose levels in both fed and fasted seabass 48h after an intraperitoneal glucose challenge and circulating lactate was shown to be an intermediate of this pathway. Copyright © 2017. Published by Elsevier Inc.

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

PubMed Central

Solis, Gonzalo P.; Hülsbusch, Nikola; Radon, Yvonne; Katanaev, Vladimir L.; Plattner, Helmut; Stuermer, Claudia A. O.

2013-01-01

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca2+ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca2+ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved. PMID:23825023

The high stability of the triple helices formed between short purine oligonucleotides and SIV/HIV-2 vpx genes is determined by the targeted DNA structure.

PubMed Central

Svinarchuk, F; Monnot, M; Merle, A; Malvy, C; Fermandjian, S

1995-01-01

In our previous works we have shown that the oligonucleotides 5'-GGGGAGGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3' give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol. Chem., 270, 14 068-14,071]. The target for the invariable part of these oligonucleotides, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here were report on the ability of four purine oligonucleotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichroism (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the triplexes. The melting temperatures of the triplexes were found to be the same as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8 degrees C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleotide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation. Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the third strand oligonucleotide: the CD spectrum shows a 'classical' A-DNA shape with the 20-mer. This is not observed with the purine/pyrimidine stretch of the HIV-1 DNA which keeps a B-like spectrum even after triplex formation. We suggest, that an A-like duplex DNA is required for the formation of a stable DNA purine(purine-pyrimidine) triplex. Images PMID:7479024

Purines and Neuronal Excitability: Links to the Ketogenic Diet

PubMed Central

Masino, SA; Kawamura, M; Ruskin, DN; Geiger, JD; Boison, D

2011-01-01

ATP and adenosine are purines that play dual roles in cell metabolism and neuronal signaling. Acting at the A1 receptor (A1R) subtype, adenosine acts directly on neurons to inhibit excitability and is a powerful endogenous neuroprotective and anticonvulsant molecule. Previous research showed an increase in ATP and other cell energy parameters when an animal is administered a ketogenic diet, an established metabolic therapy to reduce epileptic seizures, but the relationship among purines, neuronal excitability and the ketogenic diet was unclear. Recent work in vivo and in vitro tested the specific hypothesis that adenosine acting at A1Rs is a key mechanism underlying the success of ketogenic diet therapy and yielded direct evidence linking A1Rs to the antiepileptic effects of a ketogenic diet. Specifically, an in vitro mimic of a ketogenic diet revealed an A1R-dependent metabolic autocrine hyperpolarization of hippocampal neurons. In parallel, applying the ketogenic diet in vivo to transgenic mouse models with spontaneous electrographic seizures revealed that intact A1Rs are necessary for the seizure-suppressing effects of the diet. This is the first direct in vivo evidence linking A1Rs to the antiepileptic effects of a ketogenic diet. Other predictions of the relationship between purines and the ketogenic diet are discussed. Taken together, recent research on the role of purines may offer new opportunities for metabolic therapy and insight into its underlying mechanisms. PMID:21880467

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

PubMed

Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

MARK2/EMK1/Par-1Balpha phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells.

PubMed

Ducharme, Nicole A; Hales, Chadwick M; Lapierre, Lynne A; Ham, Amy-Joan L; Oztan, Asli; Apodaca, Gerard; Goldenring, James R

2006-08-01

Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Balpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.

Phloem-Specific Methionine Recycling Fuels Polyamine Biosynthesis in a Sulfur-Dependent Manner and Promotes Flower and Seed Development1[OPEN

PubMed Central

Hajirezaei, Mohammad R.

2016-01-01

The Yang or Met Cycle is a series of reactions catalyzing the recycling of the sulfur (S) compound 5′-methylthioadenosine (MTA) to Met. MTA is produced as a by-product in ethylene, nicotianamine, and polyamine biosynthesis. Whether the Met Cycle preferentially fuels one of these pathways in a S-dependent manner remained unclear so far. We analyzed Arabidopsis (Arabidopsis thaliana) mutants with defects in the Met Cycle enzymes 5-METHYLTHIORIBOSE-1-PHOSPHATE-ISOMERASE1 (MTI1) and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (DEP1) under different S conditions and assayed the contribution of the Met Cycle to the regeneration of S for these pathways. Neither mti1 nor dep1 mutants could recycle MTA but showed S-dependent reproductive failure, which was accompanied by reduced levels of the polyamines putrescine, spermidine, and spermine in mutant inflorescences. Complementation experiments with external application of these three polyamines showed that only the triamine spermine could specifically rescue the S-dependent reproductive defects of the mutant plants. Furthermore, expressing gene-reporter fusions in Arabidopsis showed that MTI1 and DEP1 were mainly expressed in the vasculature of all plant parts. Phloem-specific reconstitution of Met Cycle activity in mti1 and dep1 mutant plants was sufficient to rescue their S-dependent mutant phenotypes. We conclude from these analyses that phloem-specific S recycling during periods of S starvation is essential for the biosynthesis of polyamines required for flowering and seed development. PMID:26662272

Arf6 negatively controls the rapid recycling of the β2 adrenergic receptor.

PubMed

Macia, Eric; Partisani, Mariagrazia; Paleotti, Olivia; Luton, Frederic; Franco, Michel

2012-09-01

β2-adrenergic receptor (β2AR), a member of the GPCR (G-protein coupled receptor) family, is internalized in a ligand- and β-arrestin-dependent manner into early endosomes, and subsequently recycled back to the plasma membrane. Here, we report that β-arrestin promotes the activation of the small G protein Arf6, which regulates the recycling and degradation of β2AR. We demonstrate in vitro that the C-terminal region of β-arrestin1 interacts directly and simultaneously with Arf6GDP and its specific exchange factor EFA6, to promote Arf6 activation. Similarly, the ligand-mediated activation of β2AR leads to the formation of Arf6GTP in vivo in a β-arrestin-dependent manner. Expression of either EFA6 or an activated Arf6 mutant caused accumulation of β2AR in the degradation pathway. This phenotype could be rescued by the expression of an activated mutant of Rab4, suggesting that Arf6 acts upstream of Rab4. We propose a model in which Arf6 plays an essential role in β2AR desensitization. The ligand-mediated stimulation of β2AR relocates β-arrestin to the plasma membrane, and triggers the activation of Arf6 by EFA6. The activation of Arf6 leads to accumulation of β2AR in the degradation pathway, and negatively controls Rab4-dependent fast recycling to prevent the re-sensitization of β2AR.

Crystal Structures of the Helicobacter pylori MTAN Enzyme Reveal Specific Interactions between S-Adenosylhomocysteine and the 5'-Alkylthio Binding Subsite

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

Mishra, Vidhi; Ronning, Donald R.

2012-11-13

The bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadenosine (5'-DOA), and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for Helicobacter pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and themore » 5'-alkylthiol binding site of MTAN have never been resolved. We have determined crystal structures of an inactive mutant form of H. pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH, allowing the determination of the structure of a ternary enzyme–product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5'-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori.« less

Process for producing 8-fluoropurines

DOEpatents

Barrio, J.R.; Satyamurthy, N.; Namavari, M.; Phelps, M.E.

1999-01-19

An efficient, regio-controlled approach to the synthesis of 8-fluoropurines by direct fluorination of purines with dilute elemental fluorine, or acetyl hypofluorite, is provided. In a preferred embodiment, a purine compound is dissolved in a polar solvent and reacted with a dilute mixture of F{sub 2} in He or other inert gas.

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.

A Synthetic Alternative to Canonical One-Carbon Metabolism.

PubMed

Bouzon, Madeleine; Perret, Alain; Loreau, Olivier; Delmas, Valérie; Perchat, Nadia; Weissenbach, Jean; Taran, Frédéric; Marlière, Philippe

2017-08-18

One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO 2 . The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.

Presidential Green Chemistry Challenge: 2008 Greener Synthetic Pathways Award

EPA Pesticide Factsheets

Presidential Green Chemistry Challenge 2008 award winner, Battelle, developed a biobased soy toner for laser printers and copiers. The technology saves energy and improves de-inking, allowing more paper fiber to be recycled.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione) Blocks the Chemotaxis of Neutrophils by Inhibiting Signal Transduction through IL-8 Receptors

PubMed Central

Takahashi, Masafumi; Ishiko, Takatoshi; Kamohara, Hidenobu; Hidaka, Hideaki; Ikeda, Osamu; Ogawa, Michio; Baba, Hideo

2007-01-01

We investigated the impact of curcumin on neutrophils. Chemotactic activity via human recombinant IL-8 (hrIL-8) was significantly inhibited by curcumin. Curcumin reduced calcium ion flow induced by internalization of the IL-8 receptor. We analyzed flow cytometry to evaluate the status of the IL-8 receptor after curcumin treatment. The change in the distribution of receptors intracellularly and on the cell surface suggested that curcumin may affect the receptor trafficking pathway intracellulary. Rab11 is a low molecular weight G protein associated with the CXCR recycling pathway. Following curcumin treatment, immunoprecipitation studies showed that the IL-8 receptor was associated with larger amounts of active Rab11 than that in control cells. These data suggest that curcumin induces the stacking of the Rab11 vesicle complex with CXCR1 and CXCR2 in the endocytic pathway. The mechanism for antiinflammatory response by curcumin may involve unique regulation of the Rab11 trafficking molecule in recycling of IL-8 receptors. PMID:17710245

Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

PubMed Central

Scharbarg, Emeric; Daenens, Marion; Lemaître, Frédéric; Geoffroy, Hélène; Guille-Collignon, Manon; Gallopin, Thierry; Rancillac, Armelle

2016-01-01

Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity. PMID:26755200

Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth.

PubMed

Cox, Andrew G; Hwang, Katie L; Brown, Kristin K; Evason, Kimberley; Beltz, Sebastian; Tsomides, Allison; O'Connor, Keelin; Galli, Giorgio G; Yimlamai, Dean; Chhangawala, Sagar; Yuan, Min; Lien, Evan C; Wucherpfennig, Julia; Nissim, Sahar; Minami, Akihiro; Cohen, David E; Camargo, Fernando D; Asara, John M; Houvras, Yariv; Stainier, Didier Y R; Goessling, Wolfram

2016-08-01

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver

PubMed Central

Blaber, Elizabeth A.; Pecaut, Michael J.

2017-01-01

Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-‘omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment. PMID:28953266

Towards generating caffeine-free tea by metabolic engineering.

PubMed

Yadav, Sudesh Kumar; Ahuja, Paramvir Singh

2007-12-01

Tea is a rich source of antioxidants which are contributing substantially to the promotion of health and the prevention of various chronic diseases. Despite the fact that tea has various important compounds, it also contains a purine alkaloid, caffeine. High intake of tea leads to an increase in level of caffeine in addition to its important antioxidant constituents. Increased level of caffeine causes several health related problems. Therefore, tea can become a most useful source of beneficial compounds, if only its caffeine level is either decreased or eliminated all together from the plant itself. This could be achieved through either of the techniques; overexpressing caffeine degradative pathway genes or silencing caffeine biosynthesis pathway gene. The identification and cloning of caffeine biosynthesis in tea and degradative genes in microorganisms opens up the possibility of using genetic engineering to produce naturally decaffeinated tea. Here we review these different strategies which can be employed to make caffeine-free tea, a human health beneficial drink.

Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth

PubMed Central

Brown, Kristin K.; Evason, Kimberley; Beltz, Sebastian; Tsomides, Allison; O'Connor, Keelin; Galli, Giorgio G.; Yimlamai, Dean; Chhangawala, Sagar; Yuan, Min; Lien, Evan C.; Wucherpfennig, Julia; Nissim, Sahar; Minami, Akihiro; Cohen, David E.; Camargo, Fernando D.; Asara, John M.; Houvras, Yariv; Stainier, Didier Y.R.; Goessling, Wolfram

2016-01-01

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signaling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumor formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppresses hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis. PMID:27428308

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver.

PubMed

Blaber, Elizabeth A; Pecaut, Michael J; Jonscher, Karen R

2017-09-27

Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-'omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment.

The Intracellular Trafficking Pathway of Transferrin

PubMed Central

Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

2011-01-01

Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse

PubMed Central

Augustine, G.J.; Morgan, J.R.; Villalba-Galea, C.A.; Jin, S.; Prasad, K.; Lafer, E.M.

2015-01-01

The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions. PMID:16417485

Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance.

PubMed

Yang, Jia; Xu, Ming; Zhang, Xuezhi; Hu, Qiang; Sommerfeld, Milton; Chen, Yongsheng

2011-01-01

This research examines the life-cycle water and nutrients usage of microalgae-based biodiesel production. The influence of water types, operation with and without recycling, algal species, geographic distributions are analyzed. The results confirm the competitiveness of microalgae-based biofuels and highlight the necessity of recycling harvested water and using sea/wastewater as water source. To generate 1 kg biodiesel, 3726 kg water, 0.33 kg nitrogen, and 0.71 kg phosphate are required if freshwater used without recycling. Recycling harvest water reduces the water and nutrients usage by 84% and 55%. Using sea/wastewater decreases 90% water requirement and eliminates the need of all the nutrients except phosphate. The variation in microalgae species and geographic distribution are analyzed to reflect microalgae biofuel development in the US. The impacts of current federal and state renewable energy programs are also discussed to suggest suitable microalgae biofuel implementation pathways and identify potential bottlenecks. Copyright © 2010 Elsevier Ltd. All rights reserved.

40 CFR 721.4685 - Substituted purine metal salt (generic name).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted purine metal salt (generic name). 721.4685 Section 721.4685 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4685...

Isolation of Purines and Pyrimidines from the Murchison Meteorite Using Sublimation

NASA Technical Reports Server (NTRS)

Glavin, D. P.; Bada, J. L.

2004-01-01

The origin of life on Earth, and possibly on other planets such as Mars, would have required the presence of liquid water and a continuous supply of prebiotic organic compounds. The exogenous delivery of organic matter by asteroids, comets, and carbonaceous meteorites could have contributed to the early Earth s prebiotic inventory by seeding the planet with biologically important organic compounds. A wide variety of prebiotic organic compounds have previously been detected in the Murchison CM type carbonaceous chondrite including amino acids, purines and pyrimidines. These compounds dominate terrestrial biochemistry and are integral components of proteins, DNA and RNA. Several purines including adenine, guanine, hypoxanthine, and xanthine, as well as the pyrimidine uracil, have previously been detected in water or formic acid extracts of Murchison using ion-exclusion chromatography and ultraviolet spectroscopy. However, even after purification of these extracts, the accurate identification and quantification of nucleobases is difficult due to interfering UV absorbing compounds. In order to reduce these effects, we have developed an extraction technique using sublimation to isolate purines and pyrimidines from other non-volatile organic compounds in Murchison acid extracts.

Profiles of phenolic compounds and purine alkaloids during the development of seeds of Theobroma cacao cv. Trinitario.

PubMed

Pereira-Caro, Gema; Borges, Gina; Nagai, Chifumi; Jackson, Mel C; Yokota, Takao; Crozier, Alan; Ashihara, Hiroshi

2013-01-16

Changes occurring in phenolic compounds and purine alkaloids, during the growth of seeds of cacao (Theobroma cacao) cv. Trinitario, were investigated using HPLC-MS/MS. Extracts of seeds with a fresh weight of 125, 700, 1550, and 2050 mg (stages 1-4, respectively) were analyzed. The phenolic compounds present in highest concentrations in developing and mature seeds (stages 3 and 4) were flavonols and flavan-3-ols. Flavan-3-ols existed as monomers of epicatechin and catechin and as procyanidins. Type B procyanidins were major components and varied from dimers to pentadecamer. Two anthocyanins, cyanidin-3-O-arabinoside and cyanidin-3-O-galactoside, along with the N-phenylpropernoyl-l-amino acids, N-caffeoyl-l-aspartate, N-coumaroyl-l-aspartate, N-coumaroyl-3-hydroxytyrosine (clovamide), and N-coumaroyltyrosine (deoxyclovamide), and the purine alkaloids theobromine and caffeine, were present in stage 3 and 4 seeds. Other purine alkaloids, such as theophylline and additional methylxanthines, did not occur in detectable quantities. Flavan-3-ols were the only components to accumulate in detectable quantities in young seeds at developmental stages 1 and 2.

‘GREENER’ CHEMICAL SYNTHESES USING MICROWAVES

EPA Science Inventory

The diverse nature of chemical entities requires various green strategic pathways in our quest towards attaining sustainability. A solvent-free approach that involves microwave (MW) exposure of neat reactants (undiluted) catalyzed by the surfaces of less-expensive and recyclable ...

Materials Management: State of the Practice 2012

EPA Science Inventory

The report presents an evaluation of six materials management technologies that were assessed with respect to each technology's process, material and energy pathways, commercialization status, and associated economics. The inclusion and implementation of a materials recycling pr...

Purine ionotropic (P2X) receptors.

PubMed

Köles, L; Fürst, S; Illes, P

2007-01-01

Purinergic signaling is involved in the proper functioning of virtually all organs of the body. Although in some cases purines have a major influence on physiological functions (e.g. thrombocyte aggregation), more often they are just background modulators contributing to fine tuning of biological events. However, under pathological conditions, when a huge amount of adenosine 5'-triphosphate (ATP) can reach the extracellular space, their significance is increasing. ATP and its various degradation products activate membrane receptors divided into two main classes: the metabotropic P2Y and the ionotropic P2X family. This latter group, the purine ionotropic receptor, is the object of this review. After providing a description about the distribution and functional properties of P2X receptors in the body, their pharmacology will be summarized. In the second part of this review, the role of purines in those organ systems and body functions will be highlighted, where the (patho)physiological role of P2X receptors has been suggested or is even well established. Besides the regulation of organ systems, for instance in the cardiovascular, respiratory, genitourinary or gastrointestinal system, some special issues will also be discussed, such as the role of P2X receptors in pain, tumors, central nervous system (CNS) injury and embryonic development. Several examples will indicate that purine ionotropic receptors might serve as attractive targets for pharmacological interventions in various diseases, and that selective ligands for these receptors will probably constitute important future therapeutic tools in humans.

Antioxidant effect of naturally occurring xanthines on the oxidative damage of DNA bases

NASA Astrophysics Data System (ADS)

Vieira, A. J. S. C.; Telo, J. P.; Pereira, H. F.; Patrocínio, P. F.; Dias, R. M. B.

1999-01-01

The repair of the oxidised radicals of adenine and guanosine by several naturally occurring xanthines was studied. Each pair of DNA purine/xanthine was made to react with the sulphate radical and the decrease of the concentration of both compounds was measured by HPLC as a function of irradiation time. The results show that xanthine efficiently prevents the oxidation of the two DNA purines. Theophyline and paraxanthine repair the oxidised radical of adenine but not the one from guanosine. Theobromine and caffeine do not show any protecting effect. An order of the oxidation potentials of all the purines studied is proposed. La réparation des radicaux oxydés de l'adénine et de la guanosine par des xanthines naturelles a été étudiée en soumettant chaque paire base de l'ADN/xanthine à l'oxydation par le radical sulfate et en mesurant par HPLC la disparition des deux composés en fonction du temps d'irradiation. Les résultats montrent que la xanthine joue un rôle protecteur efficace contre l'oxydation des deux purines de l'ADN. La théophyline et la paraxanthine réparent le radical oxydé de l'adénine mais pas celui de la guanosine. La théobromine et la cafeíne n'ont pas d'effet protecteur. Un ordre de potentiels d'oxydation des purines étudiées est proposé.

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

Purines and neuronal excitability: links to the ketogenic diet.

PubMed

Masino, S A; Kawamura, M; Ruskin, D N; Geiger, J D; Boison, D

2012-07-01

ATP and adenosine are purines that play dual roles in cell metabolism and neuronal signaling. Acting at the A(1) receptor (A(1)R) subtype, adenosine acts directly on neurons to inhibit excitability and is a powerful endogenous neuroprotective and anticonvulsant molecule. Previous research showed an increase in ATP and other cell energy parameters when an animal is administered a ketogenic diet, an established metabolic therapy to reduce epileptic seizures, but the relationship among purines, neuronal excitability and the ketogenic diet was unclear. Recent work in vivo and in vitro tested the specific hypothesis that adenosine acting at A(1)Rs is a key mechanism underlying the success of ketogenic diet therapy and yielded direct evidence linking A(1)Rs to the antiepileptic effects of a ketogenic diet. Specifically, an in vitro mimic of a ketogenic diet revealed an A(1)R-dependent metabolic autocrine hyperpolarization of hippocampal neurons. In parallel, applying the ketogenic diet in vivo to transgenic mouse models with spontaneous electrographic seizures revealed that intact A(1)Rs are necessary for the seizure-suppressing effects of the diet. This is the first direct in vivo evidence linking A(1)Rs to the antiepileptic effects of a ketogenic diet. Other predictions of the relationship between purines and the ketogenic diet are discussed. Taken together, recent research on the role of purines may offer new opportunities for metabolic therapy and insight into its underlying mechanisms. Copyright © 2011 Elsevier B.V. All rights reserved.

Targeting MUC1-Mediated Tumor-Stromal Metabolic Interactions in Triple-Negative Breast Cancer

DTIC Science & Technology

2015-09-01

exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid . Lactic acid is mainly transported by two H+/lactate symporters...and ammonia recycling were most significantly altered in MDA-MB468 closely followed by citric acid cycle pathway. Mitochondrial electron transport...chain and citric acid cycle pathways were most significantly altered in BT20. Furthermore, relative comparison of the fold change in individual

Fever is not responsible for the elevated glucose kinetics in sepsis

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

Lang, C.H.; Bagby, G.J.; Blakesley, H.L.

Previous studies have suggested that alterations in the classical neuroendocrine system may not be responsible for the increased glucose metabolism observed during hypermetabolic sepsis. The purpose of the present study was to determine whether inhibition of the cyclooxygenase pathway with indomethacin, which prevents the production of arachidonic acid metabolites by this pathway and the sepsis-induced increase in body temperature, would abolish the increases in glucose appearance (Ra), recycling, and hyperlactacidemia. Sepsis was induced in chronically catheterized conscious rats by multiple injections of live Escherichia coli via a subcutaneous catheter. Septic animals received iv injections of indomethacin every 6-8 hr tomore » block the cyclooxygenase pathway. Glucose kinetics were assessed in 24-hr fasted rats using a constant iv infusion of (6-/sup 3/H)- and (U-/sup 14/C) glucose. Treatment with indomethacin prevented the 1-2/sup 0/C increase in body temperature observed in septic animals. Septic rats exhibited an elevated plasma lactate concentration and increased rates of glucose appearance and recycling. The sepsis-induced alterations in these variables were not attenuated by indomethacin. These results suggest that neither elevated body temperature nor the generation of arachidonic acid metabolites of the cyclooxygenase pathway is responsible for increasing glucose production in hypermetabolic septic rats.« less

Purines and Carotid Body: New Roles in Pathological Conditions

PubMed Central

Conde, Silvia V.; Monteiro, Emilia C.; Sacramento, Joana F.

2017-01-01

It is known that adenosine and adenosine-5′-triphosphate (ATP) are excitatory mediators involved in carotid body (CB) hypoxic signaling. The CBs are peripheral chemoreceptors classically defined by O2, CO2, and pH sensors. When hypoxia activates the CB, it induces the release of neurotransmitters from chemoreceptor cells leading to an increase in the action potentials frequency at the carotid sinus nerve (CSN). This increase in the firing frequency of the CSN is integrated in the brainstem to induce cardiorespiratory compensatory responses. In the last decade several pathologies, as, hypertension, diabetes, obstructive sleep apnea and heart failure have been associated with CB overactivation. In the first section of the present manuscript we review in a concise manner fundamental aspects of purine metabolism. The second section is devoted to the role of purines on the hypoxic response of the CB, providing the state-of-the art for the presence of adenosine and ATP receptors in the CB; for the role of purines at presynaptic level in CB chemoreceptor cells, as well as, its metabolism and regulation; at postsynaptic level in the CSN activity; and on the ventilatory responses to hypoxia. Recently, we have showed that adenosine is involved in CB hypersensitization during chronic intermittent hypoxia (CIH), which mimics obstructive sleep apnea, since caffeine, a non-selective adenosine receptor antagonist that inhibits A2A and A2B adenosine receptors, decreased CSN chemosensory activity in animals subjected to CIH. Apart from this involvement of adenosine in CB sensitization in sleep apnea, it was recently found that P2X3 ATP receptor in the CB contributes to increased chemoreflex hypersensitivity and hypertension in spontaneously hypertension rats. Therefore the last section of this manuscript is devoted to review the recent findings on the role of purines in CB-mediated pathologies as hypertension, diabetes and sleep apnea emphasizing the potential clinical importance of modulating purines levels and action to treat pathologies associated with CB dysfunction. PMID:29311923

T.C.G triplet in an antiparallel purine.purine.pyrimidine DNA triplex. Conformational studies by NMR.

PubMed

Dittrich, K; Gu, J; Tinder, R; Hogan, M; Gao, X

1994-04-12

The antiparallel purine.purine.pyrimidine DNA triplex, RRY6, which contains a T.C.G inverted triplet in the center of the sequence, was examined by proton and phosphorous two-dimensional NMR spectroscopy. The local conformation of the T.C.G triplet (T4.C11.G18) and the effect of this triplet on the global helical structure were analyzed in detail. The formation of the T.C.G triplet is confirmed by a set of cross-strand NOEs, including unusual cross-strand NOEs between the third strand and the pyrimidine strand as opposed to the purine strand of the duplex. NMR data suggest that the T.C.G triplet may be present in an equilibrium between a non-hydrogen-bonded form and a T(O4)-C(NH2) hydrogen-bonded form and that there is a distortion of the in-plane alignment of the three bases. The flanking G.G.C base triplets are well-defined on the 5'-side of T4, but somewhat interrupted on the 3'-side of T4. The effect of the third strand binding on the Watson-Crick duplex was probed by an NMR study of the free duplex RY6. NMR parameters are affected mostly around the T.C.G inversion site. The perturbations extend to at least two adjacent base triplets on either side. The binding of the third purine strand and the accommodation of a central T.C.G inversion in RRY6 does not require a readjustment in sugar pucker, which remains in the range of C2'-endo. 31P resonances of RRY6 distribute over a range of 2.2 ppm. The H-P coupling patterns of the third strand differ from those of the duplex. General spectral patterns defined by the marker protons of the RRY and YRY triplexes are compared.

Oral choline decreases brain purine levels in lithium-treated subjects with rapid-cycling bipolar disorder: a double-blind trial using proton and lithium magnetic resonance spectroscopy.

PubMed

Lyoo, In Kyoon; Demopulos, Christina M; Hirashima, Fuyuki; Ahn, Kyung Heup; Renshaw, Perry F

2003-08-01

Oral choline administration has been reported to increase brain phosphatidylcholine levels. As phospholipid synthesis for maintaining membrane integrity in mammalian brain cells consumes approximately 10-15% of the total adenosine triphosphate (ATP) pool, an increased availability of brain choline may lead to an increase in ATP consumption. Given reports of genetic studies, which suggest mitochondrial dysfunction, and phosphorus (31P) magnetic resonance spectroscopy (MRS) studies, which report dysfunction in high-energy phosphate metabolism in patients with bipolar disorder, the current study is designed to evaluate the role of oral choline supplementation in modifying high-energy phosphate metabolism in subjects with bipolar disorder. Eight lithium-treated patients with DSM-IV bipolar disorder, rapid cycling type were randomly assigned to 50 mg/kg/day of choline bitartrate or placebo for 12 weeks. Brain purine, choline and lithium levels were assessed using 1H- and 7Li-MRS. Patients received four to six MRS scans, at baseline and weeks 2, 3, 5, 8, 10 and 12 of treatment (n = 40 scans). Patients were assessed using the Clinical Global Impression Scale (CGIS), the Young Mania Rating Scale (YRMS) and the Hamilton Depression Rating Scale (HDRS) at each MRS scan. There were no significant differences in change-from-baseline measures of CGIS, YMRS, and HDRS, brain choline/creatine ratios, and brain lithium levels over a 12-week assessment period between the choline and placebo groups or within each group. However, the choline treatment group showed a significant decrease in purine metabolite ratios from baseline (purine/n-acetyl aspartate: coef = -0.08, z = -2.17, df = 22, p = 0.030; purine/choline: coef = -0.12, z = -1.97, df = 22, p = 0.049) compared to the placebo group, controlling for brain lithium level changes. Brain lithium level change was not a significant predictor of purine ratios. The current study reports that oral choline supplementation resulted in a significant decrease in brain purine levels over a 12-week treatment period in lithium-treated patients with DSM-IV bipolar disorder, rapid-cycling type, which may be related to the anti-manic effects of adjuvant choline. This result is consistent with mitochondrial dysfunction in bipolar disorder inadequately meeting the demand for increased ATP production as exogenous oral choline administration increases membrane phospholipid synthesis.

Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior.

PubMed

Gromova, Kira V; Muhia, Mary; Rothammer, Nicola; Gee, Christine E; Thies, Edda; Schaefer, Irina; Kress, Sabrina; Kilimann, Manfred W; Shevchuk, Olga; Oertner, Thomas G; Kneussel, Matthias

2018-05-29

Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

PubMed

Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Exciplexes and conical intersections lead to fluorescence quenching in π-stacked dimers of 2-aminopurine with natural purine nucleobases†

PubMed Central

Liang, JingXin; Nguyen, Quynh L.; Matsika, Spiridoula

2016-01-01

Fluorescent analogues of the natural DNA bases are useful in the study of nucleic acids’ structure and dynamics. 2-Aminopurine (2AP) is a widely used analogue with environmentally sensitive fluorescence behavior. The quantum yield of 2AP has been found to be significantly decreased when engaged in π-stacking interactions with the native bases. We present a theoretical study on fluorescence quenching mechanisms in dimers of 2AP π-stacked with adenine or guanine as in natural DNA. Relaxation pathways on the potential energy surfaces of the first excited states have been computed and reveal the importance of exciplexes and conical intersections in the fluorescence quenching process. PMID:23625036

Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

DOEpatents

Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

2003-06-03

The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

Chemical evolution. XXIX - Pyrimidines from hydrogen cyanide

NASA Technical Reports Server (NTRS)

Ferris, J. P.; Joshi, P. C.; Lawless, J. G.

1978-01-01

Compounds obtained by hydrolysis of HCN oligomers formed by allowing pH 9.2, 0.1 M cyanide to stand at room temperature for 4 to 12 months were analyzed. Hydrolysis of HCN oligomers yielded 4,5-dihydroxypyrimidine and 5-hydroxyuracil; orotic acid was detected after hydrolysis at pH 8.5. A unified pathway from diaminofumaronitrile to the pyrimidines observed is suggested. As purines, pyrimidines and amino acids are released by hydrolysis of HCN oligomers in either acidic or mildly basic aqueous solutions, they could have been formed on the primitive earth in spite of fluctuations in pH. 4,5-dihydroxypyrimidines appear to be likely candidates for incorporation into primitive nucleic acids, as they should undergo Watson-Crick hydrogen bonding with adenine.

Urinary purine derivatives as a tool to estimate dry matter intake in cattle: a meta-analysis

USDA-ARS?s Scientific Manuscript database

The objectives of this study were: 1) to investigate the relationship between dry matter intake (DMI) and urinary purine derivatives (PD) excretion in order to develop equations to predict DMI, and 2) to determine the endogenous excretion of PD for beef and dairy cattle using a meta-analytic approac...

From Purines to Basic Biochemical Concepts: Experiments for High School Students

ERIC Educational Resources Information Center

Marini, Isabella; Ipata, Piero Luigi

2007-01-01

Many high school biology courses address mainly the molecular and cellular basis of life. The complexity that underlies the most essential processes is often difficult for the students to understand; possibly, in part, because of the inability to see and explore them. Six simple practical experiments on purine catabolism as a part of a…

Catalytic carbene transfer allows the direct customization of cyclic purine dinucleotides.

PubMed

Fei, Na; Häussinger, Daniel; Blümli, Seraina; Laventie, Benoît-Joseph; Bizzini, Lorenzo D; Zimmermann, Kaspar; Jenal, Urs; Gillingham, Dennis

2014-08-11

We describe a simple method for the direct modification of nucleobases in cyclic purine dinucleotides, important signalling molecules in both prokaryotes and eukaryotes. The method tolerates all members of the cyclic dinucleotide family and could be used to modulate their function or introduce useful side-chains such as fluorophores and photo-crosslinking groups.

Chiral symmetry breaking during the self-assembly of monolayers from achiral purine molecules.

PubMed

Sowerby, S J; Heckl, W M; Petersen, G B

1996-11-01

Scanning tunneling microscopy was used to investigate the structure of the two-dimensional adsorbate formed by molecular self-assembly of the purine base, adenine, on the surfaces of the naturally occurring mineral molybdenite and the synthetic crystal highly oriented pyrolytic graphite. Although formed from adenine, which is achiral, the observed adsorbate surface structures were enantiomorphic on molybdenite. This phenomenon suggests a mechanism for the introduction of a localized chiral symmetry break by the spontaneous crystallization of these prebiotically available molecules on inorganic surfaces and may have some role in the origin of biomolecular optical asymmetry. The possibility that purine-pyrimidine arrays assembled on naturally occurring mineral surfaces might act as possible templates for biomolecular assembly is discussed.

Pathway-based discovery of genetic interactions in breast cancer

PubMed Central

Xu, Zack Z.; Boone, Charles; Lange, Carol A.

2017-01-01

Breast cancer is the second largest cause of cancer death among U.S. women and the leading cause of cancer death among women worldwide. Genome-wide association studies (GWAS) have identified several genetic variants associated with susceptibility to breast cancer, but these still explain less than half of the estimated genetic contribution to the disease. Combinations of variants (i.e. genetic interactions) may play an important role in breast cancer susceptibility. However, due to a lack of statistical power, the current tests for genetic interactions from GWAS data mainly leverage prior knowledge to focus on small sets of genes or SNPs that are known to have an association with breast cancer. Thus, many genetic interactions, particularly among novel variants, remain understudied. Reverse-genetic interaction screens in model organisms have shown that genetic interactions frequently cluster into highly structured motifs, where members of the same pathway share similar patterns of genetic interactions. Based on this key observation, we recently developed a method called BridGE to search for such structured motifs in genetic networks derived from GWAS studies and identify pathway-level genetic interactions in human populations. We applied BridGE to six independent breast cancer cohorts and identified significant pathway-level interactions in five cohorts. Joint analysis across all five cohorts revealed a high confidence consensus set of genetic interactions with support in multiple cohorts. The discovered interactions implicated the glutathione conjugation, vitamin D receptor, purine metabolism, mitotic prometaphase, and steroid hormone biosynthesis pathways as major modifiers of breast cancer risk. Notably, while many of the pathways identified by BridGE show clear relevance to breast cancer, variants in these pathways had not been previously discovered by traditional single variant association tests, or single pathway enrichment analysis that does not consider SNP-SNP interactions. PMID:28957314

Inflammation-induced abnormalities in the subcellular localization and trafficking of the neurokinin 1 receptor in the enteric nervous system

PubMed Central

Lieu, TinaMarie; Pelayo, Juan Carlos; Eriksson, Emily M.; Veldhuis, Nicholas A.; Bunnett, Nigel W.

2015-01-01

Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10−/−-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions. PMID:26138465

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

PubMed

Pissarek, M; Reichelt, C; Krauss, G J; Illes, P

1998-11-23

ATP-dependent potassium (KATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate KATP channels. In addition, the effects of the KATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N2-5% CO2 instead of 95% O2-5% CO2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%),, probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (30 microM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 MicroM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 microM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 microM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of KATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival viaprotein and DNA synthesis.

The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.

PubMed

Lee, Younghyun; Li, Huizi Keiko; Masaoka, Aya; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A; Okayasu, Ryuichi

2016-10-01

PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by γ-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of γ-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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).

Fundamental mechanisms of DNA radiosensitization: damage induced by low-energy electrons in brominated oligonucleotide trimers.

PubMed

Park, Yeunsoo; Polska, Katarzyna; Rak, Janusz; Wagner, J Richard; Sanche, Léon

2012-08-16

The replacement of nucleobases with brominated analogs enhances DNA radiosensitivity. We examine the chemistry of low-energy electrons (LEEs) in this sensitization process by experiments with thin films of the oligonucleotide trimers TBrXT, where BrX = 5-BrU (5-bromouracil), 5-BrC (5-bromocytosine), 8-BrA (8-bromoadenine), or 8-BrG (8-bromoguanine). The products induced from irradiation of thin (∼ 2.5 nm) oligonucleotide films, with 10 eV electrons, under ultrahigh vacuum (UHV) are analyzed by HPLC-UV. The number of damaged brominated trimers ranges from about 12 to 15 × 10(-3) molecules per incident electron, whereas under the identical conditions, these numbers drop to 4-7 × 10(-3) for the same, but nonbrominated oligonucleotides. The results of HPLC analysis show that the main degradation pathway of trinucleotides containing brominated bases involve debromination (i.e., loss of the bromine atom and its replacement with a hydrogen atom). The electron-induced sum of products upon bromination increases by factors of 2.1 for the pyrimidines and 3.2 for the purines. Thus, substitution of any native nucleobase with a brominated one in simple models of DNA increases LEE-induced damage to DNA and hence its radiosensitivity. Furthermore, besides the brominated pyrimidines that have already been tested in clinical trials, brominated purines not only appear to be promising sensitizers for radiotherapy, but could provide a higher degree of radiosensitization.

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea, kucha (Camellia assamica var. kucha).

PubMed

Zheng, Xin-Qiang; Ye, Chuang-Xing; Kato, Misako; Crozier, Alan; Ashihara, Hiroshi

2002-05-01

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.

GREENER ORGANIC SYNTHESES USING ALTERNATIVE REACTION CONDITIONS AND MEDIA

EPA Science Inventory

The diverse nature of chemical entities requires various green' strategic pathways in our quest towards attaining sustainability. A solvent-free approach that involves microwave (MW) exposure of neat reactants (undiluted) catalyzed by the surfaces of less-expensive and recyclable...

EFFICIENT CHEMICAL TRANSFORMATIONS USING ALTERNATIVE REACTION CONDITIONS AND MEDIA

EPA Science Inventory

The diverse nature of chemical entities requires various green' strategic pathways in our quest towards attaining sustainability. A solvent-free approach that involves microwave (MW) exposure of neat reactants (undiluted) catalyzed by the surfaces of less-expensive and recyclable...

An in vitro analysis of purine-mediated renal vasoconstriction in rat isolated kidney.

PubMed Central

Kenakin, T. P.; Pike, N. B.

1987-01-01

In the rat isolated perfused kidney, 2-chloroadenosine and L-N6-phenyl-isopropyl adenosine (L-PIA) produced a modest vasodilatation. After kidneys had been pretreated with methoxamine (to elevate vascular tone) and forskolin (to activate adenyl cyclase and reduce vascular tone), both purine agonists produced vasoconstriction at low doses and vasodilatation at higher doses. This was consistent with the working hypothesis that vasoconstriction resulted from activation of A1-purinoceptors mediating adenyl cyclase inhibition and vasodilatation from activation of A2-purinoceptors stimulating adenyl cyclase. These kidney preparations also demonstrated a marked potentiation of purine-mediated vasoconstriction in the presence of various concentrations of 8-p-sulpho-phenyltheophylline (8-SPT), a drug reported in the literature to be a competitive antagonist of A1- and A2-purinoceptors. Maximal renal vasoconstriction to 2-chloroadenosine and L-PIA was observed in the presence of 10 mM 8-SPT; the fact that this vasoconstriction was sensitive to the selective A1-receptor antagonist 8-(2-amino-4-chlorophenyl)-1,3-dipropylxanthine (PACPX) and that the order of potency of agonists for this effect was L-PIA greater than 2-chloroadenosine greater than D-PIA greater than N6-ethylcarboxamide adenosine (NECA) was consistent with activation of vascular A1-purinoceptors. While these data are consistent with the hypothesis that purines activate vascular A1- and A2-receptors in the rat isolated kidney, the nature of the results did not allow definitive classification of the receptors mediating the purine effects. PMID:3828655

Interactions between barley grain processing and source of supplemental dietary fat on nitrogen metabolism and urea-nitrogen recycling in dairy cows.

PubMed

Gozho, G N; Hobin, M R; Mutsvangwa, T

2008-01-01

The objective of this study was to determine the effects of methods of barley grain processing and source of supplemental fat on urea-N transfer to the gastrointestinal tract (GIT) and the utilization of this recycled urea-N in lactating dairy cows. Four ruminally cannulated Holstein cows (656.3 +/- 27.7 kg of BW; 79.8 +/- 12.3 d in milk) were used in a 4 x 4 Latin square design with 28-d periods and a 2 x 2 factorial arrangement of dietary treatments. Experimental diets contained dry-rolled barley or pelleted barley in combination with whole canola or whole flaxseed as supplemental fat sources. Nitrogen balance was measured from d 15 to 19, with concurrent measurements of urea-N kinetics using continuous intrajugular infusions of [15N 15N]-urea. Dry matter intake and N intake were higher in cows fed dry-rolled barley compared with those fed pelleted barley. Nitrogen retention was not affected by diet, but fecal N excretion was higher in cows fed dry-rolled barley than in those fed pelleted barley. Actual and energy-corrected milk yield were not affected by diet. Milk fat content and milk fat yield were higher in cows fed dry-rolled barley compared with those fed pelleted barley. Source of supplemental fat did not affect urea-N kinetics. Urea-N production was higher (442.2 vs. 334.3 g of N/d), and urea-N entering the GIT tended to be higher (272.9 vs. 202.0 g of N/d), in cows fed dry-rolled barley compared with those fed pelleted barley. The amount of urea-N entry into the GIT that was returned to the ornithine cycle was higher (204.1 vs. 159.5 g of N/d) in cows fed dry-rolled barley than in pelleted barley-fed cows. The amount of urea-N recycled to the GIT and used for anabolic purposes, and the amounts lost in the urine or feces were not affected by dietary treatment. Microbial nonammonia N supply, estimated using total urinary excretion of purine derivatives, was not affected by diet. These results show that even though barley grain processing altered urea-N entry into the GIT, the utilization of this recycled urea-N for microbial production was unaffected as the additional urea-N, which entered the GIT was returned to ureagenesis.

Murine lymphoma L5178Y cells resistant to purine antagonists: differences in cross-resistance to thioguanine-platinum(II) and selenoguanine-platinum(II).

PubMed

Kanzawa, F; Maeda, M; Sasaki, T; Hoshi, A; Kuretani, K

1982-02-01

To determine whether the antitumor activities of thioguanine-platinum(II) [TG-Pt(II)] and selenoguanine-platinum(II) [SeG-Pt(II)] are due to direct actions of these compounds or to the actions of their hydrolysis products, studies were made on a purine antagonist-resistant, murine lymphoma L5178Y/MP subline that lacked the anabolic enzyme hypoxanthine-guanine phosphoribosyltransferase necessary for tumor inhibition. The L5178Y/MP subline proved to be highly resistant to both TG-Pt(II) and thioguanine; the resistance ratios to the two compounds were almost identical. The subline showed high resistance to selenoguanine, but the cross-resistance to SeG-Pt(II) was negligible. Whether the compounds exhibit the delayed cytotoxicity characteristic of purine antagonists was also investigated. Delayed cytotoxicity was demonstrated for TG-Pt(II) as well as for thioguanine and other purine antagonists but not for SeG-Pt(II) or cis-dichlorodiammineplatinum(II). Experiments on cross-resistance and delayed cytotoxicity showed differences in the cytotoxicities of TG-Pt(II) and SeG-Pt(II): TG-Pt(II) exerted its activity through its hydrolysis product thioguanine, whereas SeG-Pt(II) compound was cytotoxic itself.

Technical note: Evaluation of urinary purine derivatives in comparison with duodenal purines for estimating rumen microbial protein supply in sheep.

PubMed

Kozloski, G V; Stefanello, C M; Oliveira, L; Filho, H M N Ribeiro; Klopfenstein, T J

2017-02-01

A data set of individual observations was compiled from digestibility trials to examine the relationship between the duodenal purine bases (PB) flow and urinary purine derivatives (PD) excretion and the validity of different equations for estimating rumen microbial N (Nm) supply based on urinary PD in comparison with estimates based on duodenal PB. Trials (8 trials, = 185) were conducted with male sheep fitted with a duodenal T-type cannula, housed in metabolic cages, and fed forage alone or with supplements. The amount of PD excreted in urine was linearly related to the amount of PB flowing to the duodenum ( < 0.05). The intercept of the linear regression was 0.180 mmol/(d·kg), representing the endogenous excretion of PD, and the slope was lower than 1 ( < 0.05), indicating that only 0.43% of the PB in the duodenum was excreted as PD in urine. The Nm supply estimated by either approach was linearly related ( < 0.05) to the digestible OM intake. However, the Nm supply estimated through either of 3 published PD-based equations probably underestimated the Nm supply in sheep.

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

PubMed

Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

2016-06-12

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Phosphorus cycling in Montreal's food and urban agriculture systems.

PubMed

Metson, Geneviève S; Bennett, Elena M

2015-01-01

Cities are a key system in anthropogenic phosphorus (P) cycling because they concentrate both P demand and waste production. Urban agriculture (UA) has been proposed as a means to improve P management by recycling cities' P-rich waste back into local food production. However, we have a limited understanding of the role UA currently plays in the P cycle of cities or its potential to recycle local P waste. Using existing data combined with surveys of local UA practitioners, we quantified the role of UA in the P cycle of Montreal, Canada to explore the potential for UA to recycle local P waste. We also used existing data to complete a substance flow analysis of P flows in the overall food system of Montreal. In 2012, Montreal imported 3.5 Gg of P in food, of which 2.63 Gg ultimately accumulated in landfills, 0.36 Gg were discharged to local waters, and only 0.09 Gg were recycled through composting. We found that UA is only a small sub-system in the overall P cycle of the city, contributing just 0.44% of the P consumed as food in the city. However, within the UA system, the rate of recycling is high: 73% of inputs applied to soil were from recycled sources. While a Quebec mandate to recycle 100% of all organic waste by 2020 might increase the role of UA in P recycling, the area of land in UA is too small to accommodate all P waste produced on the island. UA may, however, be a valuable pathway to improve urban P sustainability by acting as an activity that changes residents' relationship to, and understanding of, the food system and increases their acceptance of composting.

Phosphorus Cycling in Montreal’s Food and Urban Agriculture Systems

PubMed Central

Metson, Geneviève S.; Bennett, Elena M.

2015-01-01

Cities are a key system in anthropogenic phosphorus (P) cycling because they concentrate both P demand and waste production. Urban agriculture (UA) has been proposed as a means to improve P management by recycling cities’ P-rich waste back into local food production. However, we have a limited understanding of the role UA currently plays in the P cycle of cities or its potential to recycle local P waste. Using existing data combined with surveys of local UA practitioners, we quantified the role of UA in the P cycle of Montreal, Canada to explore the potential for UA to recycle local P waste. We also used existing data to complete a substance flow analysis of P flows in the overall food system of Montreal. In 2012, Montreal imported 3.5 Gg of P in food, of which 2.63 Gg ultimately accumulated in landfills, 0.36 Gg were discharged to local waters, and only 0.09 Gg were recycled through composting. We found that UA is only a small sub-system in the overall P cycle of the city, contributing just 0.44% of the P consumed as food in the city. However, within the UA system, the rate of recycling is high: 73% of inputs applied to soil were from recycled sources. While a Quebec mandate to recycle 100% of all organic waste by 2020 might increase the role of UA in P recycling, the area of land in UA is too small to accommodate all P waste produced on the island. UA may, however, be a valuable pathway to improve urban P sustainability by acting as an activity that changes residents’ relationship to, and understanding of, the food system and increases their acceptance of composting. PMID:25826256

Impact of recycled effluent on the hydrolysis during anaerobic digestion of vegetable and flower waste.

PubMed

Lü, F; He, P J; Hao, L P; Shao, L M

2008-01-01

Two trials were established to investigate the effect of recycled effluent on hydrolysis during anaerobic co-digestion of vegetable and flower waste. Trial I evaluated the effect by regulating the flow rate of recycled effluent, while Trial II regulated the ratio of hydrolytic effluent to methanogenic effluent, which were recycled to hydrolysis reactor. Results showed that the recirculation of methanogenic effluent could enhance the buffer capability and operation stability of hydrolysis reactor. Higher recycled flow rate was favourable for microbial anabolism and further promoted hydrolysis. After 9 days of hydrolysis, the cumulative SCOD in the hydrolytic effluent reached 334, 407, 413, 581 mg/g at recycled flow rates of 0.1, 0.5, 1.0, 2.0 m3/(m3 x d), respectively. It was feasible to recycling a mixture of hydrolytic and methanogenic effluent to the hydrolysis reactor. This research showed that partially introducing hydrolytic effluent into the recycled liquid could enhance hydrolysis, while excessive recirculation of hydrolytic effluent will inhibit the hydrolysis. The flow ratio 1:3 of hydrolytic to methanogenic effluent was found to provide the highest hydrolysis efficiency and degradation rate of lignocelluloses-type biomass, among four ratios of 0:1, 1:3, 1:1 and 3:1. Under this regime, after 9 days of hydrolysis, the cumulative TOC and TN in the hydrolytic effluent reached 162 mg/g and 15 mg/g, the removal efficiency of TS, VS, C and cellulose in the solid phase were 60.66%, 62.88%, 58.35% and 49.12%, respectively. The flow ratio affected fermentation pathways, i.e. lower ratio favoured propionic acid fermentation and the generation of lactic acid while higher ratio promoted butyric acid fermentation. IWA Publishing 2008.

Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

PubMed

Mallet, W G; Maxfield, F R

1999-07-26

Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J. Cell Biol.142:923-936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.

Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

NASA Astrophysics Data System (ADS)

Snoeberger, Ning-Shiuan Nicole

Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

High salt diet induces metabolic alterations in multiple biological processes of Dahl salt-sensitive rats.

PubMed

Wang, Yanjun; Liu, Xiangyang; Zhang, Chen; Wang, Zhengjun

2018-06-01

High salt induced renal disease is a condition resulting from the interactions of genetic and dietary factors causing multiple complications. To understand the metabolic alterations associated with renal disease, we comprehensively analyzed the metabonomic changes induced by high salt intake in Dahl salt-sensitive (SS) rats using GC-MS technology and biochemical analyses. Physiological features, serum chemistry, and histopathological data were obtained as complementary information. Our results showed that high salt (HS) intake for 16 weeks caused significant metabolic alterations in both the renal medulla and cortex involving a variety pathways involved in the metabolism of organic acids, amino acids, fatty acids, and purines. In addition, HS enhanced glycolysis (hexokinase, phosphofructokinase and pyruvate kinase) and amino acid metabolism and suppressed the TCA (citrate synthase and aconitase) cycle. Finally, HS intake caused up-regulation of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the ratio of NADPH/NADP + , NADPH oxidase activity and ROS production, suggesting that increased oxidative stress was associated with an altered PPP pathway. The metabolic pathways identified may serve as potential targets for the treatment of renal damage. Our findings provide comprehensive biochemical details about the metabolic responses to a high salt diet, which may contribute to the understanding of renal disease and salt-induced hypertension in SS rats. Copyright © 2018. Published by Elsevier Inc.

Synthesis and antitumor activity of seleno- and thio-purines complexed with cis-diamminoplatinum (II).

PubMed

Maeda, M; Abiko, N; Sasaki, T

1982-02-01

cis-Diamminoplatinum (II) complexes with selenoguanine, thioguanine, 6-thioxanthine, or 6-mercaptopurine were synthesized by the reaction of stoichiometric amounts of selenopurine or thiopurine with aquated cis-dichlorodimmineplatinum (II) in slightly acidic medium, and their antitumor activity was studied against L1210 cells in mice. These compounds exhibited a medium antitumor activity with very low toxicity. The antitumor activity was dependent on the nature of the purine ligand. These complexes were very stable in various aqueous solvents at 37 degrees C for 10 d but not in the presence of mouse serum. The mechanism of the action effected by the complex is not clear. However, the slow release of an antitumor active purine from the complex, SeG-Pt (NH3)2, was observed.

Purine derivatives as potent Bruton’s tyrosine kinase (BTK) inhibitors for autoimmune diseases

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

Shi, Qing; Tebben, Andrew; Dyckman, Alaric J.

Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3 & 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discovery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400 kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferation and CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystal structure with BTK shows that the high selectivity is gained from filling amore » BTK specific lipophilic pocket. However, physical and ADME properties leading to low oral exposure hindered further development.« less

Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

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

Abramchik, Yu. A., E-mail: inna@ns.crys.ras.ru; Timofeev, V. I., E-mail: espiov@ibch.ru; Zhukhlistova, N. E., E-mail: tostars@mail.ru

2015-07-15

Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamermore » is the biological active form of E. coli. purine nucleoside phosphorylase.« less

Sustainable and efficient pathways for bioenergy recovery from low-value process streams via bioelectrochemical systems in biorefineries

DOE PAGES

Borole, Abhijeet P.

2015-08-25

Conversion of biomass into bioenergy is possible via multiple pathways resulting in production of biofuels, bioproducts and biopower. Efficient and sustainable conversion of biomass, however, requires consideration of many environmental and societal parameters in order to minimize negative impacts. Integration of multiple conversion technologies and inclusion of upcoming alternatives such as bioelectrochemical systems can minimize these impacts and improve conservation of resources such as hydrogen, water and nutrients via recycle and reuse. This report outlines alternate pathways integrating microbial electrolysis in biorefinery schemes to improve energy efficiency while evaluating environmental sustainability parameters.

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

PubMed Central

Schultz, Eric P.; Vasquez, Ernesto E.; Scott, William G.

2014-01-01

The hammerhead ribozyme catalyzes RNA cleavage via acid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK a of the substituted purine; in both cases inosine, which is similar to G in pK a and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential role of G12 as a general base, indicates that an alternative hammerhead cleavage mechanism involving specific base catalysis may instead explain the observed rate dependence upon purine substitutions at G12. The crystallographic results, contrary to previous assumptions, therefore cannot be interpreted to favor the general base catalysis mecahnism over the specific base catalysis mechanism. Instead, both of these mutually exclusive mechanistic alternatives must be considered in light of the current structural and biochemical data. PMID:25195740

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

PubMed

Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

2014-09-01

The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential role of G12 as a general base, indicates that an alternative hammerhead cleavage mechanism involving specific base catalysis may instead explain the observed rate dependence upon purine substitutions at G12. The crystallographic results, contrary to previous assumptions, therefore cannot be interpreted to favor the general base catalysis mecahnism over the specific base catalysis mechanism. Instead, both of these mutually exclusive mechanistic alternatives must be considered in light of the current structural and biochemical data.

Expression and localization of exocytic and recycling Rabs from Magnaporthe oryzae in mammalian cells

PubMed Central

Qi, Yaoyao; Marlin, M. Caleb; Liang, Zhimin; Zhang, Dongmei; Zhou, Jie; Wang, Zonghua; Lu, Guodong; Li, Guangpu

2018-01-01

Rab GTPases are master regulators of intracellular membrane trafficking along endocytic and exocytic pathways. In this chapter, we began to characterize the exocytic and recycling Rabs from the filamentous fungus Magnaporthe oryzae (M. oryzae) that causes the rice blast disease. Among the 11 putative Rabs identified from the M. oryzae genome database (MoRabs), MoRab1, MoRab8, and MoRab11 appear orthologs of mammalian Rab1, Rab8, and Rab11 and likely function in exocytosis and endosomal recycling. To test this contention, we cloned, expressed, and determined intracellular localization of the three MoRabs in mammalian cells, in comparison to their human counterparts (hRabs). The MoRabs were well expressed as GFP fusion proteins and colocalized with the tdTomato-labeled hRabs on exocytic and recycling organelles, as determined by immunoblot analysis and confocal fluorescence microscopy. The colocalization supports the contention that the MoRabs are indeed Rab orthologs and may play important roles in the development and pathogenicity of M. oryzae. PMID:26360026

Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

PubMed

Brickman, Timothy J; Suhadolc, Ryan J; McKelvey, Pamela J; Armstrong, Sandra K

2017-02-01

Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis. © 2016 John Wiley & Sons Ltd.

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

PubMed Central

Coller, Kelly E.; Heaton, Nicholas S.; Berger, Kristi L.; Cooper, Jacob D.; Saunders, Jessica L.; Randall, Glenn

2012-01-01

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells. PMID:22241992

Wobble↔Watson-Crick tautomeric transitions in the homo-purine DNA mismatches: a key to the intimate mechanisms of the spontaneous transversions.

PubMed

Brovarets', Ol'ha O; Hovorun, Dmytro M

2015-01-01

The intrinsic capability of the homo-purine DNA base mispairs to perform wobble↔Watson-Crick/Topal-Fresco tautomeric transitions via the sequential intrapair double proton transfer was discovered for the first time using QM (MP2/DFT) and QTAIM methodologies that are crucial for understanding the microstructural mechanisms of the spontaneous transversions.

Accumulation of polybrominated diphenyl ethers in breast milk of women from an e-waste recycling center in China.

PubMed

Li, Xinghong; Tian, Yuan; Zhang, Yun; Ben, Yujie; Lv, Quanxia

2017-02-01

Polybrominated diphenyl ethers (PBDEs) can be transferred to infants through the ingestion of breast milk, resulting in potential health risk. In this study, PBDEs, hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and 2,2',4,4',5,6'-hexachlorobiphenyl (CB-153) in human milk from women living adjacent to e-waste recycling sites of Wenling, China, were investigated. The median level of PBDEs in samples from residents living in the e-waste recycling environment >20years (R 20 group, 19.5ng/g lipid weight (lw)) was significantly higher than that for residents living in Wenling <3years (R 3 group, 3.88ng/g lw) (p<0.05), likely ascribable to specific exposure to PBDEs from e-waste recycling activities. In the R 20 group, most congeners (except for BDE-209) were correlated with each other (p<0.05). Moreover, CB-153 showed significant association with most PBDE congeners, rather than BDE-209. The relationship indicated that most BDE congeners other than BDE-209 shared common sources and/or pathways with CB-153, e.g., dietary ingestion. The correlations between BDE-209 and other congeners were different in the two groups, likely suggesting their different exposure sources and/or pathways for PBDEs. Although estimated dietary intake of PBDEs for infants via breast milk was lower than the minimum value affecting human health, the PBDE exposure of infants should be of great concern because of their potential effect on the development of neonates over long-term exposure. OH-PBDEs were not detected in the collected samples, which is in accordance with reports in published literature, likely indicating that they were not apt to be accumulated in human milk. Copyright © 2016. Published by Elsevier B.V.

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

PubMed Central

Pereira, Liliana M. G.; Pinto, Rute D.; Silva, Daniela S.; Moreira, Ana R.; Beitzinger, Christoph; Oliveira, Pedro; Sampaio, Paula; Benz, Roland; Azevedo, Jorge E.; dos Santos, Nuno M. S.

2014-01-01

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway. PMID:25287919

ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway

PubMed Central

Mamuya, Fahmy A.; Cano-Peñalver, Jose Luis; Li, Wei; Rodriguez Puyol, Diego; Rodriguez Puyol, Manuel; Brown, Dennis; de Frutos, Sergio

2016-01-01

Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, to open a path toward effective diabetes insipidus therapeutics. A recent study has shown that integrin-linked kinase (ILK) conditional-knockdown mice developed polyuria along with decreased AQP2 expression. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway. PMID:27760768

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

PubMed Central

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

2013-01-01

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

Rab11a differentially modulates epidermal growth factor-induced proliferation and motility in immortal breast cells.

PubMed

Palmieri, Diane; Bouadis, Amina; Ronchetti, Ruban; Merino, Maria J; Steeg, Patricia S

2006-11-01

The development of cancer prevention strategies depends on the elucidation of molecular pathways underlying oncogenesis. In a previous proteomic study of matched normal breast ducts and Ductal Carcinoma in Situ (DCIS), we identified overexpression of Rab11a in DCIS. Rab11a is not well studied in cancer, but is known to regulate the recycling of internalized cell surface proteins and receptors from the early endosome through the trans-Golgi network. Using immunohistochemistry, we confirmed our observation, noting increased Rab11a expression in 19 of 22 (86%) DCIS cases compared to matched normal breast epithelium. To study the function of Rab11a, immortal, nontumorigenic MCF10A breast cells were stimulated with ligands to the EGF receptor (EGFR) after transfection with empty vector (control), Rab11a, or a S25N dominant-negative (DN) Rab11a. Using an iodinated ligand:receptor recycling assay, transfection of Rab11a accelerated, while DN-Rab11a postponed EGFR recycling in vitro. The signaling and in vitro phenotypic consequences of Rab11a expression and function were studied. Transfection of DN-Rab11a increased Erk1/2 activation downstream of EGF, but exerted no effect on the Akt pathway. Expression of DN-Rab11a inhibited MCF10A proliferation by 50-60%, and also inhibited anchorage-dependent colonization. Notably, DN-Rab11a transfection increased motility toward EGFR ligands. The data provide a first demonstration that Rab11a modulates EGFR recycling, and promotes the proliferation but inhibits the motility of an immortal breast line, consistent with the DCIS phenotype.

High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

PubMed

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

2013-01-01

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

PubMed

Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E

2010-02-01

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

A National-Scale Comparison of Resource and Nutrient Demands for Algae-Based Biofuel Production by Lipid Extraction and Hydrothermal Liquefaction

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

Venteris, Erik R.; Skaggs, Richard; Wigmosta, Mark S.

Algae’s high productivity provides potential resource advantages over other fuel crops. However, demand for land, water, and nutrients must be minimized to avoid impacts on food production. We apply our national-scale, open-pond, growth and resource models to assess several biomass to fuel technological pathways based on Chlorella. We compare resource demands between hydrothermal liquefaction (HTL) and lipid extraction (LE) to meet 1.89E+10 and 7.95E+10 L yr-1 biofuel targets. We estimate nutrient demands where post-fuel biomass is consumed as co-products and recycling by anaerobic digestion (AD) or catalytic hydrothermal gasification (CHG). Sites are selected through prioritization based on fuel value relativemore » to a set of site-specific resource costs. The highest priority sites are located along the Gulf of Mexico coast, but potential sites exist nationwide. We find that HTL reduces land and freshwater consumption by up to 46% and saline groundwater by around 70%. Without recycling, nitrogen (N) and phosphorous (P) demand is reduced 33%, but is large relative to current U.S. agricultural consumption. The most nutrient-efficient pathways are LE+CHG for N and HTL+CHG for P (by 42%). Resource gains for HTL+CHG are offset by a 344% increase in N consumption relative to LE+CHG (with potential for further recycling). Nutrient recycling is essential to effective use of alternative nutrient sources. Modeling of utilization availability and costs remains, but we find that for HTL+CHG at the 7.95E+10 L yr-1 production target, municipal sources can offset 17% of N and 40% of P demand and animal manures can generally meet demands.« less

AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

PubMed Central

Glyvuk, Nataliya; Tsytsyura, Yaroslav; Geumann, Constanze; D'Hooge, Rudi; Hüve, Jana; Kratzke, Manuel; Baltes, Jennifer; Böning, Daniel; Klingauf, Jürgen; Schu, Peter

2010-01-01

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation. PMID:20203623

Stacking of purines in water: the role of dipolar interactions in caffeine.

PubMed

Tavagnacco, L; Di Fonzo, S; D'Amico, F; Masciovecchio, C; Brady, J W; Cesàro, A

2016-05-11

During the last few decades it has been ascertained that base stacking is one of the major contributions stabilizing nucleic acid conformations. However, the understanding of the nature of the interactions involved in the stacking process remains under debate and it is a subject of theoretical and experimental studies. Structural similarity between purine bases (guanine and adenine) in DNA and the caffeine molecule makes caffeine an excellent model for the purine bases. The present study clearly shows that dipolar interactions play a fundamental role in determining stacking of purine molecules in solution. In order to reach this achievement, polarized ultraviolet Raman resonant scattering experiments have been carried out on caffeine aqueous solutions as a function of concentration and temperature. The investigation pointed out at the aggregation and solvation properties, particularly at elevated temperatures. Kubo-Anderson theory was used as a framework to investigate the non-coincidence effect (NCE) occurring in the totally symmetric breathing modes of the purine rings, and in the bending modes of the methyl groups of caffeine. The NCE concentration dependence shows that caffeine aggregation at 80 °C occurs by planar stacking of the hydrophobic faces. The data clearly indicate that dipolar interactions determine the reorientational motion of the molecules in solution and are the driving force for the stacking of caffeine. In parallel, the observed dephasing times imply a change in caffeine interactions as a function of temperature and concentration. A decrease, at low water content, of the dephasing time for the ring breathing vibration mode indicates that self-association alters the solvation structure that is detectable at low concentration. These results are in agreement with simulation predictions and serve as an important validation of the models used in those calculations.

Theoretical and experimental studies on the corrosion inhibition potentials of some purines for aluminum in 0.1 M HCl

PubMed Central

Eddy, Nnabuk O.; Momoh-Yahaya, H.; Oguzie, Emeka E.

2014-01-01

Experimental aspect of the corrosion inhibition potential of adenine (AD), guanine (GU) and, hypoxanthine (HYP) was carried out using weight loss, potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) methods while the theoretical aspect of the work was carried out by calculations of semi-empirical parameters (for AM1, MNDO, CNDO, PM3 and RM1 Hamiltonians), Fukui functions and inhibitor–metal interaction energies. Results obtained from the experimental studies were in good agreement and indicated that adenine (AD), guanine (GU) and hypoxanthine (HYP) are good adsorption inhibitors for the corrosion of aluminum in solutions of HCl. Data obtained from electrochemical experiment revealed that the studied purines functioned by adsorption on the aluminum/HCl interface and inhibited the cathodic half reaction to a greater extent and anodic half reaction to a lesser extent. The adsorption of the purines on the metal surface was found to be exothermic and spontaneous. Deviation of the adsorption characteristics of the studied purines from the Langmuir adsorption model was compensated by the fitness of Flory Huggins and El Awardy et al. adsorption models. Quantum chemical studies revealed that the experimental inhibition efficiencies of the studied purines are functions of some quantum chemical parameters including total energy of the molecules (TE), energy gap (EL–H), electronic energy of the molecule (EE), dipole moment and core–core repulsion energy (CCR). Fukui functions analysis through DFT and MP2 theories indicated slight complications and unphysical results. However, results obtained from calculated Huckel charges, molecular orbital and interaction energies, the adsorption of the inhibitors proceeded through the imine nitrogen (N5) in GU, emanine nitrogen (N7) in AD and the pyridine nitrogen (N5) in HPY. PMID:25750754

Theoretical and experimental studies on the corrosion inhibition potentials of some purines for aluminum in 0.1 M HCl.

PubMed

Eddy, Nnabuk O; Momoh-Yahaya, H; Oguzie, Emeka E

2015-03-01

Experimental aspect of the corrosion inhibition potential of adenine (AD), guanine (GU) and, hypoxanthine (HYP) was carried out using weight loss, potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) methods while the theoretical aspect of the work was carried out by calculations of semi-empirical parameters (for AM1, MNDO, CNDO, PM3 and RM1 Hamiltonians), Fukui functions and inhibitor-metal interaction energies. Results obtained from the experimental studies were in good agreement and indicated that adenine (AD), guanine (GU) and hypoxanthine (HYP) are good adsorption inhibitors for the corrosion of aluminum in solutions of HCl. Data obtained from electrochemical experiment revealed that the studied purines functioned by adsorption on the aluminum/HCl interface and inhibited the cathodic half reaction to a greater extent and anodic half reaction to a lesser extent. The adsorption of the purines on the metal surface was found to be exothermic and spontaneous. Deviation of the adsorption characteristics of the studied purines from the Langmuir adsorption model was compensated by the fitness of Flory Huggins and El Awardy et al. adsorption models. Quantum chemical studies revealed that the experimental inhibition efficiencies of the studied purines are functions of some quantum chemical parameters including total energy of the molecules (TE), energy gap (E L-H), electronic energy of the molecule (EE), dipole moment and core-core repulsion energy (CCR). Fukui functions analysis through DFT and MP2 theories indicated slight complications and unphysical results. However, results obtained from calculated Huckel charges, molecular orbital and interaction energies, the adsorption of the inhibitors proceeded through the imine nitrogen (N5) in GU, emanine nitrogen (N7) in AD and the pyridine nitrogen (N5) in HPY.

Study of Copper and Purine-Copper Complexes on Modified Carbon Electrodes by Cyclic and Elimination Voltammetry

PubMed Central

Trnkova, Libuse; Zerzankova, Lenka; Dycka, Filip; Mikelova, Radka; Jelen, Frantisek

2008-01-01

Using a paraffin impregnated graphite electrode (PIGE) and mercury-modified pyrolytic graphite electrode with basal orientation (Hg-PGEb) copper(II) and Cu(II)-DNA purine base solutions have been studied by cyclic (CV) and linear sweep voltammetry (LSV) in connection with elimination voltammetry with linear scan (EVLS). In chloride and bromide solutions (pH 6), the redox process of Cu(II) proceeded on PIGE with two cathodic and two anodic potentially separated signals. According to the elimination function E4, the first cathodic peak corresponds to the reduction Cu(II) + e- → Cu(I) with the possibility of fast disproportionation 2Cu(I) → Cu(II)+ Cu(0). The E4 of the second cathodic peak signalized an electrode process controlled by a surface reaction. The electrode system of Cu(II) on Hg-PGEb in borate buffer (pH 9.2) was characterized by one cathodic and one anodic peak. Anodic stripping voltammetry (ASV) on PIGE and cathodic stripping voltammetry (CSV) on Hg-PGEb were carried out at potentials where the reduction of copper ions took place and Cu(I)-purine complexes were formed. By using ASV and CSV in combination with EVLS, the sensitivity of Cu(I)-purine complex detection was enhanced relative to either ASV or CSV alone, resulting in higher peak currents of more than one order of magnitude. The statistical treatment of CE data was used to determine the reproducibility of measurements. Our results show that EVLS in connection with the stripping procedure is useful for both qualitative and quantitative microanalysis of purine derivatives and can also reveal details of studied electrode processes. PMID:27879715

Patterns of new versus recycled primary production in the terrestrial biosphere

USDA-ARS?s Scientific Manuscript database

Nitrogen (N) and phosphorus (P) availability regulate plant productivity throughout the terrestrial biosphere, influencing the patterns and magnitude of net primary production (NPP) by land plants both now and into the future. These nutrients enter ecosystems via geologic and atmospheric pathways, a...

Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin

PubMed Central

Lee, Sora; Tumolo, Jessica M; Ehlinger, Aaron C; Jernigan, Kristin K; Qualls-Histed, Susan J; Hsu, Pi-Chiang; McDonald, W Hayes; Chazin, Walter J

2017-01-01

Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole. PMID:29130884

Increase in urinary purines and pyrimidines in patients with methylmalonic aciduria combined with homocystinuria.

PubMed

Porcu, Simona; Corda, Marcella; Lilliu, Franco; Contini, Liliana; Era, Benedetta; Traldi, Pietro; Fais, Antonella

2010-06-03

Methylmalonic aciduria combined with homocystinuria (MMA-HC) is the biochemical trait of a metabolic disorder resulting from impaired conversion of dietary cobalamin (cbl, or vitamin B12) to its two metabolically active forms. Effects on urinary purine and pyrimidine levels have not been described for this condition. Urine samples were collected from three patients with methylmalonic aciduria combined with homocystinuria and from 70 healthy subjects. Urinary purine and pyrimidine levels were quantitated by the use of LC/UV-Vis and LC/ESI/MS. Higher urine levels of pyrimidines were detected with both methods in patients compared to controls. Methylmalonic aciduria with homocystinuria is due to deficiency of the enzyme, cobalamin reductase. The enzyme defect leads to altered hepatic metabolism, which appears to modify circulating pyrimidine levels. Copyright 2010 Elsevier B.V. All rights reserved.

Direct Isolation of Purines and Pyrimidines from Nucleic Acids Using Sublimation

NASA Technical Reports Server (NTRS)

Glavin, Daniel P.; Schubert, Michael; Bada, Jeffrey L.

2003-01-01

A sublimation technique was developed to isolate purines and pyrimidines directly from lambda-deoxyribonucleic acid (lambda-DNA) and Escherichia coli cells. The sublimation of adenine, cytosine, guanine, and thymine from lambda-DNA was tested under reduced pressure (approx. 0.5 Torr) at temperatures of >150 C. With the exception of guanine, approximately 60 -75% of each base was sublimed directly from the lambda-DNA and recovered on a coldfinger of the sublimation apparatus after heating to 450 C. Several nucleobases including adenine, cytosine, thymine, and uracil were also recovered from E. coli bacteria after heating the cells to the same temperature, although some thermal decomposition of the bases also occurred. These results demonstrate the feasibility of using sublimation to isolate purines and pyrimidines from native E. coli DNA and RNA without any chemical treatment of the cells.

Inborn Errors of Fructose Metabolism. What Can We Learn from Them?

PubMed

Tran, Christel

2017-04-03

Fructose is one of the main sweetening agents in the human diet and its ingestion is increasing globally. Dietary sugar has particular effects on those whose capacity to metabolize fructose is limited. If intolerance to carbohydrates is a frequent finding in children, inborn errors of carbohydrate metabolism are rare conditions. Three inborn errors are known in the pathway of fructose metabolism; (1) essential or benign fructosuria due to fructokinase deficiency; (2) hereditary fructose intolerance; and (3) fructose-1,6-bisphosphatase deficiency. In this review the focus is set on the description of the clinical symptoms and biochemical anomalies in the three inborn errors of metabolism. The potential toxic effects of fructose in healthy humans also are discussed. Studies conducted in patients with inborn errors of fructose metabolism helped to understand fructose metabolism and its potential toxicity in healthy human. Influence of fructose on the glycolytic pathway and on purine catabolism is the cause of hypoglycemia, lactic acidosis and hyperuricemia. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provided new understandings into pathogenesis for these frequent diseases.

Inborn Errors of Fructose Metabolism. What Can We Learn from Them?

PubMed Central

Tran, Christel

2017-01-01

Fructose is one of the main sweetening agents in the human diet and its ingestion is increasing globally. Dietary sugar has particular effects on those whose capacity to metabolize fructose is limited. If intolerance to carbohydrates is a frequent finding in children, inborn errors of carbohydrate metabolism are rare conditions. Three inborn errors are known in the pathway of fructose metabolism; (1) essential or benign fructosuria due to fructokinase deficiency; (2) hereditary fructose intolerance; and (3) fructose-1,6-bisphosphatase deficiency. In this review the focus is set on the description of the clinical symptoms and biochemical anomalies in the three inborn errors of metabolism. The potential toxic effects of fructose in healthy humans also are discussed. Studies conducted in patients with inborn errors of fructose metabolism helped to understand fructose metabolism and its potential toxicity in healthy human. Influence of fructose on the glycolytic pathway and on purine catabolism is the cause of hypoglycemia, lactic acidosis and hyperuricemia. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provided new understandings into pathogenesis for these frequent diseases. PMID:28368361

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

PubMed

Muhar, Matthias; Ebert, Anja; Neumann, Tobias; Umkehrer, Christian; Jude, Julian; Wieshofer, Corinna; Rescheneder, Philipp; Lipp, Jesse J; Herzog, Veronika A; Reichholf, Brian; Cisneros, David A; Hoffmann, Thomas; Schlapansky, Moritz F; Bhat, Pooja; von Haeseler, Arndt; Köcher, Thomas; Obenauf, Anna C; Popow, Johannes; Ameres, Stefan L; Zuber, Johannes

2018-05-18

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

A transcription factor links growth rate and metabolism in the hypersaline adapted archaeon Halobacterium salinarum.

PubMed

Todor, Horia; Dulmage, Keely; Gillum, Nicholas; Bain, James R; Muehlbauer, Michael J; Schmid, Amy K

2014-09-01

Co-ordinating metabolism and growth is a key challenge for all organisms. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. The mechanisms underlying this 'growth homeostasis' are known in bacteria and eukaryotes, but remain unexplored in archaea. In the model archaeon Halobacterium salinarum, the transcription factor TrmB regulates enzyme-coding genes in diverse metabolic pathways in response to glucose. However, H. salinarum is thought not to catabolize glucose. To resolve this discrepancy, we demonstrate that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Additionally, we show that TrmB-DNA binding correlates with instantaneous growth rate, likely because S-layer glycosylation is proportional to growth. This suggests that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Remarkably, the topology and function of this growth homeostatic network appear conserved across domains despite extensive alterations in protein components. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.

PubMed

Madji Hounoum, Blandine; Mavel, Sylvie; Coque, Emmanuelle; Patin, Franck; Vourc'h, Patrick; Marouillat, Sylviane; Nadal-Desbarats, Lydie; Emond, Patrick; Corcia, Philippe; Andres, Christian R; Raoul, Cédric; Blasco, Hélène

2017-04-01

The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592-605. © 2017 Wiley Periodicals, Inc.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi.

PubMed

Liao, Y; Williams, T J; Ye, J; Charlesworth, J; Burns, B P; Poljak, A; Raftery, M J; Cavicchioli, R

2016-11-22

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi

PubMed Central

Liao, Y.; Williams, T. J.; Ye, J.; Charlesworth, J.; Burns, B. P.; Poljak, A.; Raftery, M. J.; Cavicchioli, R.

2016-01-01

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community. PMID:27874045

One-carbon metabolism: an aging-cancer crossroad for the gerosuppressant metformin.

PubMed

Menendez, Javier A; Joven, Jorge

2012-12-01

The gerosuppressant metformin operates as an efficient inhibitor of the mTOR/S6K1 gerogenic pathway due to its ability to ultimately activate the energy-sensor AMPK. If an aging-related decline in the AMPK sensitivity to cellular stress is a crucial event for mTOR-driven aging and aging-related diseases, including cancer, unraveling new proximal causes through which AMPK activation endows its gerosuppressive effects may offer not only a better understanding of metformin function but also the likely possibility of repositioning our existing gerosuppressant drugs. Here we provide our perspective on recent findings suggesting that de novo biosynthesis of purine nucleotides, which is based on the metabolism of one-carbon compounds, is a new target for metformin's actions at the crossroads of aging and cancer.

Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops

NASA Astrophysics Data System (ADS)

Mellidou, Ifigeneia; Kanellis, Angelos K.

2017-07-01

Ascorbic acid (AsA) is an essential compound present in almost all living organisms that has important functions in several aspects of plant growth and development, hormone signalling, as well as stress defense networks. In recent years, the genetic regulation of AsA metabolic pathways has received much attention due to its beneficial role in human diet. Despite the great variability within species, genotypes, tissues and developmental stages, AsA accumulation is considered to be controlled by the fine orchestration of net biosynthesis, recycling, degradation/oxidation, and/or intercellular and intracellular transport. To date, several structural genes from the AsA metabolic pathways and transcription factors are considered to significantly affect AsA in plant tissues, either at the level of activity, transcription or translation via feedback inhibition. Yet, all the emerging studies support the notion that the steps proceeding through GDP-L-galactose phosphorylase and to a lesser extent through GDP-D-mannose-3,5-epimerase are control points in governing AsA pool size in several species. In this mini review, we discuss the current consensus of the genetic regulation of AsA biosynthesis and recycling, with a focus on horticultural crops. The aspects of AsA degradation and transport are not discussed herein. Novel insights of how this multifaceted trait is regulated are critical to prioritize candidate genes for follow-up studies towards improving the nutritional value of fruits and vegetables.

Immunoinformatics Features Linked to Leishmania Vaccine Development: Data Integration of Experimental and In Silico Studies

PubMed Central

Brito, Rory C. F.; Guimarães, Frederico G.; Velloso, João P. L.; Corrêa-Oliveira, Rodrigo; Ruiz, Jeronimo C.; Reis, Alexandre B.; Resende, Daniela M.

2017-01-01

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. There is no human vaccine available and it is considered by many studies as apotential effective tool for disease control. To discover novel antigens, computational programs have been used in reverse vaccinology strategies. In this work, we developed a validation antigen approach that integrates prediction of B and T cell epitopes, analysis of Protein-Protein Interaction (PPI) networks and metabolic pathways. We selected twenty candidate proteins from Leishmania tested in murine model, with experimental outcome published in the literature. The predictions for CD4+ and CD8+ T cell epitopes were correlated with protection in experimental outcomes. We also mapped immunogenic proteins on PPI networks in order to find Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with them. Our results suggest that non-protective antigens have lowest frequency of predicted T CD4+ and T CD8+ epitopes, compared with protective ones. T CD4+ and T CD8+ cells are more related to leishmaniasis protection in experimental outcomes than B cell predicted epitopes. Considering KEGG analysis, the proteins considered protective are connected to nodes with few pathways, including those associated with ribosome biosynthesis and purine metabolism. PMID:28208616

Immunoinformatics Features Linked to Leishmania Vaccine Development: Data Integration of Experimental and In Silico Studies.

PubMed

Brito, Rory C F; Guimarães, Frederico G; Velloso, João P L; Corrêa-Oliveira, Rodrigo; Ruiz, Jeronimo C; Reis, Alexandre B; Resende, Daniela M

2017-02-10

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. There is no human vaccine available and it is considered by many studies as apotential effective tool for disease control. To discover novel antigens, computational programs have been used in reverse vaccinology strategies. In this work, we developed a validation antigen approach that integrates prediction of B and T cell epitopes, analysis of Protein-Protein Interaction (PPI) networks and metabolic pathways. We selected twenty candidate proteins from Leishmania tested in murine model, with experimental outcome published in the literature. The predictions for CD4⁺ and CD8⁺ T cell epitopes were correlated with protection in experimental outcomes. We also mapped immunogenic proteins on PPI networks in order to find Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with them. Our results suggest that non-protective antigens have lowest frequency of predicted T CD4⁺ and T CD8⁺ epitopes, compared with protective ones. T CD4⁺ and T CD8⁺ cells are more related to leishmaniasis protection in experimental outcomes than B cell predicted epitopes. Considering KEGG analysis, the proteins considered protective are connected to nodes with few pathways, including those associated with ribosome biosynthesis and purine metabolism.

A recycling index for food and health security: urban Taipei.

PubMed

Huang, Susana Tzy-Ying

2010-01-01

The modern food system has evolved into one with highly inefficient activities, producing waste at each step of the food pathway from growing to consumption and disposal. The present challenge is to improve recyclability in the food system as a fundamental need for food and health security. This paper develops a methodological approach for a Food Recycling Index (FRI) as a tool to assess recyclability in the food system, to identify opportunities to reduce waste production and environmental contamination, and to provide a self-assessment tool for participants in the food system. The urban Taipei framework was used to evaluate resource and nutrient flow within the food consumption and waste management processes of the food system. A stepwise approach for a FRI is described: (1) identification of the major inputs and outputs in the food chain; (2) classification of inputs and outputs into modules (energy, water, nutrients, and contaminants); (3) assignment of semi-quantitative scores for each module and food system process using a matrix; (4) assessment for recycling status and recyclability potential; (5) conversion of scores into sub-indices; (6) derivation of an aggregate FRI. A FRI of 1.24 was obtained on the basis of data for kitchen waste management in Taipei, a score which encompasses absolute and relative values for a comprehensive interpretation. It is apparent that a FRI could evolve into a broader ecosystem concept with health relevance. Community end-users and policy planners can adopt this approach to improve food and health security.

Biochemistry of Trypanosomatidae of Importance in Africa.

DTIC Science & Technology

1982-12-01

product . Compounds which appear as likely candidates include the following: Formycin B Formycin-B-ronophosphate Formycin-A-monophosphate Formycin-A...tetrahydro-2-fur-yl) -purine N6-Dimenthyladenine 2 ,6-bis- Chydroxyamino) -9-B-D-ri-bofuranosy3.- purine 6- Mercaptopurine 6-iodo-9- (tetr-ahydro-2...controlled anti- parasitic vaccines. Membrane antigens differ from one species to another and during the course of infection, making the production of a usqful

Evidence for the binding of the carcinogen 3-methylcholanthrene to both the purine and the pyrimidine bases of hamster fibroblast deoxyribonucleic acid (Short Communication)

PubMed Central

Jones, Peter A.; Gevers, Wieland; Hawtrey, Arthur O.

1973-01-01

The binding of [3H]3-methylcholanthrene to the DNA of hamster fibroblasts was studied by using chemical methods for DNA degradation. DNA depurinated by mild acid hydrolysis released approximately half of the radioactivity at the same rate as the purine bases, but the resulting apurinic acid still contained radioactive carcinogen. PMID:4797167

Synthesis of carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives as new potential PET tracers for imaging of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).

PubMed

Gao, Mingzhang; Wang, Min; Zheng, Qi-Huang

2016-03-01

The target tracer carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives, N-(3-[(11)C]methoxy-4-methoxyphenyl)-2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamide (3-[(11)C]4a) and N-(4-[(11)C]methoxy-3-methoxyphenyl)-2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamide (4-[(11)C]4a); 2-((6-amino-9H-purin-8-yl)thio)-N-(3-[(11)C]methoxy-4-methoxyphenyl)acetamide (3-[(11)C]8a) and 2-((6-amino-9H-purin-8-yl)thio)-N-(4-[(11)C]methoxy-3-methoxyphenyl)acetamide (4-[(11)C]8a), were prepared by O-[(11)C]methylation of their corresponding precursors with [(11)C]CH3OTf under basic condition (2N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50-60% radiochemical yields based on [(11)C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185-555GBq/μmol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Three stages during the evolution of the genetic code. [Abstract only

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1994-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity and a small codon number those amino acids emerging later in a translation process are derived. Both criteria indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage one use purines rich codons, thus purines have been retained in their third codon position. All the amino acids introduced in the second stage, in contrast, use pyrimidines in this codon position. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non enzymatic replication and interactions of DNA hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids which gradually decreased during their evolution. Amino acids independently available form prebiotic synthesis were thus correlated to purine rich codons. Conclusions on prebiotic replication are discussed also in the light of recent codon usage data.

Catalytic Role of Manganese Oxides in Prebiotic Nucleobases Synthesis from Formamide.

PubMed

Bhushan, Brij; Nayak, Arunima; Kamaluddin

2016-06-01

Origin of life processes might have begun with the formation of important biomonomers, such as amino acids and nucleotides, from simple molecules present in the prebiotic environment and their subsequent condensation to biopolymers. While studying the prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide, the manganese oxides demonstrated not only good binding for formamide but demonstrated novel catalytic activity. A novel one pot manganese oxide catalyzed synthesis of pyrimidine nucleobases like thymine is reported along with the formation of other nucleobases like purine, 9-(hydroxyacetyl) purine, cytosine, 4(3 H)-pyrimidinone and adenine in acceptable amounts. The work reported is significant in the sense that the synthesis of thymine has exhibited difficulties especially under one pot conditions and also such has been reported only under the catalytic activity of TiO2. The lower oxides of manganese were reported to show higher potential as catalysts and their existence were favored by the reducing atmospheric conditions prevalent on early Earth; thereby confirming the hypothesis that mineral having metals in reduced form might have been more active during the course of chemical evolution. Our results further confirm the role of formamide as a probable precursor for the formation of purine and pyrimidine bases during the course of chemical evolution and origin of life.

Current Concepts of Hyperuricemia and Gout

PubMed Central

Klinenberg, James R.

1969-01-01

Recent studies have confirmed that gout is an inborn error of metabolism. It has now become evident that the hyperuricemia associated with gout might occur either due to overproduction of uric acid, underexcretion of uric acid or a combination of these processes. Furthermore, patients with excessive purine synthesis may have a specific enzyme defect resulting in altered feedback inhibition of purine synthesis. A neurological disease manifest by mental retardation, choreo-athetosis, aggressive behavior, lip-biting and self-mutilation and associated with decidedly increased purine biosynthesis serves as a prototype of this kind of disorder. Other defects in regulation of purine biosynthesis have been postulated but their existence not yet confirmed. It has been demonstrated that urate crystals which are deposited from hyperuricemic body fluids set up an acute inflammatory reaction by means of a variety of chemical mediators. Thus, acute gouty arthritis is now recognized as an example of “crystal induced” synovitis. The treatment of gout consists of (1) the control of acute gouty attacks, and (2) the maintenance of normal serum uric acid concentrations. This latter may be achieved either with uricosuric drugs or with xanthine oxidase inhibition. With these principles in mind, it is now possible to avoid many of the severe crippling effects of gout and to restore the vast majority of gouty patients to useful and productive lives. PMID:5773483

Effects of pyrimidines on the guinea-pig coronary vasculature.

PubMed Central

Vials, A. J.; Burnstock, G.

1993-01-01

1. The effects of the pyrimidines, uridine 5'-triphosphate (UTP), thymidine 5'-triphosphate (TTP) and cytidine 5'-triphosphate (CTP), were examined in the guinea-pig coronary bed, by use of a Langendorff technique. Comparisons were made with the actions of the purines adenosine 5'-triphosphate (ATP), inosine 5'-triphosphate (ITP) and guanosine 5'-triphosphate (GTP). The effect of, the nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-NAME) and, the prostaglandin synthesis inhibitor, indomethacin on the vasodilator response to these purines and pyrimidines was examined. The effects of these inhibitors were assessed on their ability to inhibit both the amplitude and the area of the vasodilator response. 2. The relative order of potency of the purines and pyrimidines studied was ATP > UTP > ITP >> GTP, TTP, CTP. 3. The maximum amplitude and area of the vasodilator response to the pyrimidines, UTP (5 x 10(-10)-5 x 10(-7) mol), TTP (5 x 10(-8)-5 x 10(-7) mol) and CTP (5 x 10(-7) mol), and purines, ITP (5 x 10(-9)-5 x 10(-7) mol) and GTP (5 x 10(-8)-5 x 10(-7) mol), were significantly reduced by L-NAME (3 x 10(-5) and 10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8298797

Use of biological fluids for the rapid diagnosis of potentially lethal inherited disorders of human purine and pyrimidine metabolism.

PubMed

Morris, G S; Simmonds, H A; Davies, P M

1986-06-01

Inherited purine and pyrimidine disorders may be associated with serious, sometimes life-threatening consequences. Early and accurate diagnosis is essential. Difficulties encountered when using existing high pressure liquid chromatographic (HPLC) methods led to the development of an improved method based on prior fractionation of urine. The advantages are as follows. 1. Production of fingerprints demonstrating altered urinary excretion patterns characteristic of any one of ten different disorders, in 30 minutes. 2. Positive identification and quantification by comparison with established methods (using conventional chromatography, electrophoresis and UV spectrophotometry) in addition to specific retention times and characteristic UV absorbance ratios at two separate wavelengths (245 and 280 nm) by HPLC. 3. Direct analysis of all the purines and pyrimidines normally found in human body fluids as well as identification of abnormal compounds. 4. Short time between successive analyses while maintaining excellent resolution between compounds of interest and column longevity. 5. Improved separation of the different adenine-based compounds encountered in some disorders, plus demonstration of potential interference by dietary or drug metabolites. 6. Applicability to the monitoring of therapy involving a variety of different purine and pyrimidine analogues. Particular attention should be paid to sample preparation. Plasma profiles will confirm the diagnosis in some, but not all, of these disorders.

DNA damage in lens epithelium of cataract patients in vivo and ex vivo.

PubMed

Øsnes-Ringen, Oyvind; Azqueta, Amaia O; Moe, Morten C; Zetterström, Charlotta; Røger, Magnus; Nicolaissen, Bjørn; Collins, Andrew R

2013-11-01

DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Transcriptional Profiling of Saccharomyces cerevisiae Reveals the Impact of Variation of a Single Transcription Factor on Differential Gene Expression in 4NQO, Fermentable, and Nonfermentable Carbon Sources

PubMed Central

Rong-Mullins, Xiaoqing; Ayers, Michael C.; Summers, Mahmoud; Gallagher, Jennifer E. G.

2017-01-01

Cellular metabolism can change the potency of a chemical’s tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO in Saccharomyces cerevisiae. To study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenic S. cerevisiae strains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression of SNQ2 (a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression of SNQ2 alone was sufficient to overcome 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci including SNQ2. In addition to a known Yrr1 target SNG1, Yrr1 also bound upstream of RPL35B; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including the de novo purine pathway, and the ribonuclease reductase pathways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of the ADE genes in the de novo purine biosynthetic pathway, the mutant Yrr1 increased expression of ADE genes even in the absence of 4NQO. These same ADE genes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integration of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization. PMID:29208650

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling.

PubMed

Bai, Zhiyong; Grant, Barth D

2015-03-24

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1-positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42-associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

PubMed Central

Bai, Zhiyong; Grant, Barth D.

2015-01-01

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

Purine uptake in Plasmodium: transport versus metabolism.

PubMed

Kirk, Kiaran; Howitt, Susan M; Bröer, Stefan; Saliba, Kevin J; Downie, Megan J

2009-06-01

In a recent paper, Quashie et al. have proposed that purine uptake into the intraerythrocytic malaria parasite involves four different plasma membrane transporters - two high affinity and two low affinity. They equate one of the two high-affinity transporters with PfNT1, a transporter reported previously to be a low-affinity system. Here, we offer an alternative interpretation of their data, suggesting that the conclusions drawn by Quashie et al. take insufficient account of metabolism.

Chemical Carcinogen (Hydrazine et al.) Induced Carcinogenesis of Human Diploid Fibroblasts in vitro.

DTIC Science & Technology

1985-06-12

compound then is converted to a carbonium ion and the radical interacts /, with the purine bases in DNA. Methylazoxymethanol acetate, ( MAMA ) in the...interacts with the purine bases in DNA. Methylazoxymethanol acetate, ( MAMA ) in the presence of colon, secum and liver homogenates reduced NAD+ to NADH...3. Human foreskin fibroblast populations blocked in G1 , released and treated with methylazoxy methanol acetate ( MAMA ) from the time of

RECEPTOR AFFINITY AND PHOSPHODIESTERASES 4B AND 10A ACTIVITY OF OCTAHYDRO- AND 6,7-DIMETHOXY-3,4-DIHYDRO- ISOQUINOLIN-2(1H)-YL-ALKYL DERIVATIVES OF IMIDAZO- AND PYRIMIDINO[2,1-f]PURINES.

PubMed

Zagórska, Agnieszka; Gryzło, Beata; Satała, Grzegorz; Bojarski, Andrzej J; Głuch-Lutwin, Monika; Mordyl, Barbara; Kazek, Grzegorz; Pawłowski, Maciej

2016-01-01

A series of octahydro- and 6,7-dimethoxy-3,4-dihydro- isoquinolin-2(1H)-yl-alkyl derivatives of imidazo- and pyrimidino[2,1-f]purines were synthesized and biologically evaluated in in vitro competition binding experiments for serotonin 5-HT(1A), 5-HT(6), 5-HT(7), and dopamine D2 receptors and inhibitory potencies for phosphodiesterases - PDE4B1 and PDE10A. The structure-activity relationships allowed to determine the structural features responsible for receptor and enzyme activity. Compound 5 (8-(4-(6,7-dimethoxy-3,4-dihydroiso- quinolin-2(1H)butyl)1,3-dimethyl-H-imidazo[2,1-f]purine-2,4(3H,8H)-dione) could be regarded as promising structure for further modification and detailed mechanistic study for obtained hybrid ligands.

S-allyl derivatives of 6-mercaptopurine are highly potent drugs against human B-CLL through synergism between 6-mercaptopurine and allicin.

PubMed

Miron, Talia; Wilchek, Meir; Shvidel, Lev; Berrebi, Alain; Arditti, Fabian D

2012-12-01

S-allylthio-6-mercaptopurine and its ribose derivative were tested for anti-leukemic activity, using a human- mouse B-CLL model. The novel prodrugs contain two components, a purine analog, which interferes with DNA synthesis, and an S-allylthio, readily engaging in thiol-disulfide exchange reactions. The latter component targets the redox homeostasis which is more sensitive in leukemic cells, than in normal B-cells. Upon administration, the prodrug permeates cells, instantly reacts with free thiol, forming S-allyl mixed disulfides and releasing purine. Several cycles of thiol-disulfide exchange reactions occur, thus extending the duration of the prodrug effects. The concerted action of 2 components, as compared with purine alone, boosted in vitro apoptotis in B-CLL cells from 10% to 38%, and decreased in vivo engraftment of B-CLL from 30% to 0.7%. Copyright © 2012 Elsevier Ltd. All rights reserved.

Determination of Caffeine and Other Purine Compounds in Food and Pharmaceuitcals by Micellar Electrokinetic Chrmoatography

NASA Astrophysics Data System (ADS)

Vogt, Carla; Contradi, S.; Rohde, E.

1997-09-01

Capillary elctrophoresis is a modern separation technique, especially the extremely high efficiencies and minimal requirements with regard to buffers, samples and solvents lead to a dramatic increase of applications in the last few years. This paper offers an introduction to the technique of micellar elektrokinetic chromatography as a special kind of capillary electrophoresis. Caffeine and other purine compounds have been determined in foodstuff (tea, coffee, cocoa) as well as in pharmaceutical formulations. Different sample preparation procedures which have been developed with regard to the special properties of the sample matrices are discussed in the paper.This preparation facilitates the separation in many cases. So students have to solve a relatively simple separation problem by variation of buffer pH, buffer components and separation parameters. By doing a calibration for the analyzed purine compounds they will learn about reproducibility in capillary electrophoresis.

Extraction of purine alkaloids from maté (Ilex paraguariensis) using supercritical CO(2).

PubMed

Saldaña, M D; Mohamed, R S; Baer, M G; Mazzafera, P

1999-09-01

Experimental data for the supercritical CO(2) extraction of purine alkaloids (caffeine, theobromine, and theophylline) from ground herbal maté tea (Ilex paraguaryensis) using a high-pressure apparatus are presented. Caffeine, theophylline, and theobromine were identified in the extracted fractions using HPLC. Results indicated a much higher CO(2) selectivity for caffeine in comparison with those for theophylline and theobromine. Solubilities of pure compounds in carbon dioxide were also determined at 313.2, 323.2, 338.2, and 343.2 K, and pressures ranging from 14 to 24 MPa. Caffeine solubility exhibited a retrograde behavior with temperature while theophylline and theobromine manifested a normal behavior at conditions explored in this study. Solubilities in binary CO(2)/purine alkaloid model systems were much higher than those obtained during extraction of maté tea, demonstrating the difficulty of using binary data in predicting complex multicomponent behavior.

Theacrine, a special purine alkaloid with sedative and hypnotic properties from Cammelia assamica var. kucha in mice.

PubMed

Xu, Jie-Kun; Kurihara, Hiroshi; Zhao, Liang; Yao, Xin-Sheng

2007-01-01

The central nervous system activities of theacrine (1,3,7,9-tetramethyluric acid), a purine alkaloid which is abundantly present in Camellia assamica var. kucha, were investigated in ambulatory activity, pentobarbital-induced sleep and forced swimming test in mice, compared with two other purine alkaloids, caffeine and theobromine. Caffeine treatment led to a marked increase in the ambulatory activity accompanied with decreasing of the immobility time in forced swimming test at both 10 and 30 mg/kg. Under the same conditions, neither theacrine nor theobromine showed obvious excited efficacy. Both doses of theacrine could significantly prolong the sleeping time induced by pentobarbital, while caffeine and theobromine exhibited an inverted effect. These results indicated that theacrine possessed potent sedative and hypnotic properties and its central nervous system effects were different from those of caffeine and theobromine.

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

PubMed

Carta, M C; Mattana, A; Camici, M; Allegrini, S; Tozzi, M G; Sgarrella, F

2001-10-03

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

PubMed Central

Brabletz, T; Pietrowski, I; Serfling, E

1991-01-01

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism. Images PMID:1707162

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

PubMed

Brabletz, T; Pietrowski, I; Serfling, E

1991-01-11

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism.

Therapeutic efficacy of atypical antipsychotic drugs by targeting multiple stress-related metabolic pathways

PubMed Central

Cai, H L; Jiang, P; Tan, Q Y; Dang, R L; Tang, M M; Xue, Y; Deng, Y; Zhang, B K; Fang, P F; Xu, P; Xiang, D X; Li, H D; Yao, J K

2017-01-01

Schizophrenia (SZ) is considered to be a multifactorial brain disorder with defects involving many biochemical pathways. Patients with SZ show variable responses to current pharmacological treatments of SZ because of the heterogeneity of this disorder. Stress has a significant role in the pathophysiological pathways and therapeutic responses of SZ. Atypical antipsychotic drugs (AAPDs) can modulate the stress response of the hypothalamic–pituitary–adrenal (HPA) axis and exert therapeutic effects on stress by targeting the prefrontal cortex (PFC) and hippocampus. To evaluate the effects of AAPDs (such as clozapine, risperidone and aripiprazole) on stress, we compared neurochemical profile variations in the PFC and hippocampus between rat models of chronic unpredictable mild stress (CUMS) for HPA axis activation and of long-term dexamethasone exposure (LTDE) for HPA axis inhibition, using an ultraperformance liquid chromatography–mass spectrometry (UPLC–MS/MS)-based metabolomic approach and a multicriteria assessment. We identified a number of stress-induced biomarkers comprising creatine, choline, inosine, hypoxanthine, uric acid, allantoic acid, lysophosphatidylcholines (LysoPCs), phosphatidylethanolamines (PEs), corticosterone and progesterone. Specifically, pathway enrichment and correlation analyses suggested that stress induces oxidative damage by disturbing the creatine–phosphocreatine circuit and purine pathway, leading to excessive membrane breakdown. Moreover, our data suggested that the AAPDs tested partially restore stress-induced deficits by increasing the levels of creatine, progesterone and PEs. Thus, the present findings provide a theoretical basis for the hypothesis that a combined therapy using adenosine triphosphate fuel, antioxidants and omega-3 fatty acids as supplements may have synergistic effects on the therapeutic outcome following AAPD treatment. PMID:28509906

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

PubMed Central

Andersen, Kasper R.; Kilstrup, Mogens; Martinussen, Jan; Switzer, Robert L.; Willemoës, Martin

2016-01-01

SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species. PMID:28031352

Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis.

PubMed

Wang, Guanglu; Shi, Ting; Chen, Tao; Wang, Xiaoyue; Wang, Yongcheng; Liu, Dingyu; Guo, Jiaxin; Fu, Jing; Feng, Lili; Wang, Zhiwen; Zhao, Xueming

2018-06-02

Commercial riboflavin production with Bacillus subtilis has been developed by combining rational and classical strain development for almost two decades, but how an improved riboflavin producer can be created rationally is still not completely understood. In this study, we demonstrate the combined use of integrated genomic and transcriptomic analysis of the genetic basis for riboflavin over-production in B. subtilis. This methodology succeeded in discerning the positive mutations in the mutagenesis derived riboflavin producer B. subtilis 24/pMX45 through whole-genome sequencing and transcriptome sequencing. These included RibC (G199D), ribD + (G+39A), PurA (P242L), CcpN(A44S), YvrH (R222Q) and two nonsense mutations YhcF (R90*) and YwaA (Q68*). Reintroducing these specific mutations into the wild-type strain recovered the riboflavin overproduction phenotype and subsequent metabolic engineering greatly improved riboflavin production, achieving an up to 3.4-fold increase of the riboflavin titer over the sequenced producer. A novel mutation, YvrH (R222Q), involved in a typical two-component regulatory system deregulated the purine de novo synthesis pathway and increased the pool of intracellular purine metabolites, which in turn increased riboflavin production. Taken together, we present a case study of combining genome and transcriptome analysis to elucidate the genetic underpinnings of a complex cellular property, which enabled the transfer of beneficial mutations to engineer a reference strain into an overproducer. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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.

Involvement of GABAA receptor in Bufo arenarum oocyte maturation.

PubMed

Toranzo, G Sánchez; Zelarayán, L; Bonilla, F; Oterino, J; Bühler, M I

2008-05-01

Amphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body. The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol. Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant. In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

PubMed

Hégarat, Nadia; Novopashina, Darya; Fokina, Alesya A; Boutorine, Alexandre S; Venyaminova, Alya G; Praseuth, Danièle; François, Jean-Christophe

2014-03-01

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells. © 2014 FEBS.

Metabolic effect of alkaline additives and guanosine/gluconate in storage solutions for red blood cells.

PubMed

D'Alessandro, Angelo; Reisz, Julie A; Culp-Hill, Rachel; Korsten, Herbert; van Bruggen, Robin; de Korte, Dirk

2018-04-06

Over a century of advancements in the field of additive solutions for red blood cell (RBC) storage has made transfusion therapy a safe and effective practice for millions of recipients worldwide. Still, storage in the blood bank results in the progressive accumulation of metabolic alterations, a phenomenon that is mitigated by storage in novel storage additives, such as alkaline additive solutions. While novel alkaline additive formulations have been proposed, no metabolomics characterization has been performed to date. We performed UHPLC-MS metabolomics analyses of red blood cells stored in SAGM (standard additive in Europe), (PAGGSM), or alkaline additives SOLX, E-SOL 5 and PAG3M for either 1, 21, 35 (end of shelf-life in the Netherlands), or 56 days. Alkaline additives (especially PAG3M) better preserved 2,3-diphosphoglycerate and adenosine triphosphate (ATP). Deaminated purines such as hypoxanthine were predictive of hemolysis and morphological alterations. Guanosine supplementation in PAGGSM and PAG3M fueled ATP generation by feeding into the nonoxidative pentose phosphate pathway via phosphoribolysis. Decreased urate to hypoxanthine ratios were observed in alkaline additives, suggestive of decreased generation of urate and hydrogen peroxide. Despite the many benefits observed in purine and redox metabolism, alkaline additives did not prevent accumulation of free fatty acids and oxidized byproducts, opening a window for future alkaline formulations including (lipophilic) antioxidants. Alkalinization via different strategies (replacement of chloride anions with either high bicarbonate, high citrate/phosphate, or membrane impermeant gluconate) results in different metabolic outcomes, which are superior to current canonical additives in all cases. © 2018 AABB.

Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes.

PubMed

Sánchez Toranzo, G; Bonilla, F; Zelarayán, L; Oterino, J; Bühler, M I

2004-08-01

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.

Isolating the metabolic pathways involved in the hepatoprotective effect of Muntingia calabura against CCl4-induced liver injury using LC/MS Q-TOF.

PubMed

Rofiee, M S; Yusof, M I M; Abdul Hisam, E E; Bannur, Z; Zakaria, Z A; Somchit, M N; Teh, L K; Salleh, M Z

2015-05-26

Muntingia calabura L. has been used in Southeast Asia and tropical America as antipyretic, antiseptic, analgesic, antispasmodic and liver tonic. This study aims to determine the acute toxicity and the metabolic pathways involved in the hepatoprotective mechanism of M. calabura. CCl4-induced hepatotoxic rat model was developed and a dose dependent effect of M. calabura was conducted. Body weight, food and water consumption were measured every day and rats were sacrificed to collect the serum samples at the end of the 10-days treatment. Liquid chromatography-mass spectrometry quadrapole time of flight (LC/MS-QTOF) combined with principal component analysis (PCA) were used to determine differentially expressed metabolites due to treatment with CCl4 and M. calabura extracts. Metabolomics Pathway Analysis (MetPA) was used for analysis and visualization of pathways involved. Body weight, food and water consumption were significantly decreased and histopathological study revealed steatosis in CCl4-induced rats. PCA score plots show distinct separation in the metabolite profiles of the normal group, CCl4-treated group and extract of M. calabura (MCME) pre-treated groups. Biomarkers network reconstruction using MetPA had identified 2 major pathways which were involved in the protective mechanism of MCME. These include the (i) biosynthesis of the primary bile acid, (ii) metabolism of arachidonic acid. This study has successfully isolated 2 major pathways involved in the hepatoprotecive effect of MCME against CCl4-induced liver injury using the LC/MS Q-TOF metabolomics approach. The involvement of archidonic acid and purine metabolism in hepatoprotection has not been reported previously and may provide new therapeutic targets and/or options for the treatment of liver injury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Development of life cycle water footprints for the production of fuels and chemicals from algae biomass.

PubMed

Nogueira Junior, Edson; Kumar, Mayank; Pankratz, Stan; Oyedun, Adetoyese Olajire; Kumar, Amit

2018-09-01

This study develops life cycle water footprints for the production of fuels and chemicals via thermochemical conversion of algae biomass. This study is based on two methods of feedstock production - ponds and photobioreactors (PBRs) - and four conversion pathways - fast pyrolysis, hydrothermal liquefaction (HTL), conventional gasification, and hydrothermal gasification (HTG). The results show the high fresh water requirement for algae production and the necessity to recycle harvested water or use alternative water sources. To produce 1 kg of algae through ponds, 1564 L of water are required. When PBRs are used, only 372 L water are required; however, the energy requirements for PBRs are about 30 times higher than for ponds. From a final product perspective, the pathway based on the gasification of algae biomass was the thermochemical conversion method that required the highest amount of water per MJ produced (mainly due to its low hydrogen yield), followed by fast pyrolysis and HTL. On the other hand, HTG has the lowest water footprint, mainly because the large amount of electricity generated as part of the process compensates for the electricity used by the system. Performance in all pathways can be improved through recycling channels. Copyright © 2018 Elsevier Ltd. All rights reserved.

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

DOE PAGES

Doyle, Siamsa M.; Haeger, Ash; Vain, Thomas; ...

2015-02-02

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering themore » polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 ( gnl1-1) and gnom ( van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. In conclusion, our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.« less

Synthesis and recycling of tetrahydrobiopterin in endothelial function and vascular disease.

PubMed

Crabtree, Mark J; Channon, Keith M

2011-08-01

Nitric oxide, generated by the nitric oxide synthase (NOS) enzymes, plays pivotal roles in cardiovascular homeostasis and in the pathogenesis of cardiovascular disease. The NOS cofactor, tetrahydrobiopterin (BH4), is an important regulator of NOS function, since BH4 is required to maintain enzymatic coupling of L-arginine oxidation, to produce NO. Loss or oxidation of BH4 to 7,8-dihydrobiopterin (BH2) is associated with NOS uncoupling, resulting in the production of superoxide rather than NO. In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR) can 'recycle' BH2, and thus regenerate BH4. It is therefore likely that net BH4 cellular bioavailability reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. Recent studies have implicated BH4 recycling in the direct regulation of eNOS uncoupling, showing that inhibition of BH4 recycling using DHFR-specific siRNA and methotrexate treatment leads to eNOS uncoupling in endothelial cells and the hph-1 mouse model of BH4 deficiency, even in the absence of oxidative stress. These studies indicate that not only BH4 level, but the recycling pathways regulating BH4 bioavailability represent potential therapeutic targets and will be discussed in this review. Copyright © 2011 Elsevier Inc. All rights reserved.

The role of DNA repair pathways in cisplatin resistant lung cancer.

PubMed

O'Grady, Shane; Finn, Stephen P; Cuffe, Sinead; Richard, Derek J; O'Byrne, Kenneth J; Barr, Martin P

2014-12-01

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

In Vitro Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units

PubMed Central

Gall, Daniel L.; Kontur, Wayne S.; Lan, Wu; Kim, Hoon; Li, Yanding; Ralph, John

2017-01-01

ABSTRACT New environmentally sound technologies are needed to derive valuable compounds from renewable resources. Lignin, an abundant polymer in terrestrial plants comprised predominantly of guaiacyl and syringyl monoaromatic phenylpropanoid units, is a potential natural source of aromatic compounds. In addition, the plant secondary metabolite tricin is a recently discovered and moderately abundant flavonoid in grasses. The most prevalent interunit linkage between guaiacyl, syringyl, and tricin units is the β-ether linkage. Previous studies have shown that bacterial β-etherase pathway enzymes catalyze glutathione-dependent cleavage of β-ether bonds in dimeric β-ether lignin model compounds. To date, however, it remains unclear whether the known β-etherase enzymes are active on lignin polymers. Here we report on enzymes that catalyze β-ether cleavage from bona fide lignin, under conditions that recycle the cosubstrates NAD+ and glutathione. Guaiacyl, syringyl, and tricin derivatives were identified as reaction products when different model compounds or lignin fractions were used as substrates. These results demonstrate an in vitro enzymatic system that can recycle cosubstrates while releasing aromatic monomers from model compounds as well as natural and engineered lignin oligomers. These findings can improve the ability to produce valuable aromatic compounds from a renewable resource like lignin. IMPORTANCE Many bacteria are predicted to contain enzymes that could convert renewable carbon sources into substitutes for compounds that are derived from petroleum. The β-etherase pathway present in sphingomonad bacteria could cleave the abundant β–O–4-aryl ether bonds in plant lignin, releasing a biobased source of aromatic compounds for the chemical industry. However, the activity of these enzymes on the complex aromatic oligomers found in plant lignin is unknown. Here we demonstrate biodegradation of lignin polymers using a minimal set of β-etherase pathway enzymes, the ability to recycle needed cofactors (glutathione and NAD+) in vitro, and the release of guaiacyl, syringyl, and tricin as depolymerized products from lignin. These observations provide critical evidence for the use and future optimization of these bacterial β-etherase pathway enzymes for industrial-level biotechnological applications designed to derive high-value monomeric aromatic compounds from lignin. PMID:29180366

Purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of Alzheimer's disease pathology.

PubMed

Alonso-Andrés, Patricia; Albasanz, José Luis; Ferrer, Isidro; Martín, Mairena

2018-01-24

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines. © 2018 International Society of Neuropathology.

Regeneration of Stevia Plant Through Callus Culture

PubMed Central

Patel, R. M.; Shah, R. R.

2009-01-01

Stevia rebaudiana Bertoni that conventionally propagated by seed or by cuttings or clump division which has a limitation of quality and quantity seed material. In present study, callus culture technique was tried to achieve rapid plant multiplication for quality seed material. Callus induction and multiplication medium was standardized from nodal as well as leaf sagments. It is possible to maintain callus on Murashige and Skoog medium supplemented with 6-benzyl amino purine and naphthalene acetic acid. Maximum callus induction was obtained on Murashige and Skoog medium incorporated with 6-benzyl amino purine (2.0-3.0 mg/l) and naphthalene acetic acid (2.0 mg/l) treatments. However, Murashige and Skoog medium containing 2.0 mg/l 6-benzyl amino purine+2.0 mg/l naphthalene acetic acid was found to be the best for callus induction. Higher regeneration frequency was noticed with Murashige and Skoog medium supplemented with 2.0 mg/l 6-benzyl amino purine+0.2 mg/l naphthalene acetic acid. Regenerated plants were rooted better on ¼ Murashige and Skoog strength supplemented with 0.1 mg/l indole-3-butyric acid. The rooted plantlets were hardened successfully in tera care medium with 63 per cent survival rate. The developed protocol can be utilized for mass production of true to type planting material on large scale independent of season, i.e. external environmental conditions. PMID:20177455

Penetration of theophylline and adenosine into excised human skin from binary and ternary vehicles: effect of a nonionic surfactant.

PubMed

Kadir, R; Stempler, D; Liron, Z; Cohen, S

1989-02-01

A nonionic surfactant, diethyleneglycol lauryl ether (PEG-2-L), increases the flux of either theophylline or adenosine by a factor of 2.2-2.7, when these are delivered from propionic acid solutions into human skin samples, with respect to propionic acid alone. At the same time, the flux of propionic acid from the same vehicles is decreased. Significant expansion of the partial molal volumes vi of both purines occurs following incorporation of PEG-2-L into their propionic acid solution. Hence, the enhancing effect of this surfactant arises mainly from an increase in the excess free energy of these solutes in the donor phase ("push" effect). Paraffin oil increases the flux of either drug from propionic acid by an entirely different mechanism. It enhances the flux of propionic acid into the skin, thus promoting the partitioning of the purine solute in the modified skin barrier ("pull" effect). Indeed, the magnitude of vi of either purine in propionic acid:paraffin oil solution gives no indication of a significant interaction between paraffin oil and the purine solute. Finally, the penetration enhancing effects of PEG-2-L and paraffin oil combined together in the same propionic acid vehicle are additive, resulting in a flux which is approximately the sum total of fluxes obtained separately with PEG-2-L or paraffin oil.

Synthesis of conformationally locked L-deoxythreosyl phosphonate nucleosides built on a bicyclo[3.1.0]hexane template.

PubMed

Saneyoshi, Hisao; Deschamps, Jeffrey R; Marquez, Victor E

2010-11-19

Two conformationally locked versions of l-deoxythreosyl phosphonate nucleosides (2 and 3) were synthesized to investigate the preference of HIV reverse transcriptase for a conformation displaying either a fully diaxial or fully diequatorial disposition of substituents. Synthesis of the enantiomeric 4-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-2-ol carbocyclic nucleoside precursors (diaxially disposed) proceeded straightforwardly from commercially available (1R,4S)-4-hydroxy-2-cyclopent-2-enyl-1-yl acetate employing a hydroxyl-directed Simmons-Smith cyclopropanation that culminated with a Mitsunobu coupling of the purine base. For the more complicated 1-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-3-ol carbocyclic nucleoside precursors (diequatorially disposed), the obligatory linear approach required the syntheses of key 1-aminobicyclo[3.1.0.]hexan-3-yl benzoate precursors that were assembled via the amide variant of the Kulinkovich reaction involving the intramolecular cyclopropanation of a substituted δ-vinylamide. Completion of the purine ring was achieved by conventional approaches but with much improved yields through the use of a microwave reactor. The syntheses of the phosphonates and the corresponding diphosphates were achieved by conventional means. None of the diphosphates, which were supposed to act as nucleoside triphosphate mimics, could compete with dATP even when present in a 10-fold excess.

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance.

PubMed

Liang, Keming; Shen, Claire R

2017-01-01

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k cat /K m ≈0.4mM -1 s -1 ) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V max ≈6U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

HIV-1 Vpu Blocks Recycling and Biosynthetic Transport of the Intrinsic Immunity Factor CD317/Tetherin To Overcome the Virion Release Restriction

PubMed Central

Schmidt, Sarah; Fritz, Joëlle V.; Bitzegeio, Julia; Fackler, Oliver T.; Keppler, Oliver T.

2011-01-01

ABSTRACT The intrinsic immunity factor CD317 (BST-2/HM1.24/tetherin) imposes a barrier to HIV-1 release at the cell surface that can be overcome by the viral protein Vpu. Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood. Here, we characterized the influence of Vpu on major CD317 trafficking pathways using quantitative antibody-based endocytosis and recycling assays as well as a microinjection/microscopy-based kinetic de novo expression approach. We report that HIV-1 Vpu inhibited both the anterograde transport of newly synthesized CD317 and the recycling of CD317 to the cell surface, while the kinetics of CD317 endocytosis remained unaffected. Vpu trapped trafficking CD317 molecules at the trans-Golgi network, where the two molecules colocalized. The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, β-TrCP. Treatment of cells with the malaria drug primaquine resulted in a CD317 trafficking defect that mirrored that induced by Vpu. Importantly, primaquine could functionally replace Vpu as a CD317 antagonist and rescue HIV-1 particle release. PMID:21610122

The early endosome: a busy sorting station for proteins at the crossroads

PubMed Central

Jovic, Marko; Sharma, Mahak; Rahajeng, Juliati; Caplan, Steve

2010-01-01

Summary Endocytosis marks the entry of internalized receptors into the complex network of endocytic trafficking pathways. Endocytic vesicles are rapidly targeted to a distinct membrane-bound endocytic organelle referred to as the early endosome. Despite the existence of numerous internalization routes, early endosomes (EE) serve as a focal point of the endocytic pathway. Sorting events initiated at this compartment determine the subsequent fate of internalized proteins and lipids, destining them either for recycling to the plasma membrane, degradation in lysosomes or delivery to the trans-Golgi network. Sorting of endocytic cargo to the latter compartments is accomplished through the formation of distinct microdomains within early endosomes, through the coordinate recruitment and assembly of the sorting machinery. An elaborate network of interactions between endocytic regulatory proteins ensures synchronized sorting of cargo to microdomains followed by morphological changes at the early endosomal membranes. Consequently, the cargo targeted either for recycling back to the plasma membrane, or for retrograde transport to the trans-Golgi network, localizes to newly-formed tubular membranes. With a high ratio of membrane surface to lumenal volume, these tubules effectively concentrate the recycling cargo, ensuring efficient transport out of the EE. Conversely, receptors sorted for degradation cluster at the flat clathrin lattices involved in invaginations of the limiting membrane, associating with newly formed intralumenal vesicles. In this review we will discuss the characteristics of early endosomes, their role in the regulation of endocytic transport, and their aberrant function in a variety of diseases. PMID:19924646

Drosophila VAMP7 regulates Wingless intracellular trafficking.

PubMed

Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

2017-01-01

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

Synthesis of some glycoside analogs and related compounds from 9-amino-6-(methylthio)-9H-purine.

PubMed

Temple, C; Kussner, C L; Montgomery, J A

1975-12-01

Additional information on the anticancer activity of 9-amino-9H-purine-6(1H)-thione and its derivatives was sought by the synthesis of some 9-(substituted amino)-6-(methylthio)-9H-purines in which the 9-substituent contained functional groups capable of either reversible or irreversible binding with an enzymatic site. Condensation of 9-amino-6-(methylthio)-9H-purine (1) with some carbonyl compounds followed by hydride reduction of the azomethine linkage in the intermediates leads to the 2-pyrrolylmethyl (8), 2,3,4-trihydroxybutyl (10), and the 1,5-dihydroxy-2- and 3-pentyl (11 and 12) compounds. A 4-hydroxybutyl derivative (13) was obtained by alkylation of 18, the 9-acetyl derivative of 1, with 4-chlorobutyl acetate followed by saponification. The cyclization of 13 and 11 with a sulfonyl chloride gave the 9-pyrrolidin-1-yl (27) and the 9-[2-(tosyloxymethyl)pyrrolidin-1-yl] (28), respectively. Acylation of 1 with ethyl L-2-pyrrolidine-5-carboxylate and ethyl 1-methyl-5-pyrrolidone-3-carboxylate, respectively, in Me2SO containing NaH gave the corresponding amides 15 and 17. Alkylation of 18 with 1-bromo-2-chloroethane and epichlorohydrin gave the N-(2-chloroethyl) and N-(1,2-epoxy-3-propyl) derivatives 19 and 20. The chloro group of the chlorobutyl derivative of 18 was displaced with KSCN and NaN3, respectively, to give the thiocyanate and azido derivatives 23 and 24. Hydrogenation of the latter gave the amine (25), which was acylated with ethyl chloroformate to give the (ethoxycarbonyl)amino compound 26. None of these compounds showed activity against L1210 leukemia cells implanted ip in mice on a single-dose schedule, suggesting that the activity observed in the simpler 9-aminopurines resulted from cleavage of the hydrazino linkage to give pH-purine-6(1H)-thione.

Viewing pre-60S maturation at a minute’s timescale

PubMed Central

Zisser, Gertrude; Ohmayer, Uli; Mauerhofer, Christina; Mitterer, Valentin; Klein, Isabella; Rechberger, Gerald N; Wolinski, Heimo; Prattes, Michael; Pertschy, Brigitte; Milkereit, Philipp

2018-01-01

Abstract The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation. PMID:29294095

Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

PubMed

Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

2015-12-01

In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

PubMed Central

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-01-01

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27–retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27–retromer–WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi. PMID:26956659

FAM21 directs SNX27-retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus.

PubMed

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-03-09

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27-retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27-retromer-WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi.

School Compost Programs: Pathways to Success

ERIC Educational Resources Information Center

Schumpert, Kary; Dietz, Cyndra

2012-01-01

After the oft-repeated three Rs (reduce, reuse, recycle) comes the lesser-known but equally important fourth R: rot. In this case, rot means compost. Classrooms, schools, and school districts can use a number of methods to establish a compost program. The finished product is a valuable soil amendment that adds fertility to local farmland, school…

Recycling signals in the neural crest.

PubMed

Taneyhill, Lisa A; Bronner-Fraser, Marianne

2005-01-01

Vertebrate neural crest cells are multipotent and differentiate into structures that include cartilage and the bones of the face, as well as much of the peripheral nervous system. Understanding how different model vertebrates utilize signaling pathways reiteratively during various stages of neural crest formation and differentiation lends insight into human disorders associated with the neural crest.

The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

PubMed

Bouchet, Jérôme; McCaffrey, Mary W; Graziani, Andrea; Alcover, Andrés

2018-07-04

Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

Biodegradation pathway of an anionic surfactant (Igepon TC-42) during recycling waste water through plant hydroponics for advanced life support during long-duration space missions

NASA Astrophysics Data System (ADS)

Levine, L. H.; Kagie, H. R.; Garland, J. L.

The degradation of an anionic surfactant (Igepon TC-42) was investigated as part of an integrated study of direct recycling of human hygiene water through hydroponic plant growth systems. Several chemical approaches were developed to characterize the degradation of Igepon and to measure the accumulation of intermediates such as fatty acids and methyl taurine. Igepon was rapidly degraded as indicated by the reduction of methylene blue active substances (MBAS) and component fatty acids. The Igepon degradation rate continued to increase over a period of several weeks following repeated daily exposure to 18 μg/l Igepon. The accumulation of free fatty acids and methyl taurine was also observed during decomposition of Igepon. The concentration of methyl taurine was below detection limit (0.2 nmol/ml) during the slow phase of Igepon degradation, and increased to 1-2 nmol/ml during the phase of rapid degradation. These findings support a degradation pathway involving initial hydrolysis of amide to release fatty acids and methyl taurine, and subsequent degradation of these intermediates.

Nitrogen recycling during phenylpropanoid metabolism in sweet potato tubers

NASA Technical Reports Server (NTRS)

Singh, S.; Lewis, N. G.; Towers, G. H.

1998-01-01

In the first step of the phenylpropanoid metabolic pathway, L-phenylalanine (L-Phe) is deaminated to form E-cinnamate, in a conversion catalyzed by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). The metabolic fate of the ammonium ion (NH4+) produced in this reaction was investigated in sweet potato (Ipomoea batatas) tuber discs. [15N]-Labeled substrates including L-Phe, in the presence or absence of specific enzyme inhibitors, were administered to sweet potato discs in light under aseptic conditions. 15N-Nuclear magnetic resonance spectroscopic analyses revealed that the 15NH4+ liberated during the PAL reaction is first incorporated into the amide nitrogen of L-glutamine (L-Gln) and then into L-glutamate (L-Glu). These results extend our previous observations in pine and potato that PAL-generated NH4+ is assimilated by the glutamine synthetase (GS; EC 6.3.1.2)/glutamate synthase (GOGAT; EC 1.4.1.13) pathway, with the NH4+ so formed ultimately being recycled back to L-Phe via L-Glu as aminoreceptor and donor.

Scaffold hopping identifies 6,8-disubstituted purines as novel anaplastic lymphoma kinase inhibitors.

PubMed

Schlütke, Laura; Immer, Markus; Preu, Lutz; Totzke, Frank; Schächtele, Christoph; Kubbutat, Michael H G; Kunick, Conrad

2018-05-01

Rearrangements of anaplastic lymphoma kinase (ALK) are associated with several cancer diseases. Due to resistance development against existing ALK-inhibitors, new, structurally unrelated inhibitors are required. By a scaffold hopping strategy, 6,8-disubstituted purines were designed as analogues of similar ALK-inhibiting thieno[3,2-d]pyrimidines. While the new title compounds indeed inhibited ALK and several ALK mutants in submicromolar concentrations, they retained poor water solubility. Copyright © 2017 Elsevier B.V. All rights reserved.

Serotonin transporter activity of imidazolidine-2,4-dione and imidazo[2,1-f]purine-2,4-dione derivatives in aspect of their acid-base properties.

PubMed

Zagórska, Agnieszka; Czopek, Anna; Pawłowski, Maciej; Dybała, Małgorzata; Siwek, Agata; Nowak, Gabriel

2012-11-01

Affinities of arylpiperazinylalkyl derivatives of imidazo[2,1-f]purine-2,4-dione and imidazolidine-2,4-dione for serotonin transporter and their acid-base properties were evaluated. The dissociation constant (pK(a)) of compounds 1-22 were determinated by potentiometric titration and calculated using pKalc 3.1 module of the Pallas system. The data from experimental methods and computational calculations were compared and suitable conclusions were reached.

Entry and Exit Mechanisms at the cis-Face of the Golgi Complex

PubMed Central

Lorente-Rodríguez, Andrés; Barlowe, Charles

2011-01-01

Vesicular transport of protein and lipid cargo from the endoplasmic reticulum (ER) to cis-Golgi compartments depends on coat protein complexes, Rab GTPases, tethering factors, and membrane fusion catalysts. ER-derived vesicles deliver cargo to an ER-Golgi intermediate compartment (ERGIC) that then fuses with and/or matures into cis-Golgi compartments. The forward transport pathway to cis-Golgi compartments is balanced by a retrograde directed pathway that recycles transport machinery back to the ER. How trafficking through the ERGIC and cis-Golgi is coordinated to maintain organelle structure and function is poorly understood and highlights central questions regarding trafficking routes and organization of the early secretory pathway. PMID:21482742

Integrative Genomic Analysis Identifies Isoleucine and CodY as Regulators of Listeria monocytogenes Virulence

PubMed Central

Lobel, Lior; Sigal, Nadejda; Borovok, Ilya; Ruppin, Eytan; Herskovits, Anat A.

2012-01-01

Intracellular bacterial pathogens are metabolically adapted to grow within mammalian cells. While these adaptations are fundamental to the ability to cause disease, we know little about the relationship between the pathogen's metabolism and virulence. Here we used an integrative Metabolic Analysis Tool that combines transcriptome data with genome-scale metabolic models to define the metabolic requirements of Listeria monocytogenes during infection. Twelve metabolic pathways were identified as differentially active during L. monocytogenes growth in macrophage cells. Intracellular replication requires de novo synthesis of histidine, arginine, purine, and branch chain amino acids (BCAAs), as well as catabolism of L-rhamnose and glycerol. The importance of each metabolic pathway during infection was confirmed by generation of gene knockout mutants in the respective pathways. Next, we investigated the association of these metabolic requirements in the regulation of L. monocytogenes virulence. Here we show that limiting BCAA concentrations, primarily isoleucine, results in robust induction of the master virulence activator gene, prfA, and the PrfA-regulated genes. This response was specific and required the nutrient responsive regulator CodY, which is known to bind isoleucine. Further analysis demonstrated that CodY is involved in prfA regulation, playing a role in prfA activation under limiting conditions of BCAAs. This study evidences an additional regulatory mechanism underlying L. monocytogenes virulence, placing CodY at the crossroads of metabolism and virulence. PMID:22969433

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

PubMed

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

2012-04-01

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

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

PubMed

Bahouth, Suleiman W; Nooh, Mohammed M

2017-08-01

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction pathway in membranes. Copyright © 2017. Published by Elsevier Inc.

The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells

PubMed Central

Cerny, Alexander C.; Altendorfer, André; Schopf, Krystina; Baltner, Karla; Maag, Nathalie; Sehn, Elisabeth; Wolfrum, Uwe; Huber, Armin

2015-01-01

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14 P75L mutant. The ttd14 P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14 P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane. PMID:26509977

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.

Chlorambucil for the treatment of patients with chronic lymphocytic leukemia (CLL) - a systematic review and meta-analysis of randomized trials.

PubMed

Vidal, Liat; Gurion, Ronit; Ram, Ron; Raanani, Pia; Bairey, Osnat; Robak, Tadeusz; Gafter-Gvili, Anat; Shpilberg, Ofer

2016-09-01

Randomized clinical trials that compared chlorambucil to different regimens, for patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) do not support an overall survival (OS) benefit. To assess the efficacy and safety of chlorambucil as frontline treatment, we conducted a systematic review and meta-analysis of randomized controlled trials. OS was the primary outcome. Meta-analysis of 18 trials that compared purine analogs, alkylators, alemtuzumab and ibrutinib to chlorambucil demonstrated no OS benefit for therapy without chlorambucil over chlorambucil (pooled HR 0.99, 95% CI 0.91-1.08; 4133 patients). PFS was longer with purine analogs compared with chlorambucil with an increased risk of infection. The risk of secondary malignancies was not increased with chlorambucil. In conclusion, our study showed that chlorambucil is an acceptable chemotherapy backbone for unfit patients with CLL. Purine analogs should be preferred in fit younger patients because of longer PFS. Future trials should focus on unfit patients who are underrepresented in clinical trials.

Nucleotide and nucleoside involvement in immunomodulation in experimental Chagas disease.

PubMed

do Carmo, Guilherme M; de Sá, Mariângela F; Baldissera, Matheus D; Grando, Thirssa H; Mendes, Ricardo E; Cardoso, Valesca V; Casali, Emerson A; Moritz, Cesar Eduardo J; Monteiro, Silvia G; Da Silva, Aleksandro S

2018-02-05

The aim of this study was to evaluate whether Trypanosma cruzi infections cause alterations in the levels of seric purines, which could contribute to host immunomodulation. Twelve mice were divided into two groups identified as control (uninfected) and infected (T. cruzi) groups. The influence of the disease on seric purine levels was verified on day 20 post-infection (PI) by HPLC. Infected mice had circulating trypomastigotes during the experiment, as well as amastigote forms in the heart associated with inflammatory infiltrates. Increases on adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine (ADO), inosine (INO), and uric acid (URIC) levels were observed in the infected animals, while the adenosine monophosphate (AMP) and xanthine (XAN) levels were reduced compared with mice of the control group, indicating a possible impairment on the purinergic system, and consequently, on the immune system during the clinical course of the disease. In summary, the T. cruzi infection alters the seric purine levels, and consequently, modulates the immune system.

Synthesis of aromatic cytokinins for plant biotechnology.

PubMed

Plíhalová, Lucie; Vylíčilová, Hana; Doležal, Karel; Zahajská, Lenka; Zatloukal, Marek; Strnad, Miroslav

2016-09-25

Cytokinins represent an important group of plant growth regulators that can modulate several biotechnological processes owing to their ability to influence almost all stages of plant development and growth. In addition, the use of purine based cytokinins with aromatic substituent in C6 position of the purine moiety in tissue culture techniques is currently experiencing a surge in interest, made possible by the ongoing systematic synthesis and study of these compounds. This review article outlines progress in the synthesis of aromatic cytokinins, the in vitro and in vivo effects of these substances and insights gleaned from their synthesis. As the purine moiety in these compounds can be substituted at several positions, we examine each of the substitution possibilities in relation to the derivatives prepared so far. The discussion highlights the gradual simplification of their preparation in relation to their application in practice and summarizes the relevant organic chemistry literature and published patents. Copyright © 2015 Elsevier B.V. All rights reserved.

Why Do Some Water Utilities Recycle More than Others? A Qualitative Comparative Analysis in New South Wales, Australia.

PubMed

Kunz, Nadja C; Fischer, Manuel; Ingold, Karin; Hering, Janet G

2015-07-21

Although the recycling of municipal wastewater can play an important role in water supply security and ecosystem protection, the percentage of wastewater recycled is generally low and strikingly variable. Previous research has employed detailed case studies to examine the factors that contribute to recycling success but usually lacks a comparative perspective across cases. In this study, 25 water utilities in New South Wales, Australia, were compared using fuzzy-set Qualitative Comparative Analysis (fsQCA). This research method applies binary logic and set theory to identify the minimal combinations of conditions that are necessary and/or sufficient for an outcome to occur within the set of cases analyzed. The influence of six factors (rainfall, population density, coastal or inland location, proximity to users; cost recovery and revenue for water supply services) was examined for two outcomes, agricultural use and "heavy" (i.e., commercial/municipal/industrial) use. Each outcome was explained by two different pathways, illustrating that different combinations of conditions are associated with the same outcome. Generally, while economic factors are crucial for heavy use, factors relating to water stress and geographical proximity matter most for agricultural reuse. These results suggest that policies to promote wastewater reuse may be most effective if they target uses that are most feasible for utilities and correspond to the local context. This work also makes a methodological contribution through illustrating the potential utility of fsQCA for understanding the complex drivers of performance in water recycling.

Decoding the similarities and differences among mycobacterial species

PubMed Central

Vedithi, Sundeep Chaitanya; Blundell, Tom L.

2017-01-01

Mycobacteriaceae comprises pathogenic species such as Mycobacterium tuberculosis, M. leprae and M. abscessus, as well as non-pathogenic species, for example, M. smegmatis and M. thermoresistibile. Genome comparison and annotation studies provide insights into genome evolutionary relatedness, identify unique and pathogenicity-related genes in each species, and explore new targets that could be used for developing new diagnostics and therapeutics. Here, we present a comparative analysis of ten-mycobacterial genomes with the objective of identifying similarities and differences between pathogenic and non-pathogenic species. We identified 1080 core orthologous clusters that were enriched in proteins involved in amino acid and purine/pyrimidine biosynthetic pathways, DNA-related processes (replication, transcription, recombination and repair), RNA-methylation and modification, and cell-wall polysaccharide biosynthetic pathways. For their pathogenicity and survival in the host cell, pathogenic species have gained specific sets of genes involved in repair and protection of their genomic DNA. M. leprae is of special interest owing to its smallest genome (1600 genes and ~1300 psuedogenes), yet poor genome annotation. More than 75% of the pseudogenes were found to have a functional ortholog in the other mycobacterial genomes and belong to protein families such as transferases, oxidoreductases and hydrolases. PMID:28854187

Design and Synthesis of Potent “Sulfur-free” Transition State Analogue Inhibitors of 5′-Methylthioadenosine Nucleosidase and 5′-Methylthioadenosine Phosphorylase

PubMed Central

Longshaw, Alistair I.; Adanitsch, Florian; Gutierrez, Jemy A.; Evans, Gary B.; Tyler, Peter C.; Schramm, Vern L.

2013-01-01

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM)-related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability we were interested in synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free” transition state analogues inhibitors, of E. coli MTAN and human MTAP that have low nano- to pico-molar dissociation constants and are potentially novel bacterial anti-infective and anti-cancer drug candidates. PMID:20718423

Regio- and Enantioselective N-Allylations of Imidazole, Benzimidazole, and Purine Heterocycles Catalyzed by Single-Component Metallacyclic Iridium Complexes

PubMed Central

Stanley, Levi M.

2010-01-01

Highly regio- and enantioselective iridium-catalyzed N-allylations of benzimidazoles, imidazoles, and purines have been developed. N-Allylated benzimidazoles and imidazoles were isolated in high yields (up to 97%) with high branched-to-linear selectivity (up to 99:1) and enantioselectivity (up to 98% ee) from the reactions of benzimidazole and imidazole nucleophiles with unsymmetrical allylic carbonates in the presence of single component, ethylene-bound, metallacyclic iridium catalysts. N-Allylated purines were also obtained in high yields (up to 91%) with high N9:N7 selectivity (up to 96:4), high branched-to-linear selectivity (98:2), and high enantioselectivity (up to 98% ee) under similar conditions. The reactions encompass a range of benzimidazole, imidazole, and purine nucleophiles, as well as a variety of unsymmetrical aryl, heteroaryl, and aliphatic allylic carbonates. Competition experiments between common amine nucleophiles and the heterocyclic nitrogen nucleophiles studied in this work illustrate the effect of nucleophile pKa on the rate of iridium-catalyzed N-allylation reactions. Kinetic studies on the allylation of benzimidazole catalyzed by metallacyclic iridium-phosphoramidite complexes, in combination with studies on the deactivation of these catalysts in the presence of heterocyclic nucleophiles, provide insight into the effects of the structure of the phosphoramidite ligands on the stability of the metallacyclic catalysts. The data obtained from these studies has led to the development of N-allylations of benzimidazoles and imidazoles in the absence of an exogenous base. PMID:19480431

Pyrimidine Biosynthesis in Lactobacillus leichmannii

PubMed Central

Hutson, Judith Y.; Downing, Mancourt

1968-01-01

Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented. PMID:5686000

Synthesis and biological evaluation of 2-fluoro and 3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential antidepressant agents.

PubMed

Zagórska, Agnieszka; Bucki, Adam; Kołaczkowski, Marcin; Siwek, Agata; Głuch-Lutwin, Monika; Starowicz, Gabriela; Kazek, Grzegorz; Partyka, Anna; Wesołowska, Anna; Słoczyńska, Karolina; Pękala, Elżbieta; Pawłowski, Maciej

2016-01-01

A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (4-21) were synthesized and evaluated for their serotonin (5-HT 1A /5-HT 7 ) receptor affinity and phosphodiesterase (PDE4B and PDE10A) inhibitor activity. The study enabled the identification of potent 5-HT 1A , 5-HT 7 and mixed 5-HT 1A /5-HT 7 receptor ligands with weak inhibitory potencies for PDE4B and PDE10A. The tests have been completed with the determination of lipophilicity and metabolic stability using micellar electrokinetic chromatography (MEKC) system and human liver microsomes (HLM) model. In preliminary pharmacological in vivo studies, selected compound 8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) behaved as a potential antidepressant in forced swim test (FST) in mice. Moreover, potency of antianxiety effects evoked by 9 (2.5 mg/kg) is greater than that of the reference anxiolytic drug, diazepam. Molecular modeling revealed that fluorinated arylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione have major significance for the provision of lead compounds for antidepressant and/or anxiolytic application.

Convergence of inhibitory neural inputs regulate motor activity in the murine and monkey stomach

PubMed Central

Shaylor, Lara A.; Hwang, Sung Jin; Sanders, Kenton M.

2016-01-01

Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase (Nos1−/−) and P2Y1 receptors (P2ry1−/−) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1−/− mice IJPs and relaxations persisted whereas in P2ry1−/− mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species. PMID:27634009

Convergence of inhibitory neural inputs regulate motor activity in the murine and monkey stomach.

PubMed

Shaylor, Lara A; Hwang, Sung Jin; Sanders, Kenton M; Ward, Sean M

2016-11-01

Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase (Nos1 -/- ) and P2Y1 receptors (P2ry1 -/- ) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1 -/- mice IJPs and relaxations persisted whereas in P2ry1 -/- mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species. Copyright © 2016 the American Physiological Society.

Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

PubMed

Perrin, Laura; Laura, Perrin; Lacas-Gervais, Sandra; Sandra, Lacas-Gervais; Gilleron, Jérôme; Jérôme, Gilleron; Ceppo, Franck; Franck, Ceppo; Prodon, François; François, Prodon; Benmerah, Alexandre; Alexandre, Benmerah; Tanti, Jean-François; Jean-François, Tanti; Cormont, Mireille; Mireille, Cormont

2013-11-01

The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

PubMed Central

McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

2016-01-01

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860