Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics.

PubMed

Poduch, Ewa; Bello, Angelica M; Tang, Sishi; Fujihashi, Masahiro; Pai, Emil F; Kotra, Lakshmi P

2006-08-10

Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

Lys314 is a nucleophile in non-classical reactions of orotidine-5'-monophosphate decarboxylase.

PubMed

Heinrich, Daniel; Diederichsen, Ulf; Rudolph, Markus Georg

2009-07-06

Orotidine-5'-monophosphate decarboxylase (OMPD) catalyzes the decarboxylation of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate (UMP) in an extremely proficient manner. The reaction does not require any cofactors and proceeds by an unknown mechanism. In addition to decarboxylation, OMPD is able to catalyze other reactions. We show that several C6-substituted UMP derivatives undergo hydrolysis or substitution reactions that depend on a lysine residue (Lys314) in the OMPD active site. 6-Cyano-UMP is converted to UMP, and UMP derivatives with good leaving groups inhibit OMPD by a suicide mechanism in which Lys314 covalently binds to the substrate. These non-classical reactivities of human OMPD were characterized by cocrystallization and freeze-trapping experiments with wild-type OMPD and two active-site mutants by using substrate and inhibitor nucleotides. The structures show that the C6-substituents are not coplanar with the pyrimidine ring. The extent of this substrate distortion is a function of the substituent geometry. Structure-based mechanisms for the reaction of 6-substituted UMP derivatives are extracted in accordance with results from mutagenesis, mass spectrometry, and OMPD enzyme activity. The Lys314-based mechanisms explain the chemodiversity of OMPD, and offer a strategy to design mechanism-based inhibitors that could be used for antineoplastic purposes for example.

Medicinal Chemistry of the Noncanonical Cyclic Nucleotides cCMP and cUMP.

PubMed

Schwede, Frank; Rentsch, Andreas; Genieser, Hans-Gottfried

2017-01-01

After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems, also the noncanonical congeners cyclic cytidine-3',5'-monophosphate (cCMP) and cyclic uridine-3',5'-monophosphate (cUMP) gained more and more interest. Until the late 1980s, only a small number of cCMP and cUMP analogs with sometimes undefined purities had been described. Moreover, most of these compounds had been rather synthesized as precursors of antitumor and antiviral nucleoside-5'-monophosphates and hence had not been tested for any second messenger activity. Along with the recurring interest in cCMP- and cUMP-related signaling in the early 2000s, it became evident that well-characterized small molecule analogs with reliable purities would serve as highly valuable tools for the evaluation of a putative second messenger role of cyclic pyrimidine nucleotides. Meanwhile, for this purpose new cCMP and cUMP derivatives have been developed, and already known analogs have been resynthesized and highly purified. This chapter summarizes early medicinal chemistry work on cCMP and cUMP and analogs thereof, followed by a description of recent synthetic developments and an outlook on potential future directions.

Dietary supplementation with uridine-5'-monophosphate (UMP), a membrane phosphatide precursor, increases acetylcholine level and release in striatum of aged rat.

PubMed

Wang, Lei; Albrecht, Meredith A; Wurtman, Richard J

2007-02-16

The biosynthesis of brain membrane phosphatides, e.g., phosphatidylcholine (PtdCho), may utilize three circulating compounds: choline, uridine (a precursor for UTP, CTP, and CDP-choline), and a PUFA (e.g., docosahexaenoic acid); moreover, oral administration of the uridine source uridine-5'-monophosphate (UMP) can significantly increase levels of the phosphatides throughout the rodent brain. Since PtdCho can provide choline for acetylcholine (ACh) synthesis, we determined whether UMP administration also affects ACh levels in striatum and striatal extracellular fluid, in aged and young rats. Among aged animals consuming a UMP-containing diet (2.5%, w/w) for 1 or 6 weeks, baseline ACh levels in striatal dialysates rose from 73 fmol/min to 148 or 197 fmol/min (P<0.05). Consuming a lower dose (0.5%) for 1 week produced a smaller but still significant increase (from 75 to 92 fmol/min, P<0.05), and elevated striatal ACh content (by 16%; P<0.05). Dietary UMP (0.5%, 1 week) also amplified the increase in ACh caused by giving atropine (10 microM in the aCSF); atropine alone increased ACh concentrations from 81 to 386 fmol/min in control rats and from 137 to 680 fmol/min in those consuming UMP (P<0.05). Young rats eating the UMP-containing diet exhibited similar increases in basal ECF ACh (from 105 to 118 fmol/min) and in the increase produced by atropine (from 489 to 560 fmol/min; P<0.05). These data suggest that giving a uridine source may enhance some cholinergic functions, perhaps by increasing brain phosphatide levels.

Some pharmacological properties of uridine nucleotides

PubMed Central

Smith, M. W.

1964-01-01

Uridine di-, tri- and monophosphates (UDP, UTP and UMP) contracted the goldfish intestine preparation in that order of decreasing potency. Adenosine triphosphate (ATP) sensitized the gut to UTP and UDP but not to UMP. The fluoro-derivatives of UMP and UTP behaved like the unsubstituted nucleotides on the goldfish intestine but the main effect of 6-azaUDP and large amounts of uracil and uridine was to cause a relaxation. Structure-action relationships are discussed on the basis of these findings. UDPglucose and UDPacetylglucosamine each contracted the goldfish intestine but they were 500-times less active than UDP. Other smooth muscle preparations (tortoise jejunum, rat uterus, guinea-pig ileum and the fowl rectal caecum) contracted to UTP and UDP, but large amounts were needed. The cardiovascular effects in rats of UMP, UDP and UTP were complex and mediated mainly through an action on the peripheral blood vessels. In rats treated with phenoxybenzamine, UMP raised the blood pressure while UDP and UTP first lowered then raised the blood pressure. The fall in blood pressure was not abolished by pronethalol or atropine. The uridine phosphates affected the rat isolated heart only under hypoxic conditions. UTP and UDP dilated the blood vessels of the rabbit ear and UTP was six-times more effective than ATP. UTP and UDP were equiactive in increasing the force of beat of the frog isolated heart. UMP also had an effect if large amounts were given. PMID:14190461

A novel procedure for purification of uridine 5'-monophosphate based on adsorption methodology using a hyper-cross-linked resin.

PubMed

Wu, Jinglan; Zhu, Hui; Liu, Yanan; Zhou, Jingwei; Zhuang, Wei; Jiao, Pengfei; Ke, Xu; Ying, Hanjie

2015-05-01

The conventional ion exchange process used for recovery of uridine 5'-monophosphate (UMP) from the enzymatic hydrolysate of RNA is environmentally harmful and cost intensive. In this work, an innovative benign process, which comprises adsorption technology and use of a hyper-cross-linked resin as a stationary phase is proposed. The adsorption properties of this kind of resin in terms of adsorption equilibrium as well as kinetics were evaluated. The influences of the operating conditions, i.e., initial UMP concentration, feed flow rate, and bed height on the breakthrough curves of UMP in the fixed bed system were investigated. Subsequently, a chromatographic column model was established and validated for the prediction of the experimentally attained breakthrough curves of UMP and the main impurity component (phosphate ion) with a real enzymatic hydrolysate of RNA as a feed mixture. At the end of this paper, the crystallization of UMP was carried out. The purity of the final product (uridine 5'-monophosphate disodium, UMPNa2) of over 99.5 % was obtained.

Supramolecular Assembly of Uridine Monophosphate (UMP) and Thymidine Monophosphate (TMP) with a Dinuclear Copper(II) Receptor.

PubMed

Rhaman, Md Mhahabubur; Powell, Douglas R; Hossain, Md Alamgir

2017-11-30

Understanding the intermolecular interactions between nucleotides and artificial receptors is crucial to understanding the role of nucleic acids in living systems. However, direct structural evidence showing precise interactions and bonding features of a nucleoside monophosphate (NMP) with a macrocycle-based synthetic molecule has not been provided so far. Herein, we present two novel crystal structures of uridine monophosphate (UMP) and thymidine monophosphate (TMP) complexes with a macrocycle-based dinuclear receptor. Structural characterization of these complexes reveals that the receptor recognizes UMP through coordinate-covalent interactions with phosphates and π-π stackings with nucleobases and TMP through coordinate-covalent interactions with phosphate groups. Furthermore, the receptor has been shown to effectively bind nucleoside monophosphates in the order of GMP > AMP > UMP > TMP > CMP in water at physiological pH, as investigated by an indicator displacement assay.

Enhanced uridine 5'-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux.

PubMed

Liu, Dong; Chen, Yong; Li, An; Xie, Jingjing; Xiong, Jian; Bai, Jianxin; Chen, Xiaochun; Niu, Huanqing; Zhou, Tao; Ying, Hanjie

2012-06-01

A whole-cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH(2)PO(4), MgCl(2) and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH(2)PO(4) 22.1 g/L; MgCl(2) 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.

Production of uridine 5'-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process.

PubMed

Wang, Xing; Wang, Xiuwen; Yin, Mengxin; Xiao, Zijun; Ma, Cuiqing; Lin, Zhixin; Wang, Peng George; Xu, Ping

2007-08-01

Attempts were made with success to develop a two-step biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both "one-factor-at-a-time" method and statistical method were performed. By "one-factor-at-a-time" optimization, orotic acid, glucose, phosphate ion (equimolar KH(2)PO(4) and K(2)HPO(4)), MgCl(2), Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett-Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l(-1)) UMP was accumulated in 24 h from 38.5 mM (6 g l(-1)) orotic acid. The yield was threefold higher than the original UMP yield before optimization.

Mild orotic aciduria in UMPS heterozygotes: a metabolic finding without clinical consequences.

PubMed

Wortmann, Saskia B; Chen, Margaret A; Colombo, Roberto; Pontoglio, Alessandro; Alhaddad, Bader; Botto, Lorenzo D; Yuzyuk, Tatiana; Coughlin, Curtis R; Descartes, Maria; Grűnewald, Stephanie; Maranda, Bruno; Mills, Philippa B; Pitt, James; Potente, Catherine; Rodenburg, Richard; Kluijtmans, Leo A J; Sampath, Srirangan; Pai, Emil F; Wevers, Ron A; Tiller, George E

2017-05-01

Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

Proton transfer from C-6 of uridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase: formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent.

PubMed

Tsang, Wing-Yin; Wood, B McKay; Wong, Freeman M; Wu, Weiming; Gerlt, John A; Amyes, Tina L; Richard, John P

2012-09-05

The exchange for deuterium of the C-6 protons of uridine 5'-monophosphate (UMP) and 5-fluorouridine 5'-monophosphate (F-UMP) catalyzed by yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 6.5-9.3 and 25 °C was monitored by (1)H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for k(cat) give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10(-5) and 0.041 s(-1), respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pK(a) of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pK(a) values for proton transfer from C-6 of uridine (pK(CH) = 28.8) and 5-fluorouridine (pK(CH) = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 10(9)-fold increase in the equilibrium constant for proton transfer from C-6, so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for k(cat)/K(m) for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M(-1) s(-1). This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 10(10) M(-1), which corresponds to a transition-state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition-state stabilization for the decarboxylation of orotidine 5'-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction.

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

PubMed Central

Tsang, Wing-Yin; Wood, B. McKay; Wong, Freeman M.; Wu, Weiming; Gerlt, John A.; Amyes, Tina L.; Richard, John P.

2012-01-01

The exchange for deuterium of the C-6 protons of uridine 5′-monophosphate (UMP) and 5-fluorouridine 5′-monophosphate (F-UMP) catalyzed by yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC) at pD 6.5 – 9.3 and 25 °C was monitored by 1H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for kcat give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10−5 and 0.041 s−1, respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pKa of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pKas for proton transfer from C-6 of uridine (pKCH = 28.8) and 5-fluorouridine (pKCH = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 109-fold increase in the equilibrium constant for proton transfer from C-6 so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for kcat/Km for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M−1 s−1. This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 1010 M−1, which corresponds to a transition state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition state stabilization for the decarboxylation of orotidine 5′-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction. PMID:22812629

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

PubMed

López-Alvarez, Arnoldo; Díaz-Pérez, Alma Laura; Sosa-Aguirre, Carlos; Macías-Rodríguez, Lourdes; Campos-García, Jesús

2012-05-01

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Supramolecular Assembly of Uridine Monophosphate (UMP) and Thymidine Monophosphate (TMP) with a Dinuclear Copper(II) Receptor

PubMed Central

2017-01-01

Understanding the intermolecular interactions between nucleotides and artificial receptors is crucial to understanding the role of nucleic acids in living systems. However, direct structural evidence showing precise interactions and bonding features of a nucleoside monophosphate (NMP) with a macrocycle-based synthetic molecule has not been provided so far. Herein, we present two novel crystal structures of uridine monophosphate (UMP) and thymidine monophosphate (TMP) complexes with a macrocycle-based dinuclear receptor. Structural characterization of these complexes reveals that the receptor recognizes UMP through coordinate–covalent interactions with phosphates and π–π stackings with nucleobases and TMP through coordinate–covalent interactions with phosphate groups. Furthermore, the receptor has been shown to effectively bind nucleoside monophosphates in the order of GMP > AMP > UMP > TMP > CMP in water at physiological pH, as investigated by an indicator displacement assay. PMID:29214233

A Unified Model of Performance for Predicting the Effects of Sleep and Caffeine.

PubMed

Ramakrishnan, Sridhar; Wesensten, Nancy J; Kamimori, Gary H; Moon, James E; Balkin, Thomas J; Reifman, Jaques

2016-10-01

Existing mathematical models of neurobehavioral performance cannot predict the beneficial effects of caffeine across the spectrum of sleep loss conditions, limiting their practical utility. Here, we closed this research gap by integrating a model of caffeine effects with the recently validated unified model of performance (UMP) into a single, unified modeling framework. We then assessed the accuracy of this new UMP in predicting performance across multiple studies. We hypothesized that the pharmacodynamics of caffeine vary similarly during both wakefulness and sleep, and that caffeine has a multiplicative effect on performance. Accordingly, to represent the effects of caffeine in the UMP, we multiplied a dose-dependent caffeine factor (which accounts for the pharmacokinetics and pharmacodynamics of caffeine) to the performance estimated in the absence of caffeine. We assessed the UMP predictions in 14 distinct laboratory- and field-study conditions, including 7 different sleep-loss schedules (from 5 h of sleep per night to continuous sleep loss for 85 h) and 6 different caffeine doses (from placebo to repeated 200 mg doses to a single dose of 600 mg). The UMP accurately predicted group-average psychomotor vigilance task performance data across the different sleep loss and caffeine conditions (6% < error < 27%), yielding greater accuracy for mild and moderate sleep loss conditions than for more severe cases. Overall, accounting for the effects of caffeine resulted in improved predictions (after caffeine consumption) by up to 70%. The UMP provides the first comprehensive tool for accurate selection of combinations of sleep schedules and caffeine countermeasure strategies to optimize neurobehavioral performance. © 2016 Associated Professional Sleep Societies, LLC.

Ultra-mini-percutaneous nephrolithotomy in pediatric nephrolithiasis: both low pressure and high efficiency.

PubMed

Dede, Onur; Sancaktutar, Ahmet Ali; Dağguli, Mansur; Utangaç, Mazhar; Baş, Okan; Penbegul, Necmettin

2015-10-01

The management of urolithiasis in children differs from adults because of anatomic and metabolic abnormalities. At present, various minimally invasive and invasive treatment strategies have been recommended to treat urinary tract Stones, including shock wave lithotripsy (SWL), standard percutaneous nephrolithotomy (PNL), mini-PNL, retrograde intrarenal surgery (RIRS), micro-PNL, and a new technique termed ultra-mini PNL (UMP). UMP is a new method for the treatment of kidney stones. The main difference between UMP and standard PNL or mini-PNL lies in the small access sheath and in its design. A 9.5F pediatric compact cystoscope was used (Figure). A major advantage of UMP is that it provides similar stone-free rates when compared with standard PNL, with lower intrarenal pressure than micro-PNL. The aim of the study was to determine the applicability and safety of UMP in the treatment of pediatric kidney stones. We retrospectively reviewed the files of 39 kidney stones in children who had undergone UMP between May 2011 and October 2014. The indications for UMP included stones resistant to SWL, stones requiring repeated sessions of SWL, and stones size larger than 20 mm. The study included 17 females and 22 males, with a mean age of 5.8 ± 4.6 years. The mean hemoglobin loss was 0.9 ± 0.6 mg/dL and none of the patients required a blood transfusion. The complication rate of the PNL procedure was 15.3% (n = 6). Complete clearance was achieved in 32 patients (82%) with UMP monotherapy, which increased to 34% (87.1%) 4 weeks after the operation. Management of urolithiasis necessitates a balance between stone clearance and morbidity related to the procedure. SWL often leads to persistent residual stones. The developing RIRS can minimize the risks associated with bleeding and visceral injury, but sometimes the abnormal pelvicaliceal anatomy and poor imaging of the flexible ureteroscopy may impact its success rate and applications. It has been reported that PNL can be performed safely and effectively to achieve a higher stone-free rate; however, it has serious complications such as bleeding that requires blood transfusion in 11-14% of the cases with increased risk of kidney loss. The recent development of smaller sheaths allows tract formation with minimal damage to the renal parenchyma, thereby reducing procedure-related morbidity without diminishing its therapeutic efficacy. The most important advantage of this surgical technique that has been developed is similar stone-free rates to standard PNL and lower intrarenal pressure than micro-PNL. Our experience supports that UMP is safe and effective for the management of renal stones in children. Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families

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

Suchi, Mariko; Mizuno, Haruo; Tsuboi, Takashi

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5{prime}-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a {lambda}EMBL-3 human genomic library and report a single-copy gene spanning {approximately}15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AGmore » rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5{prime} flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A- to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A ({nu} = .26) and 440 Gpoly ({nu} = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function. 76 refs., 5 figs., 7 tabs.« less

Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access.

PubMed

Fritz, Timothy A; Liu, Lu; Finer-Moore, Janet S; Stroud, Robert M

2002-06-04

Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.

Structure of the Varicella Zoster Virus Thymidylate Synthase Establishes Functional and Structural Similarities as the Human Enzyme and Potentiates Itself as a Target of Brivudine

PubMed Central

Hew, Kelly; Dahlroth, Sue-Li; Veerappan, Saranya; Pan, Lucy Xin; Cornvik, Tobias; Nordlund, Pär

2015-01-01

Varicella zoster virus (VZV) is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS), which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TSVZV with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TSHS) in an open conformation. The interactions between TSVZV with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF), suggesting that TSVZV binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TSVZV and in vitro phosphorylated brivudine (BVDUP), a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDUP to TSVZV was further confirmed by the complex structure of TSVZV and BVDUP solved at a resolution of 2.9 Å. BVDUP binds similarly as dUMP in the TSHS but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDUP is likely to inhibit TSVZV by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TSVZV and BVDUP are consistent with that TSVZV is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TSVZV inhibitors. PMID:26630264

A Unified Model of Performance for Predicting the Effects of Sleep and Caffeine

PubMed Central

Ramakrishnan, Sridhar; Wesensten, Nancy J.; Kamimori, Gary H.; Moon, James E.; Balkin, Thomas J.; Reifman, Jaques

2016-01-01

Study Objectives: Existing mathematical models of neurobehavioral performance cannot predict the beneficial effects of caffeine across the spectrum of sleep loss conditions, limiting their practical utility. Here, we closed this research gap by integrating a model of caffeine effects with the recently validated unified model of performance (UMP) into a single, unified modeling framework. We then assessed the accuracy of this new UMP in predicting performance across multiple studies. Methods: We hypothesized that the pharmacodynamics of caffeine vary similarly during both wakefulness and sleep, and that caffeine has a multiplicative effect on performance. Accordingly, to represent the effects of caffeine in the UMP, we multiplied a dose-dependent caffeine factor (which accounts for the pharmacokinetics and pharmacodynamics of caffeine) to the performance estimated in the absence of caffeine. We assessed the UMP predictions in 14 distinct laboratory- and field-study conditions, including 7 different sleep-loss schedules (from 5 h of sleep per night to continuous sleep loss for 85 h) and 6 different caffeine doses (from placebo to repeated 200 mg doses to a single dose of 600 mg). Results: The UMP accurately predicted group-average psychomotor vigilance task performance data across the different sleep loss and caffeine conditions (6% < error < 27%), yielding greater accuracy for mild and moderate sleep loss conditions than for more severe cases. Overall, accounting for the effects of caffeine resulted in improved predictions (after caffeine consumption) by up to 70%. Conclusions: The UMP provides the first comprehensive tool for accurate selection of combinations of sleep schedules and caffeine countermeasure strategies to optimize neurobehavioral performance. Citation: Ramakrishnan S, Wesensten NJ, Kamimori GH, Moon JE, Balkin TJ, Reifman J. A unified model of performance for predicting the effects of sleep and caffeine. SLEEP 2016;39(10):1827–1841. PMID:27397562

Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

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

Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed thatmore » hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.« less

Structural Characterization of the Molecular Events during a Slow Substrate-Product Transition in Orotidine 5'-Monophosphate Decarboxylase

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

Fujihashi, Masahiro; Wei, Lianhu; Kotra, Lakshmi P

2009-04-06

Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyano-uridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6more » of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis.« less

Structural characterization of the molecular events during a slow substrate-product transition in orotidine 5'-monophosphate decarboxylase.

PubMed

Fujihashi, Masahiro; Wei, Lianhu; Kotra, Lakshmi P; Pai, Emil F

2009-04-17

Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyano-uridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6 of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis.

Identification of cytidine 2',3'-cyclic monophosphate and uridine 2',3'-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture.

PubMed

Bordeleau, Emily; Oberc, Christopher; Ameen, Eve; da Silva, Amanda Mendes; Yan, Hongbin

2014-09-15

Cytidine 2',3'-cyclic monophosphate (2',3'-cCMP) and uridine 2',3'-cyclic monophosphate (2',3'-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2',3'-cCMP and 2',3'-cUMP were determined. Addition of 2',3'-cCMP and 2',3'-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures. Copyright © 2014 Elsevier Ltd. All rights reserved.

Coordinating properties of uridine 5'-monophosphate with selected Ln(3+) ions in ionic micellar media.

PubMed

Sudhiranjan Singh, M; Homendra, Naorem; Lonibala, R K

2012-12-01

Coordinating properties of uridine 5'-monophosphate (UMP) towards trivalent La, Pr, Nd, Sm, Eu and Gd ions in presence of cationic and anionic micelles have been investigated by potentiometric pH-titration and spectroscopic methods. Stability constants of the 2:1 complexes have been determined and the change in free energy, enthalpy and entropy associated with the complexation are also calculated. Nd(III) complexes isolated from aqueous and aqueous-micellar media do not show any significant structural difference. Formation of Ln(III) complexes in all cases completes below pH 7.5 showing that UMP best interacts with Ln(3+) ions at the physiological pH range 7.3-7.5. The nucleobase is not involved in the complexation and the metal ion coordination of UMP is through the phosphate moiety only. Coordinating tendency of UMP with lanthanides, Nd(III) ion in particular, at different pH is also discussed. Luminescent properties of Eu(III) complex and its decay lifetime are also presented. This information may prove helpful regarding the use of lanthanides as biological probes for calcium/magnesium ions.

The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

PubMed

Garavaglia, Marco; Rossi, Elio; Landini, Paolo

2012-01-01

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

Stress predictors in two Asian dental schools with an integrated curriculum and traditional curriculum.

PubMed

Nguyen, T T T; Seki, N; Morio, I

2018-05-01

This study explored stress predictors and the role of instructional methods and institutional differences in perceived stress levels amongst students at two Asian dental schools. An anonymous questionnaire was distributed to undergraduate dental students at Tokyo Medical and Dental University (TMDU), Japan and the University of Medicine and Pharmacy (UMP), Hochiminh City, Vietnam in 2016. Data concerning the students' demographic information and grades, and responses to the Perceived Stress Scale (PSS) and Dental Environment Stress questionnaire (DES) were collected. The questionnaires were prepared in English and translated into Japanese and Vietnamese following a forward-backward translation process. Altogether 684 students answered the questionnaire with a response rate of 97% for TMDU and 89% for UMP. The mean DES score of UMP students was significantly higher than TMDU (P < .001). TMDU students with dentistry as their first programme choice had significantly lower PSS and DES scores in several categories than other TMDU students, whilst UMP clinical students reported higher stress scores in several areas than UMP preclinical students. Having dentistry as their first choice of educational programme was a significant stress predictor for Japanese students whilst the clinical practicum was a significant stress predictor for Vietnamese students. Previous academic performance was not a significant stress predictor for students at either dental school. Dental students of an integrated, active-learning curriculum reported lower stress levels than students of a traditional, discipline-based curriculum. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5'-monophosphate by Saccharomyces cerevisiae.

PubMed

Chen, Yong; Li, Shuya; Xiong, Jian; Li, Zhenjiang; Bai, Jianxin; Zhang, Lei; Ye, Qi; Ouyang, Pingkai; Ying, Hanjie

2010-03-01

A whole cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l(-1) sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.

A Unified Model of Performance: Validation of its Predictions across Different Sleep/Wake Schedules.

PubMed

Ramakrishnan, Sridhar; Wesensten, Nancy J; Balkin, Thomas J; Reifman, Jaques

2016-01-01

Historically, mathematical models of human neurobehavioral performance developed on data from one sleep study were limited to predicting performance in similar studies, restricting their practical utility. We recently developed a unified model of performance (UMP) to predict the effects of the continuum of sleep loss-from chronic sleep restriction (CSR) to total sleep deprivation (TSD) challenges-and validated it using data from two studies of one laboratory. Here, we significantly extended this effort by validating the UMP predictions across a wide range of sleep/wake schedules from different studies and laboratories. We developed the UMP on psychomotor vigilance task (PVT) lapse data from one study encompassing four different CSR conditions (7 d of 3, 5, 7, and 9 h of sleep/night), and predicted performance in five other studies (from four laboratories), including different combinations of TSD (40 to 88 h), CSR (2 to 6 h of sleep/night), control (8 to 10 h of sleep/night), and nap (nocturnal and diurnal) schedules. The UMP accurately predicted PVT performance trends across 14 different sleep/wake conditions, yielding average prediction errors between 7% and 36%, with the predictions lying within 2 standard errors of the measured data 87% of the time. In addition, the UMP accurately predicted performance impairment (average error of 15%) for schedules (TSD and naps) not used in model development. The unified model of performance can be used as a tool to help design sleep/wake schedules to optimize the extent and duration of neurobehavioral performance and to accelerate recovery after sleep loss. © 2016 Associated Professional Sleep Societies, LLC.

The Effect of Uncertainty Management Program on Quality of Life Among Vietnamese Women at 3 Weeks Postmastectomy.

PubMed

Ha, Xuan Thi Nhu; Thanasilp, Sureeporn; Thato, Ratsiri

2018-05-10

In Vietnam, breast cancer is a top contributor to cancer-related deaths in women. Evidence shows that, after mastectomy, women in Vietnam have a lower quality of life than women in other countries. In addition, high uncertainty is a predictor of low quality of life postmastectomy. Therefore, if nurses can manage uncertainty, the quality of life postmastectomy can improve. This study examined the effect of the Uncertainty Management Program (UMP) on quality of life at 3 weeks postmastectomy in Vietnamese women. This research was a quasi-experimental study using a "posttest only with control group" design. There were 115 subjects assigned to either the experimental group (n = 57), who participated in the UMP and routine care, or the control group (n = 58), who received only routine care. Participants were assessed 2 times postmastectomy using the modified Quality of Life Index Scale-Vietnamese version. The experimental group exhibited low uncertainty before discharge and significantly higher quality of life than the control group at 1 and 3 weeks postmastectomy, respectively (P < .05). Women's physical well-being, psychological well-being, body image concerns, and social concerns were significantly increased with UMP. The UMP was considered as a promising program that might benefit the QoL of women with breast cancer 3 weeks postmastectomy. The UMP appears feasible to apply for women with breast cancer to improve their QoL postmastectomy in various settings. Nurses can flexibility instruct women in their holistic care attention both in the hospital and at home.

Restorative Effects of Uridine Plus Docosahexaenoic Acid in a Rat Model of Parkinson’s Disease

PubMed Central

Cansev, Mehmet; Ulus, Ismail H.; Wang, Lei; Maher, Timothy J.; Wurtman, Richard J.

2008-01-01

Summary Administering uridine-5’-monophosphate (UMP) and docosahexaenoic acid (DHA) increases synaptic membranes (as characterized by pre-and post-synaptic proteins) and dendritic spines in rodents. We examined their effects on rotational behavior and dopaminergic markers in rats with partial unilateral 6-hydroxydopamine (6-OHDA)-induced striatal lesions. Rats receiving UMP, DHA, both, or neither, daily, and intrastriatal 6-OHDA 3 days after treatment onset, were tested for d-amphetamine-induced rotational behavior and dopaminergic markers after 24 and 28 days, respectively. UMP/DHA treatment reduced ipsilateral rotations by 57% and significantly elevated striatal dopamine, tyrosine hydroxylase (TH) activity, TH protein and Synapsin-1 on the lesioned side. Hence, giving uridine and DHA may partially restore dopaminergic neurotransmission in this model of Parkinson’s Disease. PMID:18761383

Restorative effects of uridine plus docosahexaenoic acid in a rat model of Parkinson's disease.

PubMed

Cansev, Mehmet; Ulus, Ismail H; Wang, Lei; Maher, Timothy J; Wurtman, Richard J

2008-11-01

Administering uridine-5'-monophosphate (UMP) and docosahexaenoic acid (DHA) increases synaptic membranes (as characterized by pre- and post-synaptic proteins) and dendritic spines in rodents. We examined their effects on rotational behavior and dopaminergic markers in rats with partial unilateral 6-hydroxydopamine (6-OHDA)-induced striatal lesions. Rats receiving UMP, DHA, both, or neither, daily, and intrastriatal 6-OHDA 3 days after treatment onset, were tested for d-amphetamine-induced rotational behavior and dopaminergic markers after 24 and 28 days, respectively. UMP/DHA treatment reduced ipsilateral rotations by 57% and significantly elevated striatal dopamine, tyrosine hydroxylase (TH) activity, TH protein and synapsin-1 on the lesioned side. Hence, giving uridine and DHA may partially restore dopaminergic neurotransmission in this model of Parkinson's disease.

Cyclin D1 in well differentiated thyroid tumour of uncertain malignant potential.

PubMed

Lamba Saini, Monika; Weynand, Birgit; Rahier, Jacques; Mourad, Michel; Hamoir, Marc; Marbaix, Etienne

2015-04-18

Encapsulated follicular tumours with equivocal papillary thyroid carcinoma (PTC) type nuclear features continue to remain a challenge despite the recent attempts to classify these borderline lesions. The term 'well differentiated tumour of uncertain malignant potential (WDT-UMP)' was introduced to classify these tumours. The present study aimed to evaluate the role of a cell cycle regulator like cyclin D1 in these tumours along with assessment of other well established PTC markers like galectin-3, HBME-1, CK19. Thirteen cases of metastatic PTC, papillary microcarcinoma and follicular variant of PTC (FVPTC) were identified from a histological review of 510 cases. In addition, 13 cases of a subset of follicular adenomatoid nodules with focal areas showing nuclear features characteristic of PTC, identified as WDT-UMP, were also analyzed. Immunohistochemical analysis of galectin-3, HBME-1, CK19 and the proliferation markers Ki67 and cyclin D1 was performed. Lesions were analyzed for cyclin D1 gene amplification by fluorescent in-situ hybridization. All WDT-UMP lesions showed immunolabelling of cyclin D1, Ki67; 11/ 13 cases showed immunolabelling of CK19; 10/13 cases showed immunolabelling of HBME-1 and 4/13 cases showed immunolabelling of galectin-3. Surrounding benign adenomatoid areas showed no to faint focal staining in all thirteen cases of cyclin D1, HBME-1 and galectin-3. A low rate of cyclin D1 gene amplification was identified in a significant proportion of cells in the WDT-UMP lesions as compared to surrounding benign adenomatoid areas. Increased expression of cyclin D1 and amplification of its gene along with immunolabelling of HBME-1 in WDT-UMP lesions showing cytological features of papillary thyroid carcinoma within follicular adenomatoid nodules suggest that these areas could correspond to a precursor lesion of follicular variant of PTC. Overexpression of cyclin D1, associated with the amplification of the gene suggests that these WDT-UMP lesions are an intermediate between the benign and malignant groups making this group of lesions a reliable precursor of FVPTC. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1851820807142117.

Chemical shift imprint of intersubunit communication in a symmetric homodimer

PubMed Central

Falk, Bradley T.; Sapienza, Paul J.; Lee, Andrew L.

2016-01-01

Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein–ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state (“lig1”) of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers. PMID:27466406

Using logic programming for modeling the one-carbon metabolism network to study the impact of folate deficiency on methylation processes.

PubMed

Gnimpieba, Etienne Z; Eveillard, Damien; Guéant, Jean-Louis; Chango, Abalo

2011-08-01

Dynamical modeling is an accurate tool for describing the dynamic regulation of one-carbon metabolism (1CM) with emphasis on the alteration of DNA methylation and/or dUMP methylation into dTMP. Using logic programming we present a comprehensive and adaptative mathematical model to study the impact of folate deficiency, including folate transport and enzymes activities. 5-Methyltetrahydrofolate (5mTHF) uptake and DNA and dUMP methylation were studied by simulating nutritional 5mTHF deficiency and methylenetetrahydrofolate reductase (MTHFR) gene defects. Both conditions had distinct effects on 1CM metabolite synthesis. Simulating severe 5mTHF deficiency (25% of normal levels) modulated 11 metabolites. However, simulating a severe decrease in MTHFR activity (25% of normal activity) modulated another set of metabolites. Two oscillations of varying amplitude were observed at the steady state for DNA methylation with severe 5mTHF deficiency, and the dUMP/dTMP ratio reached a steady state after 2 h, compared to 2.5 h for 100% 5mTHF. MTHFR activity with 25% of V(max) resulted in an increased methylated DNA pool after half an hour. We observed a deviation earlier in the profile compared to 50% and 100% V(max). For dUMP methylation, the highest level was observed with 25%, suggesting a low rate of dUMP methylation into dTMP with 25% of MTHFR activity. In conclusion, using logic programming we were able to construct the 1CM for analyzing the dynamic system behavior. This model may be used to refine biological interpretations of data or as a tool that can provide new hypotheses for pathogenesis.

24-h urine metabolic profile: is it necessary in all kidney stone formers?

PubMed

Abu-Ghanem, Yasmin; Shvero, Asaf; Kleinmann, Nir; Winkler, Harry Z; Zilberman, Dorit E

2018-06-06

A 24-h urine metabolic profile (24-UMP) is an integral part of nephrolithiasis work-up. We aimed to explore whether it can be waived under certain circumstances. We reviewed our prospective registry database of patients seen at our outpatient clinic for nephrolithiasis between the years 2010 and 2017. Data included: gender, age at first stone, body mass index (BMI), self-reported comorbidities and family history of nephrolithiasis. A 24-UMP was obtained from each patient under random diet. The following were recorded: urine volume, urinary levels of sodium, calcium, uric acid, oxalate and citrate. Presence of at least one comorbidity (i.e., hypertension/diabetes/hyperlipidemia) was defined as "associated comorbidities" (AC). Their absence was defined as "no comorbidities" (NC). Subjects were divided into two subgroups: first-time and recurrent stone formers, which were further divided into two subgroups: 1st + AC; 1st + NC; recurrent + AC; recurrent + NC. 24-UMPs have been compared between the four groups. Four hundred and fifty-seven patients were included in the study. In the AC groups, patients demonstrated higher BMI levels (p = 0.001), and were statistically significantly obese (BMI > 30, p = 0.001) and older at first stone event (p = 0.001). First formers, either with AC or NC were more likely to have low urine volume (LUV) compared with recurrent formers (72.5 vs. 59.5%, p = 0.005). In the remaining metabolic abnormalities, no such differences were observed. First-time stone formers, either with or without AC are likely to demonstrate LUV as their primary metabolic abnormality in 24-UMP. Therefore, 24-UMP may be postponed until recurrent stone event.

Dietary uridine-5'-monophosphate supplementation increases potassium-evoked dopamine release and promotes neurite outgrowth in aged rats.

PubMed

Wang, Lei; Pooler, Amy M; Albrecht, Meredith A; Wurtman, Richard J

2005-01-01

Membrane phospholipids like phosphatidylcholine (PC) are required for cellular growth and repair, and specifically for synaptic function. PC synthesis is controlled by cellular levels of its precursor, cytidine-5'-diphosphate choline (CDP-choline), which is produced from cytidine triphosphate (CTP) and phosphocholine. In rat PC12 cells exogenous uridine was shown to elevate intracellular CDP-choline levels, by promoting the synthesis of uridine triphosphate (UTP), which was partly converted to CTP. In such cells uridine also enhanced the neurite outgrowth produced by nerve growth factor (NGF). The present study assessed the effect of dietary supplementation with uridine-5'-monophosphate disodium (UMP-2Na+, an additive in infant milk formulas) on striatal dopamine (DA) release in aged rats. Male Fischer 344 rats consumed either a control diet or one fortified with 2.5% UMP for 6 wk, ad libitum. In vivo microdialysis was then used to measure spontaneous and potassium (K+)-evoked DA release in the right striatum. Potassium (K+)-evoked DA release was significantly greater among UMP-treated rats, i.e., 341+/-21% of basal levels vs. 283+/-9% of basal levels in control rats (p<0.05); basal DA release was unchanged. In general, each animal's K+-evoked DA release correlated with its striatal DA content, measured postmortem. The levels of neurofilament-70 and neurofilament-M proteins, biomarkers of neurite outgrowth, increased to 182+/-25% (p<0.05) and 221+/-34% (p<0.01) of control values, respectively, with UMP consumption. Hence, UMP treatment not only enhances membrane phosphatide production but also can modulate two membrane-dependent processes, neurotransmitter release and neurite outgrowth, in vivo.

A Unified Model of Performance: Validation of its Predictions across Different Sleep/Wake Schedules

PubMed Central

Ramakrishnan, Sridhar; Wesensten, Nancy J.; Balkin, Thomas J.; Reifman, Jaques

2016-01-01

Study Objectives: Historically, mathematical models of human neurobehavioral performance developed on data from one sleep study were limited to predicting performance in similar studies, restricting their practical utility. We recently developed a unified model of performance (UMP) to predict the effects of the continuum of sleep loss—from chronic sleep restriction (CSR) to total sleep deprivation (TSD) challenges—and validated it using data from two studies of one laboratory. Here, we significantly extended this effort by validating the UMP predictions across a wide range of sleep/wake schedules from different studies and laboratories. Methods: We developed the UMP on psychomotor vigilance task (PVT) lapse data from one study encompassing four different CSR conditions (7 d of 3, 5, 7, and 9 h of sleep/night), and predicted performance in five other studies (from four laboratories), including different combinations of TSD (40 to 88 h), CSR (2 to 6 h of sleep/night), control (8 to 10 h of sleep/night), and nap (nocturnal and diurnal) schedules. Results: The UMP accurately predicted PVT performance trends across 14 different sleep/wake conditions, yielding average prediction errors between 7% and 36%, with the predictions lying within 2 standard errors of the measured data 87% of the time. In addition, the UMP accurately predicted performance impairment (average error of 15%) for schedules (TSD and naps) not used in model development. Conclusions: The unified model of performance can be used as a tool to help design sleep/wake schedules to optimize the extent and duration of neurobehavioral performance and to accelerate recovery after sleep loss. Citation: Ramakrishnan S, Wesensten NJ, Balkin TJ, Reifman J. A unified model of performance: validation of its predictions across different sleep/wake schedules. SLEEP 2016;39(1):249–262. PMID:26518594

[The effect of uridine and uridine nucleotides on isolated rat heart performance in regional myocardial ischemia].

PubMed

Eliseev, V V; Rodionova, O M; Sapronov, N S; Selizarova, N O

2002-01-01

We studied the effects of uridine, uridine-5'-monophosphate (UMP), uridine-5'-diphosphate (UDP) and uridine-5'-triphosphate on contractility, coronary flow and heart rate in isolated perfused rat hearts under 60-minute regional ischemia of the left ventricle. All the compounds (50 mumol/l) induced a positive inotropic effect but had no effect on the heart rate. Uridine and UMP prevented the development of the contracture. UDP and especially UTP increased coronary flow. Probably, a protective effect of uridine and UMP is due to activation of myocardial glycogen synthesis while favourable effects of UDP and UTP on contractility and coronary flow are explained by their influence on P2U-receptors of cardiomyocytes. In addition, coronary dilatation induced by UDP and UTP promoted the reduction of the damaged zone.

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

PubMed Central

Wang, Kuifeng; Wang, Qi; Chen, Jing; Chen, Lili; Jiang, Hualiang; Shen, Xu

2011-01-01

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and Km values of the two substrates dUMP and CH2H4folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery. PMID:21633987

The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 5'-monophosphate decarboxylase.

PubMed

Takashima, Yasuhide; Mizohata, Eiichi; Krungkrai, Sudaratana R; Fukunishi, Yoshifumi; Kinoshita, Takayoshi; Sakata, Tsuneaki; Matsumura, Hiroyoshi; Krungkrai, Jerapan; Horii, Toshihiro; Inoue, Tsuyoshi

2012-08-01

Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 Å resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.

Purification and characterization of the bifunctional uridylyltransferase and the signal transducing proteins GlnB and GlnK from Herbaspirillum seropedicae.

PubMed

Bonatto, Ana C; Couto, Gustavo H; Souza, Emanuel M; Araújo, Luiza M; Pedrosa, Fabio O; Noindorf, Lilian; Benelli, Elaine M

2007-10-01

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme that has a central role in the general nitrogen regulatory system NTR. In enterobacteria, GlnD uridylylates the PII proteins GlnB and GlnK under low levels of fixed nitrogen or ammonium. Under high ammonium levels, GlnD removes UMP from these proteins (deuridylylation). The PII proteins are signal transduction elements that integrate the signals of nitrogen, carbon and energy, and transduce this information to proteins involved in nitrogen metabolism. In Herbaspirillum seropedicae, an endophytic diazotroph isolated from grasses, several genes coding for proteins involved in nitrogen metabolism have been identified and cloned, including glnB, glnK and glnD. In this work, the GlnB, GlnK and GlnD proteins of H. seropedicae were overexpressed in their native forms, purified and used to reconstitute the uridylylation system in vitro. The results show that H. seropedicae GlnD uridylylates GlnB and GlnK trimers producing the forms PII (UMP)(1), PII (UMP)(2) and PII (UMP)(3), in a reaction that requires 2-oxoglutarate and ATP, and is inhibited by glutamine. The quantification of these PII forms indicates that GlnB was more efficiently uridylylated than GlnK in the system used.

Chemical models and their mechanistic implications for the transformation of 6-cyanouridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase.

PubMed

Chien, Tun-Cheng; Jen, Cheng-Hung; Wu, Yuen-Jen; Liao, Chen-Chieh

2008-01-01

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes an unprecedented transformation of 6- cyanouridine 5'-monophosphate (6-CN-UMP) into barbiturate nucleoside 5'-monophosphate (6-hydroxyuridine 5'-monophosphate, BMP). The reactions of 6- cyano-1,3-dimethyluracil toward various nucleophilic conditions have been studied as chemical models in order to understand the possible mechanism for the ODCase-catalyzed transformation of 6-CN-UMP.

The isotopic and chemical evolution of planets: Mars as a missing link

NASA Technical Reports Server (NTRS)

Depaolo, D. J.

1988-01-01

The study of planetary bodies has advanced to a stage where it is possible to contemplate general models for the chemical and physical evolution of planetary interiors, which might be referred to as UMPES (Unified Models of Planetary Evolution and Structure). UMPES would be able to predict the internal evolution and structure of a planet given certain input parameters such as mass, distance from the sun, and a time scale for accretion. Such models are highly dependent on natural observations because the basic material properties of planetary interiors, and the processes that take place during the evolution of planets are imperfectly understood. The idea of UMPES was particularly unrealistic when the only information available was from the earth. However, advances have been made in the understanding of the general aspects of planetary evolution now that there is geochemical and petrological data available for the moon and for meteorites.

Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

PubMed

Jastreboff, M; Kedzierska, B; Rode, W

1982-01-15

Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.

Stabilities of lead(II) complexes formed in aqueous solution with methyl thiophosphate (MeOPS(2-)), uridine 5'- O-thiomonophosphate (UMPS(2-)) or adenosine 5'- O-thiomonophosphate (AMPS(2-)).

PubMed

Da Costa, Carla P; Krajewska, Danuta; Okruszek, Andrzej; Stec, Wojciech J; Sigel, Helmut

2002-04-01

The acidity constants of twofold protonated methyl thiophosphate (MeOPS(2-)) and of monoprotonated uridine 5'- O-thiomonophosphate (UMPS(2-)) have been determined in aqueous solution (25 degrees C; I= 0.1 M, NaNO(3)) by potentiometric pH titration. The stability constants of their 1:1 complexes formed with Pb(2+), i.e. Pb(MeOPS) and Pb(UMPS), have also been measured. The results show that replacement of a phosphate oxygen by a sulfur atom increases the acidity by about 1.4 p K units. On the basis of recently established log versus plots ( = simple phosphate or phosphonate ligands where R is a non-coordinating residue), it is shown that the stability of the Pb(thiophosphate) complexes is by log Delta= 2.43+/-0.09 larger than expected for a Pb(2+)-phosphate interaction. The identity of the stability increase (log Delta) observed for Pb(MeOPS) and Pb(UMPS) shows that the nucleobase residue in the Pb(UMPS) complex has no influence on complex formation. To be able to carry out the mentioned comparisons, we have also determined the stability constant of the complex formed between Pb(2+) and methyl phosphate; the corresponding data for Pb(UMP) were already known from our earlier studies. The present results allow an evaluation of other Pb(2+) complexes formed with thiophosphate derivatives and they are applied now to the Pb(2+) complexes of adenosine 5'- O-thiomonophosphate (AMPS(2-)). The stability constants of the Pb(H;AMPS)(+) and Pb(AMPS) complexes were measured and it is shown that, within the error limits, the stability of the Pb(AMPS) complex is determined by the basicity of the thiophosphate group of AMPS(2-); in other words, no hint for macrochelate formation involving N7 was observed. More important, with the aid of micro-stability-constant considerations it is concluded that the structure of the dominating isomer of the Pb(H;AMPS)(+) species is the one where the proton is located at the N1 site of the adenine residue and Pb(2+) is coordinated to the deprotonated thiophosphate group. The insights gained from this study with regard to thiophosphate-altered single-stranded nucleic acids and their affinity towards Pb(2+) are discussed.

The cardioprotective effect of uridine and uridine-5'-monophosphate: the role of the mitochondrial ATP-dependent potassium channel.

PubMed

Krylova, Irina B; Kachaeva, Evgeniya V; Rodionova, Olga M; Negoda, Alexander E; Evdokimova, Nataliya R; Balina, Maria I; Sapronov, Nikolay S; Mironova, Galina D

2006-07-01

The activity of mitochondrial ATP-dependent potassium channel (mitoKATP) of rat heart and liver mitochondria was shown to decrease during aging. This partially explains the increase of risk of ischemia at a mature age since mitoKATP activation provides cardioprotection. We demonstrated that uridine-5'-diphosphate (UDP) possesses the property to activate mitoKATP. At a concentration of 30 microM, it reactivated mitoKATP in mitochondria, and 5-hydroxydecanoate (5-HD) eliminated this effect. In experimental animals, UDP precursors uridine and uridine-5'-monophosphate (UMP) (both 30 mg/kg, administered intravenously 5 min before coronary occlusion) decreased the myocardium ischemic alteration index (1.9 and 3.5 times, respectively) and the T-wave amplitude within 60 min after occlusion. Both effects were inhibited by Glibenclamide (Glib) and 5-HD. UMP and uridine decreased the number of premature ventricular beats 5.6 and 1.9 times and the duration of ventricular tachycardia 9.4 and 4.1 times, respectively. Glib and 5-HD inhibited the anti-arrhythmic parameters, 5-HD being less effective. Uridine and UMP decreased the duration of fibrillation 10.8 and 3.6 times, respectively, and this effect was not abolished by Glib and 5-HD. Thus, uridine and UMP, which are the precursors of UDP in the cell, possess cardioprotective properties. MitoKATP prevents mainly ischemic injuries and partially rhythm disorders.

Prospecting for Precious Metals in Ultra-Metal-Poor Stars

NASA Astrophysics Data System (ADS)

French, R. S.

2000-05-01

The chemical compositions of the most metal-poor halo stars are living records of the very early nucleosynthetic history of the Galaxy. Only a few prior generations, if not a single one, of element-donating supernovae could have been responsible for the heavy elements observed in ultra-metal-poor (UMP; [Fe/H] < --2.5) stars. Abundances of the heavy neutron-capture elements (Z > 30) can yield direct information about the supernova progenitors to UMP stars, and abundances of unstable thorium and uranium (Z = 90, 92) can potentially provide age estimates for the Galactic halo. Already, many studies have demonstrated that abundances of rare-earth elements (56 <= Z <= 72) in UMP stars are completely consistent with their production in rapid neutron-capture synthesis (r-process) events, usually believed to occur during supernovae explosions. Therefore, mapping the entire abundance pattern of UMP stars is of significant interest. In particular, abundances of the most massive stable elements (Os -> Pb or 76 <= Z <= 82) could provide crucial information about the so-called ``third r-process peak,'' and are critical to the radioactive-dating technique that uses unstable thorium as a chronometer. Until recently, abundance determinations for these elements have been virtually non-existent, as the strongest relevant transitions lay in the vacuum UV, inaccessible to ground-based observation. The availability of high-resolution space-based spectrometers has opened up new regions of spectral coverage, including precisely the range in wavelength needed to make these sensitive measurements. We have undertaken a study of about 10 metal-poor halo giants to determine the abundances of several of the heaviest neutron-capture elements including platinum, osmium, lead, and gold. Preliminary results indicate that the abundance pattern of heavy neutron-capture elements (56 <= Z <= 82) in UMP stars does mimic a scaled solar system r-process. Thus, the ability to estimate the initial abundances of thorium and uranium is greatly reinforced.

Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase

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

Maley, F.; Maley, G.F.

1983-01-01

It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A preventsmore » the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.« less

Effect of unbalanced magnetic pull and hydraulic seal force on the vibration of large rotor-bearing systems

NASA Astrophysics Data System (ADS)

Song, Z.; Guo, P.; Liu, Y.

2014-03-01

The influence of unbalanced magnetic pull (UMP) and hydraulic seal force on the vibration of large rotor-bearing systems is studied. The UMP caused by rotor eccentricity imposes important effects on rotating machinery, especially for large generators such as water turbine generator sets, because these machines operate above their first critical speed in some instances and are supported by oil film bearings. A magnetic stiffness matrix for studying the effects of the UMP is proposed. The magnetic stiffness matrix can be generated by decomposing the expression of air gap magnetic field energy. Two vibration models are constructed using the Lagrange equation. The difference between the two models lies in the boundary support condition: one has rigid support and the other has elastic bearing support. The influence of the magnetic stiffness and elastic support on the critical speed of the rotor is studied using Lyapunov nonlinear vibration stability theory. The vibration amplitude of the rotor is calculated, taking the magnetic stiffness and horizontal centrifugal force into account. The unbalanced hydraulic seal force is produced because of the asymmetry of seal clearance. This imbalance is one of the factors that causes self-excited vibration in rotating machinery, and is as important as the UMP for large water turbine generator sets. The rotor-bearing system is supported by an oil film journal bearing, whose characteristic also impose considerable influence on vibration. On the basis of the above-mentioned conditions, a three-dimensional finite element model of the rotating system that includes the oil film journal bearing is constructed. The effect of the UMP and unbalanced hydraulic seal force is considered in the construction, and studied in relation to the magnetic parameters, seal parameters, journal bearing stiffness, and outer diameter of the rotating machine critical speed. Conclusions may benefit the dynamic design and optimized operation of large rotating machinery.

Erythrocyte arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N) as indices of lead exposure.

PubMed Central

Cook, L R; Angle, C R; Stohs, S J

1986-01-01

The activities of three erythrocyte (rbc) enzymes, arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N), were compared in 16 lead workers and 14 age matched controls as correlates of blood lead (PbB) and unextracted zinc protoporphyrin (EP) concentrations. Subjects with PbB of 0.9-2.5 microM (19-52 micrograms/dl) had 6.5 +/- 0.6 IU of P5N activity with uridine monophosphate (UMP) as substrate, significantly less (p less than 0.001) than the 12.0 +/- 0.7 IU activity of controls with PbB 0.3-0.6 microM (6-12 micrograms/dl). The mean activity of rbc dP5N with either deoxyuridine monophosphate (dUMP) or thymidine monophosphate as substrate, and of rbc arginase, did not differentiate the two groups. The correlation coefficients of ln PbB with the selected substrates for P5N and dP5N were: UMP, r = -0.75; dUMP, r = -0.61; TMP, r = -0.23. The correlation coefficient of ln PbB and arginase activity was -0.03. Rbc P5N (UMPase) is a significant correlate of PbB, equivalent to rbc protoporphyrin. HPLC assay of rbc UMPase activity is a sensitive and rapid assay that appears to meet criteria for a reliable clinical laboratory index of blood lead concentrations. PMID:3013277

Message from Vice-Chancellor

NASA Astrophysics Data System (ADS)

Nasir Ibrahim, Daing

2013-12-01

Salam Malaysia! First and foremost, I want to thank all 2nd International Conference on Mechanical Engineering Research (ICMER 2013) organisers for inviting me to address and officiate this conference. This 2nd ICMER 2013 provides a platform to bring together not only researchers, postgraduate students but also industrial people. With this platform, ICMER will embark on a whole process of making new discoveries and then translating them into products and services for the marketplace and this is only made possible with people like all of you. I would like to congratulate the ICMER 2013 organisers for the achievement of collecting 208 papers for this conference. Submissions received from 17 local universities, 7 industrial companies and 9 different countries is a great achievement for UMP. I am very happy to welcome all of you from Malaysia, Iran, Turkey, Japan, India, Australia, Ireland, Saudi Arabia and Bangladesh to this conference. As one of Malaysia's Public Universities, UMP's main challenge is to remain competitive and relevant by offering high quality technical academic programmes and research activities, focusing on its niche areas. New knowledge and findings cannot be generated without any research and development (R&D) activities. These efforts will undoubtedly generate lots of interesting results and new knowledge either bring further commercialisation activities. The Malaysian government has invested a huge sum of Ringgits in R&D over the years. Therefore, UMP must produce more quality researchers and graduates to ensure Malaysia reaps the returns from these investments vice versa of progressive economic growth for the country. UMP's 2011-2015 Strategic Plan determines to strengthen and sustained its financial support by allocating research grants and industry collaboration and consultations through its business and commercial unit. On behalf of UMP, I would like to express my appreciation to all committee members of ICMER 2013 from Faculty of Mechanical Engineering for their hardwork and relentless effort. Without their commitment and contributions, this event would not been possible and successfully delivered at this time. I wish you all a fruitful discourse. Thank you. Professor Dato' Dr Daing Nasir Ibrahim Vice-Chancellor, Universiti Malaysia Pahang

Implementation of Ultramini Percutaneous Nephrolithotomy for Treatment of 2-3 cm Kidney Stones: A Preliminary Report.

PubMed

Shah, Arvind K; Xu, Kewei; Liu, Hao; Huang, Hai; Lin, Tianxin; Bi, Liangkuan; Jinli, Han; Fan, Xinxiang; Shrestha, Rujan; Huang, Jian

2015-11-01

Miniatured percutaneous nephrolithotomy (PCNL) techniques such as micro-PCNL (microperc) and ultramini-PCNL (UMP) are usually indicated for renal stones <2 cm. We present our preliminary report of treating patients with 2 to 3 cm renal stones using UMP in a semisupine combined lithotomy position associated with a retrograde ureteral access sheath (UAS). From April 2013 to January 2014, we implemented 13F UMP for 22 patients with renal stones that were 2 to 3 cm with the patient positioned in a 45-degree semisupine combined lithotomy position. A retrograde 9.5/11.5F UAS was placed for maintaining low intrarenal pressure and debris drainage. Flexible ureteroscopy was used for stones inaccessible through the primary percutaneous tract in two patients. A 200-μ holmium laser was used for stone disintegration. Intrapelvic pressure was measured using an open end 5F ureteral catheter inserted through the UAS. All 22 cases were completed successfully. The mean preoperative stone size was 26.6 ± 4.7 mm (range 21-32 mm), mean operative time was 85.7 ± 18.0 minutes (range 47-112 min), and mean hemoglobin drop was 1.2 ± 0.3 g/dL (range 0.5-2.2 g/dL). Intrapelvic pressure during the surgical procedure ranged 5 to 10 cm H2O. The mean hospital stay was 3.1 ± 1.8 days (range 2-5 d). Complete stone clearance was 18/22 (81.8%) with solo UMP and 20/22 (90.9%) when associated with retrograde intrarenal surgery (RIRS). No major intra- or postoperative complications occurred. Implementation of UMP for the treatment of patients with renal stones 2-3 cm is feasible and safe. The procedure is less invasive and has a faster recovery period. Intraoperative retrograde UAS decreases intrarenal pressure, facilitates removal of stone fragments, and also allows simultaneous RIRS for stones in an inaccessible calix.

Computational model for calculating the dynamical behaviour of generators caused by unbalanced magnetic pull and experimental validation

NASA Astrophysics Data System (ADS)

Pennacchi, Paolo

2008-04-01

The modelling of the unbalanced magnetic pull (UMP) in generators and the experimental validation of the proposed method are presented in this paper. The UMP is one of the most remarkable effects of electromechanical interactions in rotating machinery. As a consequence of the rotor eccentricity, the imbalance of the electromagnetic forces acting between rotor and stator generates a net radial force. This phenomenon can be avoided by means of a careful assembly and manufacture in small and stiff machines, like electrical motors. On the contrary, the eccentricity of the active part of the rotor with respect to the stator is unavoidable in big generators of power plants, because they operate above their first critical speed and are supported by oil-film bearings. In the first part of the paper, a method aimed to calculate the UMP force is described. This model is more general than those available in literature, which are limited to circular orbits. The model is based on the actual position of the rotor inside the stator, therefore on the actual air-gap distribution, regardless of the orbit type. The closed form of the nonlinear UMP force components is presented. In the second part, the experimental validation of the proposed model is presented. The dynamical behaviour in the time domain of a steam turbo-generator of a power plant is considered and it is shown that the model is able to reproduce the dynamical effects due to the excitation of the magnetic field in the generator.

Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens.

PubMed

Fang, Haitian; Liu, Huiyan; Chen, Ning; Zhang, Chenglin; Xie, Xixian; Xu, Qingyang

2013-06-01

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP's feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5'-phosphate synthesis and pathogenesis of Magnaporthe oryzae.

PubMed

Qi, Zhongqiang; Liu, Muxing; Dong, Yanhan; Yang, Jie; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

2016-04-01

Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5'-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae.

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

PubMed Central

Arvizu-Flores, Aldo A.; Sugich-Miranda, Rocio; Arreola, Rodrigo; Garcia-Orozco, Karina D.; Velazquez-Contreras, Enrique F.; Montfort, William R.; Maley, Frank; Sotelo-Mundo, Rogerio R.

2008-01-01

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH2THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The kcat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the Km for the (R)-stereoisomer of CH2THF was 300 µM, about 30 fold larger than Km from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH2THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group. PMID:18403248

Towards a formal genealogical classification of the Lezgian languages (North Caucasus): testing various phylogenetic methods on lexical data.

PubMed

Kassian, Alexei

2015-01-01

A lexicostatistical classification is proposed for 20 languages and dialects of the Lezgian group of the North Caucasian family, based on meticulously compiled 110-item wordlists, published as part of the Global Lexicostatistical Database project. The lexical data have been subsequently analyzed with the aid of the principal phylogenetic methods, both distance-based and character-based: Starling neighbor joining (StarlingNJ), Neighbor joining (NJ), Unweighted pair group method with arithmetic mean (UPGMA), Bayesian Markov chain Monte Carlo (MCMC), Unweighted maximum parsimony (UMP). Cognation indexes within the input matrix were marked by two different algorithms: traditional etymological approach and phonetic similarity, i.e., the automatic method of consonant classes (Levenshtein distances). Due to certain reasons (first of all, high lexicographic quality of the wordlists and a consensus about the Lezgian phylogeny among Caucasologists), the Lezgian database is a perfect testing area for appraisal of phylogenetic methods. For the etymology-based input matrix, all the phylogenetic methods, with the possible exception of UMP, have yielded trees that are sufficiently compatible with each other to generate a consensus phylogenetic tree of the Lezgian lects. The obtained consensus tree agrees with the traditional expert classification as well as some of the previously proposed formal classifications of this linguistic group. Contrary to theoretical expectations, the UMP method has suggested the least plausible tree of all. In the case of the phonetic similarity-based input matrix, the distance-based methods (StarlingNJ, NJ, UPGMA) have produced the trees that are rather close to the consensus etymology-based tree and the traditional expert classification, whereas the character-based methods (Bayesian MCMC, UMP) have yielded less likely topologies.

Towards a Formal Genealogical Classification of the Lezgian Languages (North Caucasus): Testing Various Phylogenetic Methods on Lexical Data

PubMed Central

Kassian, Alexei

2015-01-01

A lexicostatistical classification is proposed for 20 languages and dialects of the Lezgian group of the North Caucasian family, based on meticulously compiled 110-item wordlists, published as part of the Global Lexicostatistical Database project. The lexical data have been subsequently analyzed with the aid of the principal phylogenetic methods, both distance-based and character-based: Starling neighbor joining (StarlingNJ), Neighbor joining (NJ), Unweighted pair group method with arithmetic mean (UPGMA), Bayesian Markov chain Monte Carlo (MCMC), Unweighted maximum parsimony (UMP). Cognation indexes within the input matrix were marked by two different algorithms: traditional etymological approach and phonetic similarity, i.e., the automatic method of consonant classes (Levenshtein distances). Due to certain reasons (first of all, high lexicographic quality of the wordlists and a consensus about the Lezgian phylogeny among Caucasologists), the Lezgian database is a perfect testing area for appraisal of phylogenetic methods. For the etymology-based input matrix, all the phylogenetic methods, with the possible exception of UMP, have yielded trees that are sufficiently compatible with each other to generate a consensus phylogenetic tree of the Lezgian lects. The obtained consensus tree agrees with the traditional expert classification as well as some of the previously proposed formal classifications of this linguistic group. Contrary to theoretical expectations, the UMP method has suggested the least plausible tree of all. In the case of the phonetic similarity-based input matrix, the distance-based methods (StarlingNJ, NJ, UPGMA) have produced the trees that are rather close to the consensus etymology-based tree and the traditional expert classification, whereas the character-based methods (Bayesian MCMC, UMP) have yielded less likely topologies. PMID:25719456

Cytosine to uracil conversion through hydrolytic deamination of cytidine monophosphate hydroxy-alkylated on the amino group: a liquid chromatography--electrospray ionization--mass spectrometry investigation.

PubMed

Losito, I; Angelico, R; Introna, B; Ceglie, A; Palmisano, F

2012-10-01

A novel pathway for cytosine to uracil conversion performed in a micellar environment, leading to the generation of uridine monophosphate (UMP), was evidenced during the alkylation reaction of cytidine monophosphate (CMP) by dodecyl epoxide. Liquid chromatography-electrospray ionization - ion trap - mass spectrometry was used to separate and identify the reaction products and to follow their formation over time. The detection of hydroxy-amino-dodecane, concurrently with free UMP, in the reaction mixture suggested that, among the various alkyl-derivatives formed, CMP alkylated on the amino group of cytosine could undergo tautomerization to an imine and hydrolytic deamination, generating UMP. Interestingly, no evidence for this peculiar conversion pathway was obtained when guanosine monophosphate (GMP), the complementary ribonucleotide of CMP, was also present in the reaction mixture, due to the fact that NH(2)-alkylated CMP was not formed in this case. The last finding emphasized the role played by CMP-GMP molecular interactions, mediated by a micellar environment, in hindering the alkylation reaction at the level of the cytosine amino group. Copyright © 2012 John Wiley & Sons, Ltd.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

Dietary uridine enhances the improvement in learning and memory produced by administering DHA to gerbils.

PubMed

Holguin, Sarah; Martinez, Joseph; Chow, Camille; Wurtman, Richard

2008-11-01

This study examined the effects on cognitive behaviors of giving normal adult gerbils three compounds, normally in the circulation, which interact to increase brain phosphatides, synaptic proteins, dendritic spines, and neurotransmitter release. Animals received supplemental uridine (as its monophosphate, UMP; 0.5%) and choline (0.1%) via the diet, and docosahexaenoic acid (DHA; 300 mg/kg/day) by gavage, for 4 wk, and then throughout the subsequent period of behavioral training and testing. As shown previously, giving all three compounds caused highly significant (P<0.001) increases in total brain phospholipids and in each major phosphatide; giving DHA or UMP (plus choline) produced smaller increases in some of the phosphatides. DHA plus choline improved performance on the four-arm radial maze, T-maze, and Y-maze tests; coadministering UMP further enhanced these increases. (Uridine probably acts by generating both CTP, which can be limiting in phosphatide synthesis, and UTP, which activates P2Y receptors coupled to neurite outgrowth and protein synthesis. All three compounds also act by enhancing the substrate-saturation of phosphatide-synthesizing enzymes.) These findings demonstrate that a treatment that increases synaptic membrane content can enhance cognitive functions in normal animals.

Direct evidence for tautomerization of the uracil moiety within the Pb2+/uridine-5'-monophosphate complex: a combined tandem mass spectrometry and IRMPD study.

PubMed

Salpin, Jean-Yves; Guillaumont, Sébastien; Ortiz, Daniel; Tortajada, Jeanine; Maître, Philippe

2011-08-15

The structure of the [Pb(UMP)-H](+) (UMP = uridine-5'-monophosphate) complex was studied in the gas phase by combining electrospray ionization (ESI), tandem mass spectrometry, and mid-infrared multiple photon dissociation (IRMPD) spectroscopy. The results obtained show that Pb(2+) ions interact not only with the deprotonated phosphate group but also with a carbonyl group of the nucleobase moiety by folding of the mononucleotide, resulting in macrochelates that are not likely to be present in solution. Comparison between the IRMPD and DFT-computed spectra suggests that the ESI-generated complex likely corresponds to a mixture of several structures, and establishes the enolic tautomers as the most abundant species for the [Pb(UMP)-H](+) ion, while the very weak IRMPD signal observed at ∼1763 cm(-1) points to a minor population of oxo forms. Our data also suggest that losing the nucleobase residue under CID conditions does not necessarily mean a lack of interaction between the metal and the nucleobase moiety, as commonly reported in the literature for large oligonucleotides. © 2011 American Chemical Society

The novel fluorescent CDP-analogue (Pbeta)MABA-CDP is a specific probe for the NMP binding site of UMP/CMP kinase.

PubMed

Rudolph, M G; Veit, T J; Reinstein, J

1999-12-01

Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N-methylanthraniloyl- (mant) group to the beta-phosphate of CDP via a butyl linker, a CDP analogue [(Pbeta)MABA-CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments.

The novel fluorescent CDP-analogue (Pbeta)MABA-CDP is a specific probe for the NMP binding site of UMP/CMP kinase.

PubMed Central

Rudolph, M. G.; Veit, T. J.; Reinstein, J.

1999-01-01

Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N-methylanthraniloyl- (mant) group to the beta-phosphate of CDP via a butyl linker, a CDP analogue [(Pbeta)MABA-CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments. PMID:10631985

Phosphorylation of uridine and cytidine by uridine-cytidine kinase.

PubMed

Qian, Yahui; Ding, Qingbao; Li, Yanyu; Zou, Zhi; Yan, Bingkun; Ou, Ling

2014-10-20

Uridine 5'-monophosphate (5'-UMP) and cytidine 5'-monophosphate (5'-CMP) were biosynthesized by recombinant uridine-cytidine kinase (UCK) and acetate kinase (ACK). The ack and uck genes from Escherichia coli K12 and the uck1, uck2 and ack genes from Lactobacillus bulgaricus ATCC 11842 were cloned and inserted into the plasmid pET-28a. All of the recombinant E. coli strains were capable of overexpressing UCK and ACK, which catalyzed the reaction using guanosine 5'-triphosphate (GTP) as a phosphate intermediate that was regenerated by ACK from acetyl phosphate. The effect of several parameters, including the substrate concentration, the GTP concentration, the temperature and the reaction pH, were optimized. High efficiency was achieved if uridine or cytidine was phosphorylated by UCK encoded by uck from E. coli and ACK encoded by ack from L. bulgaricus. The maximum conversion yield of 5'-UMP and 5'-CMP was 97% at 37 °C and pH 7.5 when 30 mM uridine/cytidine and 0.5mM GTP in a total of 1 mL were used. In addition, the 5'-UMP and 5'-CMP products were very stable in the reaction system and did not undergo significant degradation. Copyright © 2014 Elsevier B.V. All rights reserved.

Unified multiphase modeling for evolving, acoustically coupled systems consisting of acoustic, elastic, poroelastic media and septa

NASA Astrophysics Data System (ADS)

Lee, Joong Seok; Kang, Yeon June; Kim, Yoon Young

2012-12-01

This paper presents a new modeling technique that can represent acoustically coupled systems in a unified manner. The proposed unified multiphase (UMP) modeling technique uses Biot's equations that are originally derived for poroelastic media to represent not only poroelastic media but also non-poroelastic ones ranging from acoustic and elastic media to septa. To recover the original vibro-acoustic behaviors of non-poroelastic media, material parameters of a base poroelastic medium are adjusted depending on the target media. The real virtue of this UMP technique is that interface coupling conditions between any media can be automatically satisfied, so no medium-dependent interface condition needs to be imposed explicitly. Thereby, the proposed technique can effectively model any acoustically coupled system having locally varying medium phases and evolving interfaces. A typical situation can occur in an iterative design process. Because the proposed UMP modeling technique needs theoretical justifications for further development, this work is mainly focused on how the technique recovers the governing equations of non-poroelastic media and expresses their interface conditions. We also address how to describe various boundary conditions of the media in the technique. Some numerical studies are carried out to demonstrate the validity of the proposed modeling technique.

Theoretical studies of damage to 3'-uridine monophosphate induced by electron attachment.

PubMed

Zhang, Ru Bo; Zhang, Ke; Eriksson, Leif A

2008-01-01

Low-energy electrons (LEE) are well known to induce nucleic acid damage. However, the damage mechanisms related to charge state and structural features remain to be explored in detail. In the present work, we have investigated the N1-glycosidic and C3'-O(P) bond ruptures of 3'-UMP (UMP=uridine monophosphate) and the protonated form 3'-UMPH with -1 and zero charge, respectively, based on hybrid density functional theory (DFT) B3 LYP together with the 6-31+G(d,p) basis set. The glycosidic bond breakage reactions of the 3'UMP and 3'UMPH electron adducts are exothermic in both cases, with barrier heights of 19-20 kcal mol(-1) upon inclusion of bulk solvation. The effects of the charge state on the phosphate group are marginal, but the C2'-OH group destabilizes the transition structure of glycosidic bond rupture of 3'-UMPH in the gas phase by approximately 5.0 kcal mol(-1). This is in contrast with the C3'-O(P) bond ruptures induced by LEE in which the charge state on the phosphate influences the barrier heights and reaction energies considerably. The barrier towards C3'-O(P) bond dissociation in the 3'UMP electron adduct is higher in the gas phase than the one corresponding to glycosidic bond rupture and is dramatically influenced by the C2'-OH group and bulk salvation, which decreases the barrier to 14.7 kcal mol(-1). For the C3'-O(P) bond rupture of the 3'UMPH electron adduct, the reaction is exothermic and the barrier is even lower, 8.2 kcal mol(-1), which is in agreement with recent results for 3'-dTMPH and 5'-dTMPH (dTMPH=deoxythymidine monophosphate). Both the Mulliken atomic charges and unpaired-spin distribution play significant roles in the reactions.

A phase II study of preoperative chemoradiation with tegafur-uracil plus leucovorin for locally advanced rectal cancer with pharmacogenetic analysis.

PubMed

Kim, Sun Young; Baek, Ji Yeon; Oh, Jae Hwan; Park, Sung Chan; Sohn, Dae Kyung; Kim, Min Ju; Chang, Hee Jin; Kong, Sun-Young; Kim, Dae Yong

2017-03-27

This study aimed to evaluate the efficacy of a high dose of oral tegafur-uracil (400 mg/m 2 ) plus leucovorin with preoperative chemoradiation of locally advanced rectal cancer and to explore the impact of polymorphisms of cytochrome P 2A6 (CYP2A6), uridine monophosphate synthetase (UMPS), and ATP-binding cassette B1 (ABCB1) on clinical outcome. Patients with cT3 or cT4 rectal cancer were enrolled and were given tegafur-uracil 400 mg/m 2 /day and leucovorin 90 mg/m 2 /day for 7 days a week during preoperative chemoradiation (50.4 Gy/28 fractions) in this phase II trial. Primary endpoint was pathologic complete response rate, and the secondary endpoint was to explore the association between clinical outcomes and genetic polymorphisms CYP2A6 (*4, *7, *9 and *10), UMPS G638C, and three ABCB1 genotypes (C1236T, C3435T, and G2677T). Ninety-one patients were given study treatment, and 90 underwent surgery. Pathologic complete response was noted in 10 patients (11.1%). There was no grade 4 or 5 toxicity; 20 (22.0%) experienced grade 3 toxicities, including diarrhea (10, 11.0%), abdominal pain (2, 2.2%), and anemia (2, 2.2%). Relapse-free survival and overall survival at 5 years were 88.6% and 94.2%, respectively. Patients with the UMPS 638 CC genotype experienced significantly more frequent grade 2 or 3 diarrhea (p for trend = 0.018). Preoperative chemoradiation with tegafur-uracil 400 mg/m 2 /day with leucovorin was feasible, but did not meet the expected pathologic complete response rate. The UMPS 638 CC genotype might be a candidate biomarker predicting toxicity in patients receiving tegafur-uracil/leucovorin-based preoperative chemoradiation for locally advanced rectal cancer. ISRCTN11812525 , registered on 25 July 2016. Retrospectively registered.

Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry.

PubMed

Qiao, Yuanbiao; Jian, Fangfang; Bai, Qian

2008-03-14

Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility.

Femtosecond laser spectroscopy of the rhodopsin photochromic reaction: a concept for ultrafast optical molecular switch creation (ultrafast reversible photoreaction of rhodopsin).

PubMed

Smitienko, Olga; Nadtochenko, Victor; Feldman, Tatiana; Balatskaya, Maria; Shelaev, Ivan; Gostev, Fedor; Sarkisov, Oleg; Ostrovsky, Mikhail

2014-11-11

Ultrafast reverse photoreaction of visual pigment rhodopsin in the femtosecond time range at room temperature is demonstrated. Femtosecond two-pump probe experiments with a time resolution of 25 fs have been performed. The first рump pulse at 500 nm initiated cis-trans photoisomerization of rhodopsin chromophore, 11-cis retinal, which resulted in the formation of the primary ground-state photoproduct within a mere 200 fs. The second pump pulse at 620 nm with a varying delay of 200 to 3750 fs relative to the first рump pulse, initiated the reverse phototransition of the primary photoproduct to rhodopsin. The results of this photoconversion have been observed on the differential spectra obtained after the action of two pump pulses at a time delay of 100 ps. It was found that optical density decreased at 560 nm in the spectral region of bathorhodopsin absorption and increased at 480 nm, where rhodopsin absorbs. Rhodopsin photoswitching efficiency shows oscillations as a function of the time delay between two рump pulses. The quantum yield of reverse photoreaction initiated by the second pump pulse falls within the range 15%±1%. The molecular mechanism of the ultrafast reversible photoreaction of visual pigment rhodopsin may be used as a concept for the development of an ultrafast optical molecular switch.

Chapter 21: Estimating Net Savings - Common Practices. The Uniform Methods Project: Methods for Determining Energy Efficiency Savings for Specific Measures

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

Kurnik, Charles W; Violette, Daniel M.; Rathbun, Pamela

This chapter focuses on the methods used to estimate net energy savings in evaluation, measurement, and verification (EM and V) studies for energy efficiency (EE) programs. The chapter provides a definition of net savings, which remains an unsettled topic both within the EE evaluation community and across the broader public policy evaluation community, particularly in the context of attribution of savings to a program. The chapter differs from the measure-specific Uniform Methods Project (UMP) chapters in both its approach and work product. Unlike other UMP resources that provide recommended protocols for determining gross energy savings, this chapter describes and comparesmore » the current industry practices for determining net energy savings but does not prescribe methods.« less

Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells.

PubMed

Jastreboff, M M; Kedzierska, B; Rode, W

1983-07-15

Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.

Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis.

PubMed

James, G T; Yeoman, L C; Matsui, S i; Goldberg, A H; Busch, H

1977-05-31

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.

Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

PubMed

Gibson, Marc W; Dewar, Simon; Ong, Han B; Sienkiewicz, Natasha; Fairlamb, Alan H

2016-05-01

Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.

Complex formation of divalent metal ions with uridine 5'-O-thiomonophosphate or methyl thiophosphate: comparison of complex stabilities with those of the parent phosphate ligands.

PubMed

Da Costa, Carla P; Okruszek, Andrzej; Sigel, Helmut

2003-07-07

The stability constants of the 1:1 complexes formed in aqueous solution between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Zn2+, or Cd2+ (M2+) and methyl thiophosphate (MeOPS(2-)) or uridine 5'-O-thiomonophosphate (UMPS(2-)) (PS(2-)=MeOPS(2-) or UMPS(2-)) have been determined (potentiometric pH titrations; 25 degrees C; I = 0.1 M, NaNO(3)). Comparison of these results for M(PS) complexes with those known for the parent M(PO) phosphate species, where PO(2-)=CH(3)OPO(2-)(3) or UMP(2-) (uridine 5'-monophosphate), shows that the alkaline earth metal ions, as well as Mn2+, Co2+, and Ni2+ have a higher affinity for phosphate groups than for their thio analogues. However, based on the linear log K(M)(M(R-PO3)) versus pK(H)(H(R-PO3)) relationships (R-PO(2-)(3) simple phosphate monoester or phosphonate ligands with a non-interacting residue R) it becomes clear that the indicated observation is only the result of the lower basicity of the thiophosphate residue. In contrast, the thio complexes of Zn2+ and Cd2+ are more stable than their parent phosphate ones, and this despite the lower basicity of the PS(2-) ligands. This stability increase is identical for M(MeOPS) and M(UMPS) species and amounts to about 0.6 and 2.4 log units for Zn(PS) and Cd(PS), respectively. Since no other binding site is available in MeOPS(2-), this enhanced stability has to be attributed to the S atom. Indeed, from the mentioned stability differences it follows that Cd2+ in Cd(PS) is coordinated by more than 99% to the thiophosphate S atom; the same value holds for Pb(PS), which was studied earlier. The formation degree of the Sbonded isomer amounts to 76+/-6 % for Zn(PS) and is close to zero for the corresponding Mg2+, Ca2+, and Mn2+ species. It is further shown that Zn(MeOPS)(aq)(2+) releases a proton from a coordinated water molecule with pK(a) approximately 6.9; i.e., this deprotonation occurs at a lower pH value than that for the same reaction in Zn(aq)(2+). Since Mg2+, Ca2+, Mn2+, and Cd2+ have a relatively low tendency for hydroxo complex formation, it was possible, for these M2+, to also quantify the stability of the binuclear complexes, M(2)(UMPS-H)+, where one M2+ is thiophosphate-coordinated and the other is coordinated at (N3)(-) of the uracil residue. The impact of the results presented herein regarding M2+/nucleic acid interactions, including those of ribozymes (rescue experiments), is briefly discussed.

Nitrogen K-edge x-ray absorption near edge structure of pyrimidine-containing nucleotides in aqueous solution

NASA Astrophysics Data System (ADS)

Shimada, Hiroyuki; Minami, Hirotake; Okuizumi, Naoto; Sakuma, Ichiro; Ukai, Masatoshi; Fujii, Kentaro; Yokoya, Akinari; Fukuda, Yoshihiro; Saitoh, Yuji

2015-05-01

X-ray absorption near edge structure (XANES) was measured at energies around the N K-edge of the pyrimidine-containing nucleotides, cytidine 5'-monophosphate (CMP), 2'-deoxythymidine 5'-monophosphate (dTMP), and uridine 5'-monophosphate (UMP), in aqueous solutions and in dried films under various pH conditions. The features of resonant excitations below the N K-edge in the XANES spectra for CMP, dTMP, and UMP changed depending on the pH of the solutions. The spectral change thus observed is systematically explained by the chemical shift of the core-levels of N atoms in the nucleobase moieties caused by structural changes due to protonation or deprotonation at different proton concentrations. This interpretation is supported by the results of theoretical calculations using density functional theory for the corresponding nucleobases in the neutral and protonated or deprotonated forms.

Nitrogen K-edge x-ray absorption near edge structure of pyrimidine-containing nucleotides in aqueous solution

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

Shimada, Hiroyuki, E-mail: hshimada@cc.tuat.ac.jp; Minami, Hirotake; Okuizumi, Naoto

2015-05-07

X-ray absorption near edge structure (XANES) was measured at energies around the N K-edge of the pyrimidine-containing nucleotides, cytidine 5′-monophosphate (CMP), 2′-deoxythymidine 5′-monophosphate (dTMP), and uridine 5′-monophosphate (UMP), in aqueous solutions and in dried films under various pH conditions. The features of resonant excitations below the N K-edge in the XANES spectra for CMP, dTMP, and UMP changed depending on the pH of the solutions. The spectral change thus observed is systematically explained by the chemical shift of the core-levels of N atoms in the nucleobase moieties caused by structural changes due to protonation or deprotonation at different proton concentrations.more » This interpretation is supported by the results of theoretical calculations using density functional theory for the corresponding nucleobases in the neutral and protonated or deprotonated forms.« less

The possible role of human milk nucleotides as sleep inducers.

PubMed

Sánchez, Cristina L; Cubero, Javier; Sánchez, Javier; Chanclón, Belén; Rivero, Montserrat; Rodríguez, Ana B; Barriga, Carmen

2009-02-01

Breast-milk contains a potent mixture of diverse components, such as the non-protein nitrogen fraction which includes nucleotides, whose variation in levels is evident throughout lactation. In addition, these substances play an important role in sleep homeostasis. In the present study, human milk samples were analyzed using a capillary electrophoresis system. The rhythmicity of each nucleotide was studied by cosinor analysis. It was found that the nucleotides 5'AMP, 5'GMP, 5'CMP, and 5'IMP have significant (P < 0.05) circadian rhythms, the acrophases of the first two being during the night, and of the latter two during the day. While 5'UMP did not show a clear circadian rhythm, there was an increase in its levels at night. In conclusion, the rise in nocturnal levels of 5'AMP, 5'GMP, and 5'UMP could be involved in inducing the 'hypnotic' action of breast-milk at night in the infant.

Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS.

PubMed

Jarmuła, Adam; Wilk, Piotr; Maj, Piotr; Ludwiczak, Jan; Dowierciał, Anna; Banaszak, Katarzyna; Rypniewski, Wojciech; Cieśla, Joanna; Dąbrowska, Magdalena; Frączyk, Tomasz; Bronowska, Agnieszka K; Jakowiecki, Jakub; Filipek, Sławomir; Rode, Wojciech

2017-10-01

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzymatic Characterization of AMP Phosphorylase and Ribose-1,5-Bisphosphate Isomerase Functioning in an Archaeal AMP Metabolic Pathway

PubMed Central

Aono, Riku; Sato, Takaaki; Yano, Ayumu; Yoshida, Shosuke; Nishitani, Yuichi; Miki, Kunio; Imanaka, Tadayuki

2012-01-01

AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis. PMID:23065974

Comparison of postoperative pain and inflammation reaction in dogs undergoing preventive laparoscopic-assisted and incisional gastropexy.

PubMed

Haraguchi, Tomoya; Kimura, Shiho; Itoh, Harumichi; Nishikawa, Shimpei; Hiyama, Masato; Tani, Kenji; Iseri, Toshie; Itoh, Yoshiki; Nakaichi, Munekazu; Taura, Yasuho; Itamoto, Kazuhito

2017-09-12

This study compared the effects of postoperative pain and inflammation reaction after preventive laparoscopic-assisted gastropexy (LAG) and incisional gastropexy (IG) in 10 clinically normal Beagles. Surgical time, incision length, visual analog scale (VAS) score, University of Melbourne Pain Scale (UMPS) score, and plasma C-reactive protein (CRP), plasma cortisol (COR), and serum interleukin-6 (IL-6) levels were evaluated. The VAS and UMPS scores and COR and IL-6 levels were recorded at 0.5, 1, 2, 4, 8, 12, 18 and 24 hr after surgery. CRP level was recorded at 12, 24 and 48 hr after surgery. The VAS and UMPS scores showed no significant intergroup differences. Compared to IG, LAG had significantly lower surgical time (45 ± 9.91 min vs 64 ± 5.30 min; P<0.05), incision length (46 ± 8.21 mm vs 129 ± 19.49 mm; P<0.05), CRP level (12 hr after surgery; 4.58 ± 1.58 mg/dl vs 12.4 ± 1.34 mg/dl; P<0.01), and COR level (1 hr after surgery; 10.79 ± 3.07 µg/dl vs 15.9 ± 3.77 µg/dl; P<0.05). IL-6 levels showed no significant intergroup differences at any time point. However, LAG resulted in lower IL-6 levels than did IG at all postoperative time points. Neither procedure resulted in significant surgical complications. LAG produced lower surgical stress than did IG, suggesting that LAG is a safe, minimally invasive, and highly useful technique for preventing canine gastric dilatation-volvulus. Nevertheless, since this study used experimental models, its usefulness should be evaluated in future cases.

Copper(II) complexes with uridine, uridine 5'-monophosphate, spermidine, or spermine in aqueous solution.

PubMed

Lomozik, Lechoslaw; Jastrzab, Renata

2003-01-15

Molecular complexes of the types (Urd)H(x)(PA) and (UMP)H(x)(PA) are formed in the uridine (Urd) or uridine 5'-monophosphate (UMP) plus spermidine or spermine systems, as shown by the results of equilibrium and spectral studies. Overall stability constants of the adducts and equilibrium constants of their formation have been determined. An increase in the efficiency of the reaction between the bioligands is observed with increasing length of the polyamine. The pH range of adduct formation is found to coincide with that in which the polyamine is protonated while uridine or its monophosphate is deprotonated. The -NH(x)(+) groups from PA and the N(3) atom of the purine base as well as phosphate groups from the nucleotides have been identified as the significant centres of non-covalent interactions. Compared to cytidine, the pH range of Urd adduct formation is shifted significantly higher due to differences in the protonation constants of the endocyclic N(3) donor atoms of particular nucleosides. Overall stability constants of the Cu(II) complexes with uridine and uridine 5'-monophosphate in ternary systems with spermidine or spermine have been determined. It has been found from spectral data that in the Cu(II) ternary complexes with nucleosides and polyamines the reaction of metallation involves mainly N(3) atoms from the pyrimidine bases, as well as the amine groups of PA. This unexpected type of interaction has been evidenced in the coordination mode of the complexes forming in the Cu-UMP systems including spermidine or spermine. Results of spectral and equilibrium studies indicate that the phosphate groups taking part in metallation are at the same time involved in non-covalent interaction with the protonated polyamine.

Effect of the combination of uridine nucleotides, folic acid and vitamin B12 on the clinical expression of peripheral neuropathies.

PubMed

Negrão, Luis; Almeida, Pedro; Alcino, Sérgio; Duro, Helena; Libório, Teresa; Melo Silva, Ulisses; Figueira, Ricardo; Gonçalves, Sílvia; Neto Parra, Laura

2014-05-01

Peripheral neuropathy (PN) is a common condition whose incidence is approximately 8% in elderly persons. Neuropathic pain (NeP) has a significant incidence in the general population and affects more than half of all patients with PN. The pathophysiology of PN is characterized by lesions of myelin-producing Schwann cells in peripheral nerves. Regeneration/protection of the myelin sheath after a nerve lesion is a fundamental element of repair in PN. Nucleotides such as uridine monophosphate (UMP) have proven to be efficacious in treating the cause of the myelin sheath lesion in several experimental and clinical models. Our objective was to evaluate clinical improvement in patients with PN and NeP treated with a combination of UMP+folic acid+vitamin B12 (Keltican). We performed an exploratory, open-label, multicenter, study of 212 patients followed for 2 months. Pain was assessed using the painDETECT questionnaire (PDQ). The intensity of the NeP assessed at the time of the consultation progressed favorably and decreased significantly (p<0.001) in all the types of PN included. The global score for pain assessed using PDQ decreased from 17.5 points to 8.8 points at the final evaluation (p<0.001). Nonsteroidal anti-inflammatory drugs were decreased/withdrawn in 77.4% of patients. The combination of UMP+folic acid+vitamin B12 is effective against NeP associated with PN. It leads to statistically significant reductions not only in the total PDQ score but also in the intensity of pain, number of areas affected, and pain radiation. Furthermore, it makes it possible to reduce the dosage of concomitant medication.

Comparison of postoperative pain and inflammation reaction in dogs undergoing preventive laparoscopic-assisted and incisional gastropexy

PubMed Central

HARAGUCHI, Tomoya; KIMURA, Shiho; ITOH, Harumichi; NISHIKAWA, Shimpei; HIYAMA, Masato; TANI, Kenji; ISERI, Toshie; ITOH, Yoshiki; NAKAICHI, Munekazu; TAURA, Yasuho; ITAMOTO, Kazuhito

2017-01-01

This study compared the effects of postoperative pain and inflammation reaction after preventive laparoscopic-assisted gastropexy (LAG) and incisional gastropexy (IG) in 10 clinically normal Beagles. Surgical time, incision length, visual analog scale (VAS) score, University of Melbourne Pain Scale (UMPS) score, and plasma C-reactive protein (CRP), plasma cortisol (COR), and serum interleukin-6 (IL-6) levels were evaluated. The VAS and UMPS scores and COR and IL-6 levels were recorded at 0.5, 1, 2, 4, 8, 12, 18 and 24 hr after surgery. CRP level was recorded at 12, 24 and 48 hr after surgery. The VAS and UMPS scores showed no significant intergroup differences. Compared to IG, LAG had significantly lower surgical time (45 ± 9.91 min vs 64 ± 5.30 min; P<0.05), incision length (46 ± 8.21 mm vs 129 ± 19.49 mm; P<0.05), CRP level (12 hr after surgery; 4.58 ± 1.58 mg/dl vs 12.4 ± 1.34 mg/dl; P<0.01), and COR level (1 hr after surgery; 10.79 ± 3.07 µg/dl vs 15.9 ± 3.77 µg/dl; P<0.05). IL-6 levels showed no significant intergroup differences at any time point. However, LAG resulted in lower IL-6 levels than did IG at all postoperative time points. Neither procedure resulted in significant surgical complications. LAG produced lower surgical stress than did IG, suggesting that LAG is a safe, minimally invasive, and highly useful technique for preventing canine gastric dilatation-volvulus. Nevertheless, since this study used experimental models, its usefulness should be evaluated in future cases. PMID:28717065

Exploring the Early Chemical Evolution of the Milky Way with LAMOST and Subaru

NASA Astrophysics Data System (ADS)

Li, Haining; Aoki, Wako; Honda, Satoshi; Zhao, Gang; Suda, Takuma; Christlieb, Norbert

Extremely Metal-Poor (EMP) stars ([Fe/H] < -3.0) provide fundamental evidence on the nucleosynthesis and enrichment of the first stars and supernovae. LAMOST will observe 6 million Galactic stars through a 5-year spectroscopic survey, and thus provide an unprecedented chance to enlarge the EMP star sample. In 2014, a joint project on EMP stars was initiated with the LAMOST survey and Subaru follow-up observation. So far, more than 70 EMP stars have been found and confirmed, including identifications of a number of chemically interesting objects: three UMP (ultra metal-poor) stars with [Fe/H] ˜ -4.0, including the second UMP turnoff star with Li detection; a super Li-rich (A(Li) = +3) EMP giant, which is the most extreme example of Li enhancement in red giants known to date; a few EMP stars showing extreme enhancements in neutron-capture elements. Statistics of a large sample of EMP stars will constrain formation of the Milky Way halo.

Synthesis and hybridization of a series of biotinylated oligonucleotides.

PubMed Central

Cook, A F; Vuocolo, E; Brakel, C L

1988-01-01

A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin substituents were subsequently attached to the allylamino sidearms by reaction with N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester. These oligomers were hybridized to target DNA immobilized on microtiter wells (ELISA plates), and were detected with a streptavidin-biotinylated horseradish peroxidase complex using hydrogen peroxide as substrate and o-phenylenediamine as chromogen. We found that the sensitivity of detection of target DNA by biotin-labeled oligonucleotide probes was strongly dependent upon the position of the biotin label. Oligonucleotides containing biotin labels near or off the ends of the hybridizing sequence were more effective probes than oligonucleotides containing internal biotin labels. An additive effect of increasing numbers of biotin-dUMP residues was found for some labeling configurations. PMID:3375076

75 FR 61959 - Georgia-Alabama-South Carolina System

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-06

... the period specified shall be: Capacity Charge: $4.19 Per kilowatt of total contract demand per month..., for a period ending September 30, 2012 (123 FERC ] 62,022). Public Notice and Comment Notice of... Judgment quotes the Fourth Circuit Court of Appeals: ``(p)umped storage poses a major environmental concern...

Combined Heat and Power Protocol for Uniform Methods Project | Advanced

Science.gov Websites

Manufacturing Research | NREL Combined Heat and Power Protocol for Uniform Methods Project Combined Heat and Power Protocol for Uniform Methods Project NREL developed a protocol that provides a ; is consistent with the scope and other protocols developed for the Uniform Methods Project (UMP

The structure of a protein primer-polymerase complex in the initiation of genome replication.

PubMed

Ferrer-Orta, Cristina; Arias, Armando; Agudo, Rubén; Pérez-Luque, Rosa; Escarmís, Cristina; Domingo, Esteban; Verdaguer, Nuria

2006-02-22

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix alpha8 of the fingers domain and helix alpha13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.

One-electron oxidation of individual DNA bases and DNA base stacks.

PubMed

Close, David M

2010-02-04

In calculations performed with DFT there is a tendency of the purine cation to be delocalized over several bases in the stack. Attempts have been made to see if methods other than DFT can be used to calculate localized cations in stacks of purines, and to relate the calculated hyperfine couplings with known experimental results. To calculate reliable hyperfine couplings it is necessary to have an adequate description of spin polarization which means that electron correlation must be treated properly. UMP2 theory has been shown to be unreliable in estimating spin densities due to overestimates of the doubles correction. Therefore attempts have been made to use quadratic configuration interaction (UQCISD) methods to treat electron correlation. Calculations on the individual DNA bases are presented to show that with UQCISD methods it is possible to calculate hyperfine couplings in good agreement with the experimental results. However these UQCISD calculations are far more time-consuming than DFT calculations. Calculations are then extended to two stacked guanine bases. Preliminary calculations with UMP2 or UQCISD theory on two stacked guanines lead to a cation localized on a single guanine base.

Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N sup 4 -hydroxy-dCMP, N sup 4 -hydroxy-5-fluoro-dCMP, and related analogues

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

Rode, W.; Zielinski, Z.; Dzik, J.M.

1990-12-01

N{sup 4}-Hydroxy-dCMP (N{sup 4}-OH-dCMP), N{sup 4}-methoxy-dCMP (N{sup 4}-OMe-dCMP), and their 5-fluoro congeners were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N{sup 4}-OH-dCMP was not a substrate and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based. K{sub i} values for N{sup 4}-OH-dCMP and its 5-fluoro analogue were in the range 10{sup {minus}7}-10{sup {minus}8} M, 2-3 orders of magnitude higher for the corresponding N{sup 4}-OMe analogues. The 5-methyl analogue of N{sup 4}-OHdCMP was 10{sup 4}-fold less potent, pointing to the anti rotamermore » of the imino form of exocyclic N{sup 4}-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N{sup 4}-OH-dCMP, suggesting interaction of both N{sup 4}-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N{sup 4}-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources. With the Ehrlich and tapeworm enzymes, N{sup 4}-OH-FdCMP and FdUMP were almost equally effective inhibitors.« less

Committee for International Conference on Mechanical Engineering Research (ICMER 2011)

NASA Astrophysics Data System (ADS)

Yusoff, Ahmad Razlan Bin

2012-09-01

Scientific Advisory Committee: 1) Prof. Dr. Ahmad Kamal Ariffin (UKM) 2) Prof. Dr. Hj. Rosli Abu Bakar (UMP) 3) Prof. Dr. Hanafi Ismail (USM) 4) Prof. Ir. Dr. Mohd Jailani Mohd Nor (MoHE) 5) Prof. Dr. Zahari Taha (UMP) 6) Prof. Dr. Masjuki Haji Hassan 7) Prof. Ir. Dr. Ramesh Singh (UNITEN) 8) Prof. Dr. Razali Ayob (UTEM) 9) Prof. Dr. Wan Khairuddin (UTM) 10) Prof. Dr. Sulaiman Hj. Hasan (UTHM) 11) Prof. Dr. Zuraidah Mohd. Zain (UniMAP) 12) Prof. Dr. Horizon Gitano (USM) 13) Prof. Dr. K.V Sharma (UMP) 14) Prof. Dr. Shahrani Anuar (UMP) 15) Assoc. Prof. Dr. Abd Rashid Abd. Aziz (UTP) 16) Assoc. Prof. Dr. Aidy Ali (UPM) 17) Assoc. Prof. Dr. Saidur Rahman (UM) 18) Assoc. Prof. Dr. Md Abdul Maleque (UIA) Organizing Committee Chairman: Prof. Dr. Hj. Rosli Abu Bakar Co-Chair: Prof. Dr. Zahari Taha Co-Chair: Prof. Ir. Dr. Jailani Salihon Secretary: Dr. Rizalman Mamat Committee on Keynote Speaker 1) Kumaran Kadirgama (Chair) 2) Prof. Dr. K.V. Sharma 3) Haji Amirruddin Abdul Kadir 4) Miminorazeansuhaila Loman 5) Mohd Akramin Mohd Romlay Technical Committee (Peer Review & Proceedings) 1) Dr. Abdul Adam Abdullah (Chair) 2) Dr. Ahmad Razlan Yusoff 3) Mohd Yusof Taib 4) Dr. Md. Mustafizur Rahman 5) Dr. Hjh. Yusnita Rahayu 6) Dr. Gigih Priyandoko 7) Dr. Agung Sudrajad 8) Muhammad Hatifi Mansor 9) Mohd Fadzil Abdul Rahim Technical Committee (Panels & Session Chairs) 1) Dr. Mahadzir Ishak (Chair) 2) Prof. Dr. Shahrani Anuar 3) Dr. Maisara Mohyeldin Gasim Mohamed 4) Muhammad Ammar Nik Mu'tasim 5) Ahmad Basirul Subha bin Alias Technical Committee (Journal Publication) 1) Dr. Ahmad Razlan bin Yusoff (Chair) 2) Mohd Yusof Taib 3) Dr. Mahadzir Ishak 4) Dr. Abdul Adam Abdullah 5) Hj. Amirruddin Abdul Kadir 6) Hadi Abdul Salaam Bureau of Publicity & Website 1) Dr. Muhamad Arifpin Mansor (Chair) 2) Amir Abdul Razak 3) Idris Mat Sahat 4) Prof. Dr. Hj. Rosli Abu Bakar 5) Muhamad Zuhairi Sulaiman 6) Dr. Sugeng Ariyono 7) Asnul Hadi Ahmad 8) Mohd Tarmizy Che Kar 9) Mohd Padzly Radzi Bureau of Special Task & Poster 1) Lee Giok Chui (Chair) 2) Mohd Fadzil Abdul Rahim 3) Che Ku Eddy Nizwan 4) Hazami Che Hussain 5) Mohd Fazli Ismail 6) Mahdhir Mohd Yusof 7) Mohd Padzly Radzi 8) Rahimah Che Ramli Secretariats 1) Ir. Ahmad Rasdan Ismail (Chair) 2) Mohd Shahri Mohd Akhir 3) Luqman Hakim Ahmad Shah 4) Juliawati Alias 5) Nurazima Ismail 6) Mohamad Faizal Mohamed Zahri 7) Raja Allen Jordan Izzuddin Shah 8) Rosidah Mohd Norsat 9) Norshalawati Mat Yusof 10) Zainab Daud 11) Nur Sufiah Jamaludin 12) Azslinda Ibrahim 13) Nurul Azreen Zainal Abidin 14) Nurul Ashikin Mohd Khalil 15) Mohd Zaki Mohd Ali

Crosslinking of Chitosan with Dialdehyde Derivatives of Nucleosides and Nucleotides. Mechanism and Comparison with Glutaraldehyde.

PubMed

Mikhailov, Sergey N; Zakharova, Alexandra N; Drenichev, Mikhail S; Ershov, Andrey V; Kasatkina, Mariya A; Vladimirov, Leonid V; Novikov, Valentin V; Kildeeva, Natalia R

2016-01-01

In medical and pharmaceutical applications, chitosan is used as a component of hydrogels-macromolecular networks swollen in water. Chemical hydrogels are formed by covalent links between the crosslinking reagents and amino functionalities of chitosan. To date, the most commonly used chitosan crosslinkers are dialdehydes, such as glutaraldehyde (GA). We have developed novel GA like crosslinkers with additional functional groups-dialdehyde derivatives of uridine (oUrd) and nucleotides (oUMP and oAMP)-leading to chitosan-based biomaterials with new properties. The process of chitosan crosslinking was investigated in details and compared to crosslinking with GA. The rates of crosslinking with oUMP, oAMP, and GA were essentially the same, though much higher than in the case of oUrd. The remarkable difference in the crosslinking properties of nucleoside and nucleotide dialdehydes can be clearly attributed to the presence of the phosphate group in nucleotides that participates in the gelation process through ionic interactions with the amino groups of chitosan. Using NMR spectroscopy, we have not observed the formation of aldimine bonds. It can be concluded that the real number of crosslinks needed to cause gelation of chitosan chains may be less than 1%.

Message from Vice Chancellor, UMP

NASA Astrophysics Data System (ADS)

Nasir Ibrahim, Daing

2012-09-01

Assalamualaikumwarahmatullahiwabarakatuh and Salam i Malaysia First and foremost, I want to thank the International Conference Mechanical Engineering Research (ICMER) organisers for inviting me to address and officiate at this conference. It is a privilege and an honour for me on this momentous occasion to grace the ceremony. The ICMER provides a platform to bring together not only researchers but also postgraduate students in Mechanical Engineering, Automotive Engineering, Manufacturing Engineering, Biomechanical Engineering, Material Engineering and Industrial Engineering. With this platform, ICMER will embark on a whole process of making new discoveries and then translating them into products and services for the marketplace; this is only made possible by people like all of you. It might be only a starting point but with hard work and perseverance I am sure you will succeed with flying colours. As one of Malaysia's Public Universities, UMP's main challenge is to remain competitive and relevant by offering high quality technical academic programmes and research activities, focusing on its niche areas. New knowledge and findings cannot be generated without research and development (R&D) therefore, Malaysia has had substantial investment in research and development facilities. These efforts will undoubtedly generate lots of interesting results and new knowledge as either further investigation or commercial activities. Therefore, researchers like you must see this as the generator of new knowledge to extend your research outcomes from laboratory experiments to the marketplace and towards commercialisation. Naybe this doesn't appear significant in the short term but it may make a tremendous impact in the future. The Malaysian government has invested a huge sum of Ringgits in R&D over the years. Therefore, public universities such as UMP must produce more quality researchers and graduates to ensure Malaysia reaps the returns from these investments and consequently progressive economic growth for the country. Commensurate with these efforts, the results of research must be commercialised effectively. That is why our 2nd KRA UMP's 2011-2015 Strategic Plan determines that we strengthen and sustain its financial support by allocating research grants and industry collaboration and consultations through its business and commercialisation unit. Commercialisation of research outcomes doesn't happen naturally. It may need many man-hours and effort to be invested rather than just purchasing new tools and equipment for laboratories. Moreover, it would be unfortunate if many research results remain in the form of technical publications and scientific journals rather than actual products. These may of course be valuable in their own right but the impact on society and economies must be the main agenda and one of the objectives of the research. The process of taking technology from laboratories to the marketplace will face challenges and pitfalls. In that case we as researchers must be responsible and willing to create a supportive environment for those who undertake this risky business of commercialising R&D especially for innovative entrepreneurship that carries out a new technological approach out from the laboratories to high-tech ventures. We have achieved many accolades from numerous competitors since we first embarked on our journey to make R&D activities play a more significant role in the economy in Malaysia. Therefore, we must keep up the pace of this journey and maintain our investment in R&D despite facing the unfavourable global financial situation because ultimately we must to remain a top and competitive player in the global economy. This can be done by continuing to produce high quality research and technology which will set a milestone in transforming our economy. On behalf of UMP, I would like to express my appreciation to the all the committee members from the Faculty of Mechanical Engineering and Automotive Engineering Centre for their hard work and relentless effort. Without their commitment and contributions, this event would not have been possible at this time. And with that, thank you again and wassalam. YH Profesor Dato' Dr Daing Nasir Ibrahim Vice Chancellor Universiti Malaysia Pahang

A novel thymidylate synthase from the Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) clade with altered nucleotide and folate binding sites.

PubMed

Lopez-Zavala, Alonso A; Guevara-Hernandez, Eduardo; Vazquez-Lujan, Luz H; Sanchez-Paz, Arturo; Garcia-Orozco, Karina D; Contreras-Vergara, Carmen A; Lopez-Leal, Gamaliel; Arvizu-Flores, Aldo A; Ochoa-Leyva, Adrian; Sotelo-Mundo, Rogerio R

2018-01-01

Thymidylate synthase (TS, E.C. 2.1.1.45) is a crucial enzyme for de novo deoxythymidine monophosphate (dTMP) biosynthesis. The gene for this enzyme is thyA , which encodes the folate-dependent TS that converts deoxyuridine monophosphate group (dUMP) into (dTMP) using the cofactor 5,10-methylenetetrahydrofolate (mTHF) as a carbon donor. We identified the thyA gene in the genome of the Vibrio parahaemolyticus strain FIM-S1708+ that is innocuous to humans but pathogenic to crustaceans. Surprisingly, we found changes in the residues that bind the substrate dUMP and mTHF, previously postulated as invariant among all TSs known (Finer-Moore, Santi & Stroud, 2003). Interestingly, those amino acid changes were also found in a clade of microorganisms that contains Vibrionales , Alteromonadales , Aeromonadales , and Pasteurellales (VAAP) from the Gammaproteobacteria class. In this work, we studied the biochemical properties of recombinant TS from V. parahemolyticus FIM-S1708+ (VpTS) to address the natural changes in the TS amino acid sequence of the VAAP clade. Interestingly, the K m for dUMP was 27.3 ± 4.3 µM, about one-fold larger compared to other TSs. The K m for mTHF was 96.3 ± 18 µM, about three- to five-fold larger compared to other species, suggesting also loss of affinity. Thus, the catalytic efficiency was between one or two orders of magnitude smaller for both substrates. We used trimethoprim, a common antibiotic that targets both TS and DHFR for inhibition studies. The IC 50 values obtained were high compared to other results in the literature. Nonetheless, this molecule could be a lead for the design antibiotics towards pathogens from the VAAP clade. Overall, the experimental results also suggest that in the VAAP clade the nucleotide salvage pathway is important and should be investigated, since the de novo dTMP synthesis appears to be compromised by a less efficient thymidylate synthase.

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

PubMed

Kasai, Yuki; Oshima, Kohei; Ikeda, Fukiko; Abe, Jun; Yoshimitsu, Yuya; Harayama, Shigeaki

2015-01-01

Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

Vapor Jet Ejector Used to Generate Free Waste Heat Driven Cooling in Military Environmental Cooling Units

DTIC Science & Technology

2012-07-01

vap erant vapor is or by a J-tub essure side of using similar pressure incre ump in order o the diesel-e per heat excha recovered at to the exhaus...top of the a tering the com at exchanger. g of the conve id flow. A nit essure pulsati tor where the ed in the cool erature level. ger where wa

UNO DMRG CASCI calculations of effective exchange integrals for m-phenylene-bis-methylene spin clusters

NASA Astrophysics Data System (ADS)

Kawakami, Takashi; Sano, Shinsuke; Saito, Toru; Sharma, Sandeep; Shoji, Mitsuo; Yamada, Satoru; Takano, Yu; Yamanaka, Shusuke; Okumura, Mitsutaka; Nakajima, Takahito; Yamaguchi, Kizashi

2017-09-01

Theoretical examinations of the ferromagnetic coupling in the m-phenylene-bis-methylene molecule and its oligomer were carried out. These systems are good candidates for exchange-coupled systems to investigate strong electronic correlations. We studied effective exchange integrals (J), which indicated magnetic coupling between interacting spins in these species. First, theoretical calculations based on a broken-symmetry single-reference procedure, i.e. the UHF, UMP2, UMP4, UCCSD(T) and UB3LYP methods, were carried out with a GAUSSIAN program code under an SR wave function. From these results, the J value by the UHF method was largely positive because of the strong ferromagnetic spin polarisation effect. The J value by the UCCSD(T) and UB3LYP methods improved an overestimation problem by correcting the dynamical electronic correlation. Next, magnetic coupling among these spins was studied using the CAS-based method of the symmetry-adapted multireference methods procedure. Thus, the UNO DMRG CASCI (UNO, unrestricted natural orbital; DMRG, density matrix renormalised group; CASCI, complete active space configuration interaction) method was mainly employed with a combination of ORCA and BLOCK program codes. DMRG CASCI calculations in valence electron counting, which included all orbitals to full valence CI, provided the most reliable result, and support the UB3LYP method for extended systems.

Oral supplementation with docosahexaenoic acid and uridine-5'-monophosphate increases dendritic spine density in adult gerbil hippocampus.

PubMed

Sakamoto, Toshimasa; Cansev, Mehmet; Wurtman, Richard J

2007-11-28

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating or brain DHA are associated with various neurocognitive disorders including Alzheimer's disease (AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA's behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic structure in living animals. Here we show that oral supplementation with DHA substantially increases the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-supplemented with a uridine source, uridine-5'-monophosphate (UMP), which increases brain levels of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (>30%) is accompanied by parallel increases in membrane phosphatides and in pre- and post-synaptic proteins within the hippocampus. Hence, oral DHA may promote neuronal membrane synthesis to increase the number of synapses, particularly when co-administered with UMP. Our findings provide a possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive disorders resulting from synapse loss.

Nonlinear dynamic behaviors of permanent magnet synchronous motors in electric vehicles caused by unbalanced magnetic pull

NASA Astrophysics Data System (ADS)

Xiang, Changle; Liu, Feng; Liu, Hui; Han, Lijin; Zhang, Xun

2016-06-01

Unbalanced magnetic pull (UMP) plays a key role in nonlinear dynamic behaviors of permanent magnet synchronous motors (PMSM) in electric vehicles. Based on Jeffcott rotor model, the stiffness characteristics of the rotor system of the PMSM are analyzed and the nonlinear dynamic behaviors influenced by UMP are investigated. In free vibration study, eigenvalue-based stability analysis for multiple equilibrium points is performed which offers an insight in system stiffness. Amplitude modulation effects are discovered of which the mechanism is explained and the period of modulating signal is carried out by phase analysis and averaging method. The analysis indicates that the effects are caused by the interaction of the initial phases of forward and backward whirling motions. In forced vibration study, considering dynamic eccentricity, frequency characteristics revealing softening type are obtained by harmonic balance method, and the stability of periodic solution is investigated by Routh-Hurwitz criterion. The frequency characteristics analysis indicates that the response amplitude is limited in the range between the amplitudes of the two kinds of equilibrium points. In the vicinity of the continuum of equilibrium points, the system hardly provides resistance to bending, and hence external disturbances easily cause loss of stability. It is useful for the design of the PMSM with high stability and low vibration and acoustic noise.

The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

PubMed Central

Adlard, B. P. F.; Lathe, G. H.

1970-01-01

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis–Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The Km for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca2+ were inhibitory in the absence of Mg2+ but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3β-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis. PMID:4251180

Acupuncture and pharmacopuncture are as effective as morphine or carprofen for postoperative analgesia in bitches undergoing ovariohysterectomy.

PubMed

Luna, Stelio Pacca Loureiro; Martino, Irene Di; Lorena, Silvia Elaine Rodolfo de Sá; Capua, Maria Luisa Buffo de; Lima, Alfredo Feio da Maia; Santos, Bianca Paiva Costa Rodrigues dos; Brondani, Juliana Tabarelli; Vesce, Giancarlo

2015-12-01

To investigate the analgesic effect of acupuncture (AP) or micro-dose pharmacopuncture (PA), using carprofen or morphine, in bitches undergoing ovariohysterectomy (OHE). Thirty five dogs were randomly assigned to five groups after sedation with acepromazine IM: AP, 0.5 mg.kg(-1) of morphine subcutaneously (SC), 4 mg.kg(-1) of carprofen SC, and PA with 0.05 mg.kg(-1) of morphine or 0.4 mg.kg(-1) of carprofen. Anaesthesia was induced with propofol and maintained with isoflurane. Pain was assessed after OHE by a blind observer for 24h, by dynamic visual analogue scale (DIVAS), Glasgow (CMPS-SF), Melbourne (UMPS) and Colorado University pain scale (CSU). Animals reaching 33% of the UMPS score received rescue analgesia with morphine IM. Non parametric data were analysed by Kruskal-Wallis or Friedman tests where applicable, followed by Dunn's test. Parametric data were analysed by two way ANOVA, followed by Tukey test. There were no differences among groups in number of rescue analgesia. Except for the DIVAS score where animals treated with morphine had the lowest score compared with AP and carprofen, at 1h after surgery, there were no other differences among groups. Acupuncture or pharmacopuncture were equally effective as morphine or carprofen to control postoperative pain in bitches undergoing ovariohysterectomy.

Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with hsa-coupled, uridine-containing, monophosphoric ribodinucleotides.

PubMed Central

Alarcón-Segovia, D; Fishbein, E; Estrada-Parra, S; García-Ortigoza, E

1976-01-01

Sera from patients with scleroderma have been found to have anti-RNA antibodies which react with human serum albumin (HSA)-coupled uridine and uridine monophosphate (UMP) and are inhibited by uracil, uridine and UMP. Scleroderma sera react uniformly with 5'-polyuridylic acid (poly(U)) and fail to react with polyadenylic, polyuridylic acid poly(A) - poly(U)) which is also indicative of their uracil specificity. Anti-RNA antibodies found in systemic lupus erythematosus (SLE) are immunochemically different from those found in scleroderma in that, instead of being uniformly specific to uracil, they are markedly heterogeneous and may react with uracil, uridine and/or UMP. SLE sera frequently react with poly(A) - poly(U), indicating also their ability to recognize the double helical structure of double-stranded RNA. Thirty-seven scleroderma and thirty-four SLE sera from as many patients with either of these conditions were tested against HSA-coupled, uridine-containing monophosphoric dinucleotides in an attempt to characterize further their anti-RNA antibodies. Scleroderma sera were found to react primarily with dinucleotides in which uridine was the base proximal to the carrier protein and, except for sera that also contained antibodies to adenosine which reacted with UpA, they failed to react with dinucleotides in which uridine was in a terminal position only. Reaction with dinucleotides in which uridine was proximal to the carrier protein could be inhibited by uracil but not by the corresponding terminal base. Some lupus sera were found to react with both dinucleotides that contain the same bases in opposite sequence, e.g. ApU and UpA, while others were found to react with only one of the sequences. They were also found to react more frequently with dinucleotides in which HSA was coupled to a base other than uridine, suggesting that the reaction is primarily due to anti-DNA antibodies. Because immunization with dinucleotides coupled to protein prepared by the same method we have used, yields higher specificity to the base attached to the carrier protein, our findings suggest that, in scleroderma, a single event, akin to that of immunization with a purified antigen, gives rise to the anti-RNA antibodies, whereas in systemic lupus erythematosus there is a considerably wider immunological aberration. PMID:1082854

Atmospheric parameters and magnesium and calcium NLTE abundances for a sample of 16 ultra metal-poor stars

NASA Astrophysics Data System (ADS)

Sitnova, Tatyana; Mashonkina, Lyudmila; Ezzeddine, Rana; Frebel, Anna

2018-06-01

The most metal-poor stars provide important observational clues to the astrophysical objects that enriched the primordial gas with heavy elements. Accurate atmospheric parameters is a prerequisite of determination of accurate abundances. We present atmospheric parameters and abundances of calcium and magnesium for a sample of 16 ultra-metal poor (UMP) stars. In spectra of UMP stars, iron is represented only by lines of Fe I, while calcium is represented with lines of Ca I and Ca II, which can be used for determination/checking of effective temperature and surface gravity. Accurate calculations of synthetic spectra of UMP stars require non-local thermodynamic equilibrium (NLTE) treatment of line formation, since deviations from LTE grow with metallicity decreasing. The method of atmospheric parameter determination is based on NLTE analysis of lines of Ca I and Ca II, multi-band photometry, and isochrones. The method was tested in advance with the ultra metal-poor giant CD-38 245, where, in addition, trigonometric parallax measurements from Gaia DR1 and lines of Fe I and Fe II are available. Using photometric Teff = 4900 K and distance based log g = 2.0 for CD-38 245, we derived consistent within error bars NLTE abundances from Fe I and Fe II and Ca I and Ca II, while LTE leads to a discrepancy of 0.6 dex between Ca I and Ca II. We determined NLTE and LTE abundances of magnesium and calcium in 16 stars of the sample. For the majority of stars, as expected, [Ca/Mg] NLTE abundance ratios are close to 0, while LTE leads to systematically higher [Ca/Mg], by up to 0.3 dex, and larger spread of [Ca/Mg] for different stars. Three stars of our sample are strongly enhanced in magnesium, with [Mg/Ca] of 1.3 dex. It is worth noting that, for these three stars, we got very similar [Mg/Ca] of 1.30, 1.45, and 1.29, in contrast to the data from the literature, where, for the same stars, [Mg/Ca] vary from 0.7 to 1.4. Very similar [Mg/Ca] abundance ratios of these stars argue that their abundances originate from a similar nucleosynthetic event.

Chemical models and their mechanistic implications for the transformation of 6-cyanouridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase.

PubMed

Wu, Yuen-Jen; Liao, Chen-Chieh; Jen, Cheng-Hung; Shih, Yu-Chiao; Chien, Tun-Cheng

2010-07-14

The reactions of 6-cyano-1,3-dimethyluracil have been studied as chemical models to illustrate the mechanism for the transformation of 6-cyanouridine 5'-monophosphate (6-CN-UMP) to barbiturate ribonucleoside 5'-monophosphate (BMP) catalyzed by orotidine 5'-monophosphate decarboxylase (ODCase). The results suggest that the Asp residue in the ODCase active site plays the role of a general base in the transformation.

Automatic rendezvous and docking systems functional and performance requirements

NASA Technical Reports Server (NTRS)

1985-01-01

A generalized mission design scheme which utilizes a standard mission profile for all OMV rendezvous operations, recognizes typical operational constraints, and minimizes propellant penalties due to nodal regression effects was developed. This scheme has been used to demonstrate a unified guidance and navigation maneuver processor (the UMP), which supports all mission phases through station-keeping. The initial demonstration version of the Orbital Rendezvous Mission Planner (ORMP) was provided for evaluation purposes, and program operation was discussed.

Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells

PubMed Central

Guo, Jianru; Chen, QianQian; Lam, Christopher Wai Kei; Wang, Caiyun; Wong, Vincent Kam Wai; Xu, Fengguo; Jiang, ZhiHong; Zhang, Wei

2015-01-01

Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance. PMID:26578061

Evolution of Dust in Primordial Supernova Remnants and Its Influence on the Elemental Composition of Hyper-Metal-Poor Stars

NASA Astrophysics Data System (ADS)

Nozawa, Takaya; Kozasa, Takashi; Habe, Asao; Dwek, Eli; Umeda, Hideyuki; Tominaga, Nozomu; Maeda, Keiichi; Nomoto, Ken'ichi

2008-05-01

The calculations for the evolution of dust within Population III supernova remnants (SNRs) are presented, based on the models of dust formed in the unmixed ejecta of Type II SNe. We show that once dust grains collide with the reverse shock penetrating into the ejecta, their fates strongly depend on the initial radius aini. For SNRs expanding into the interstellar medium (ISM) with nH,0 = 1 cm-3, grains of aini<0.05 μm are trapped in the hot gas to be completely destroyed; grains of aini = 0.05-0.2 μm are piled up in the dense shell formed behind the forward shock; grains of aini>0.2 μm are injected into the ISM without being eroded significantly. The total mass of surviving dust is 0.01 to 0.8 Msolar for nH,0 = 10 to 0.1 cm-3. We also investigate the influence of the piled-up dust on the elemental abundances of the second-generation stars formed in the dense shell of Population III SNRs. The comparison of the calculated elemental abundances with those observed in hyper-metal-poor (HMP) and ultra-metal-poor (UMP) stars indicates that the transport of dust separated from metal-rich gas can be an important process in determining the abundance patterns of Mg and Si in HMP and UMP stars.

Aerospace Medicine and Biology. A Continuing Bibliography with Indexes

DTIC Science & Technology

1987-12-01

GLYCERALDEHYDE AS A SOURCE OF SIMULATION ABOUT THE ORIGIN OF LIFE ENERGY AND MONOMERS FOR PREBIOTIC CONDENSATION URSULA NIESERT (Freiburg, Universitaet, Freiburg...primitive prebiotic replication system. The adsorption of Poly-C, Poly-U, Poly-A, Poly-G, and 5’-AMP, 5’-GMP, 5’-CMP and 5’-UMP onto gypsum was studied. It...Cambridge University Press. 1986. 378 p. For individual titles see A87-49035 to A87-49049. Topics discussed include prebiotic systems and evolutionary

Tm:germanate Fiber Laser: Tuning And Q-switching

NASA Technical Reports Server (NTRS)

Barnes, Norman P.; Walsh, Brian M.; Reichle, Donald J.; DeYoung, R. J.; Jiang, Shibin

2007-01-01

A Tm:germanate fiber laser produced >0.25 mJ/pulse in a 45 ns pulse. It is capable of producing multiple Q-switched pulses from a single p ump pulse. With the addition of a diffraction grating, Tm:germanate f iber lasers produced a wide, but length dependent, tuning range. By s electing the fiber length, the tuning range extends from 1.88 to 2.04 ?m. These traits make Tm:germanate lasers suitable for remote sensin g of water vapor.

Development of a Gene Cloning System in Methanogens

DTIC Science & Technology

1988-07-01

ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION fr (if aplicable ) University of Oklahoma NA Office of Naval Research , ADDRESS...sulfanilamide and folate analogues on the growth of Methanomicrobium mobile . Abstr. Ann Meeting Amer. Soc. Microbiol. 1 34, p. 178. Nagle, D.P., Jr., V.E...we obtained UMP syn- proof that the resistance of the strain was not due to a -t thesis levels 2 to 5 times higher than those shown in ’able 2

Improvements on ν-Twin Support Vector Machine.

PubMed

Khemchandani, Reshma; Saigal, Pooja; Chandra, Suresh

2016-07-01

In this paper, we propose two novel binary classifiers termed as "Improvements on ν-Twin Support Vector Machine: Iν-TWSVM and Iν-TWSVM (Fast)" that are motivated by ν-Twin Support Vector Machine (ν-TWSVM). Similar to ν-TWSVM, Iν-TWSVM determines two nonparallel hyperplanes such that they are closer to their respective classes and are at least ρ distance away from the other class. The significant advantage of Iν-TWSVM over ν-TWSVM is that Iν-TWSVM solves one smaller-sized Quadratic Programming Problem (QPP) and one Unconstrained Minimization Problem (UMP); as compared to solving two related QPPs in ν-TWSVM. Further, Iν-TWSVM (Fast) avoids solving a smaller sized QPP and transforms it as a unimodal function, which can be solved using line search methods and similar to Iν-TWSVM, the other problem is solved as a UMP. Due to their novel formulation, the proposed classifiers are faster than ν-TWSVM and have comparable generalization ability. Iν-TWSVM also implements structural risk minimization (SRM) principle by introducing a regularization term, along with minimizing the empirical risk. The other properties of Iν-TWSVM, related to support vectors (SVs), are similar to that of ν-TWSVM. To test the efficacy of the proposed method, experiments have been conducted on a wide range of UCI and a skewed variation of NDC datasets. We have also given the application of Iν-TWSVM as a binary classifier for pixel classification of color images. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ergonomics Risk Assessment among support staff in Universiti Malaysia Pahang

NASA Astrophysics Data System (ADS)

Jusoh, Faisal; Nafis Osman Zahid, Muhammed

2018-03-01

Awareness of ergonomic risk assessment among workers are getting intense in many industries nowadays. It is essential since most of the workers spend 7 to 8 hours of their time in the workplaces. Previous study shown that spending too much time with static posture in sitting at workplace leads to the problem of Musculoskeletal Disorders (MSDs). The implications are not only harmful to human body but also effect the productivity. Currently, there are no scientific study conducted to assess the conditions of workers in Universiti Malaysia Pahang (UMP). Therefore, the problem of MSDs could not be justified clearly and the top management did not acknowledge this issue. This study aims to present current scenario of ergonomic risk level at UMP by using structured model. It focuses on operational staff from faculties and Human Resources Department (HRD). Initially, three types of assessments are executed based on general working condition, Cornell Muscokeletal Discomfort Questionnaire (CMDQ) and Rapid Office Strain Assessment (ROSA). Based on the findings, 90% of the respondents felt discomfort at workplace but prefer to rectify the issues by themselves. Almost 50% of them evaluated themselves in level 4-5 of discomfort level. The CMDQ result shown the discomfort area at faculties and HRD. The workplace at faculties and HRD had been assessed through ROSA and the overall result shown the risk level is medium level respectively. Therefore, further investigation is requires and improvement of workplace need to be proposed to establish good working condition.

Experimental study of the vehicle dynamics behavior during lane changing in different speeds

NASA Astrophysics Data System (ADS)

Heerwan, P. M.; Asyraf, S. M.; Efistein, A. N.; Seah, C. H.; Zikri, J. M.; Syawahieda, J. N.

2017-10-01

During lane changing, the speed of the vehicle is related to the stability of the vehicle. If the driver changes the lane at a high speed, the vehicle will lose its stability and it can increase the possibility of an accident. In this study, the experiment has been developed to analyse how the speed of the vehicle can affect the vehicle dynamics behavior. To achieve this objective, the UMP Test Car which employed with global positioning system (GPS), steering torque and angle sensor, displacement sensor and gyro sensor is used in the experiment. The experiment is run at the UMP test track and the track has 2 lanes which can allows the vehicle to change the position from the left to the right. In the experiment, when the GPS monitor shows 30 km/h, the driver will maintain the speed and start to turn the steering just after the test car reaches to the first skittle. Then, the driver will turn again the steering when the test car reaches to the second skittle. This method is repeated two times and the same methods is used for the speed 50 km/h. The data from the sensors is recorded in the Dewetron software and the graph is plotted. From the experimental results, the steering angle, steering torque, yaw rate and displacement for the speed 30 km/h is smaller than 50 km/h. It means that during lane changing, the speed 30 km/h is more stable compared with 50 km/h.

Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

PubMed

McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

2017-07-07

NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Welcoming speech from Dean Faculty of Mechanical Engineering, UMP

NASA Astrophysics Data System (ADS)

Taha, Zahari

2012-09-01

In the Name of Allah, the Most Beneficent, the Most Merciful. It is with great pleasure that I welcome the participants of the International Conference of Mechanical Engineering Research 2011. The Prophet Muhammad (peace be upon him) said 'Acquire knowledge and impart it to the people.' (Al Tirmidhi). The quest for knowledge has been from the beginning of time but knowledge only becomes valuable when it is disseminated and applied to benefit humankind. It is hoped that ICMER 2011 will be a platform to gather and disseminate the latest knowledge in mechanical engineering. Academicians, Scientist, Researchers and practitioners of mechanical engineering will be able to share and discuss new findings and applications of mechanical engineering. It is envisaged that the intellectual discourse will result in future collaborations between universities, research institutions and industry both locally and internationally. In particular it is expected that focus will be given to issues on environmental and energy sustainability. Researchers in the mechanical engineering faculty at UMP have a keen interest in technology to harness energy from the ocean. Lowering vehicle emissions has been a primary goal of researchers in the mechanical engineering faculty and the automotive engineering centre as well including developing vehicles using alternative fuels such as biodiesel and renewable sources such as solar driven electric vehicles. Finally I would like to congratulate the organizing committee for their tremendous efforts in organizing the conference. As I wrote this in the Holy Land of Makkah, I pray to Allah swt that the conference will be a success. Prof. Dr. Zahari Taha CEng, MIED, FASc Dean, Faculty of Mechanical Engineering Universiti Malaysia Pahang

Real-time individualization of the unified model of performance.

PubMed

Liu, Jianbo; Ramakrishnan, Sridhar; Laxminarayan, Srinivas; Balkin, Thomas J; Reifman, Jaques

2017-12-01

Existing mathematical models for predicting neurobehavioural performance are not suited for mobile computing platforms because they cannot adapt model parameters automatically in real time to reflect individual differences in the effects of sleep loss. We used an extended Kalman filter to develop a computationally efficient algorithm that continually adapts the parameters of the recently developed Unified Model of Performance (UMP) to an individual. The algorithm accomplishes this in real time as new performance data for the individual become available. We assessed the algorithm's performance by simulating real-time model individualization for 18 subjects subjected to 64 h of total sleep deprivation (TSD) and 7 days of chronic sleep restriction (CSR) with 3 h of time in bed per night, using psychomotor vigilance task (PVT) data collected every 2 h during wakefulness. This UMP individualization process produced parameter estimates that progressively approached the solution produced by a post-hoc fitting of model parameters using all data. The minimum number of PVT measurements needed to individualize the model parameters depended upon the type of sleep-loss challenge, with ~30 required for TSD and ~70 for CSR. However, model individualization depended upon the overall duration of data collection, yielding increasingly accurate model parameters with greater number of days. Interestingly, reducing the PVT sampling frequency by a factor of two did not notably hamper model individualization. The proposed algorithm facilitates real-time learning of an individual's trait-like responses to sleep loss and enables the development of individualized performance prediction models for use in a mobile computing platform. © 2017 European Sleep Research Society.

Hazardous Chemical Pump Tests.

DTIC Science & Technology

1980-07-01

hydraulic flow rate is the product of the pump speed and the pump displacement. The pump displacement for each respective pump was constant throughout...speed - rpm T - torque - ft lbs 7= 3.1416 By substituting the product of pump speed and pump displacement for the hydraulic flow rate (Q=NO) in the above...FF:iipr’: iL 40 H FLUID F-’UMPED; FPl H FVIi T’E1l ’HJO I...S Lu FL: H KFITE C F~~:ri FIGURE 2 CC E MT 2, Fi C F . c ;E’C F11 *:;_cl PF fog O ~ \\ 4 1

Density functional Gaussian-type-orbital approach to theoretical study of nitric oxide dimers

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

Jursic, B.S.; Zdravkovski, Z.

Structure and total energies of the cis NO dimer, the trans NO dimer, and the NO monomer were calculated by ab initio methods (UHF, UMP2, and MP3) and density functional theory methods (LSDA and BLYP) with different basis sets [from 3-21G* to 6-311++(3df,3pd)]. The system is especially hard to model because two NO molecules are weakly associated in a dimer that has very long N-N bond. The results obtained by different methods are compared and the necessity of correlational methods for studying these systems is discussed.

In vitro synthesis of intermediates involved in the assembly of enterobacterial common antigen (ECA)

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

Barr, K.; Wolski, S.; Kroto, J.

1986-05-01

ECA is a cell surface antigen found in all bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of trisaccharide repeat units; the component sugars are N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-D-fucose (Fuc4NAc). In vivo studies have suggested that GlcNAc-pyrophosphorylundecaprenol (GlcNAc-PP-lipid) is an intermediate in ECA synthesis. More recently, they have demonstrated UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase activity in cell envelope preparations of E. coli. Radioactivity from UDP-(/sup 3/H)Glc-NAc was incorporated into endogenous lipid acceptor, and the labeled product was characterized as GlcNAc-PP-lipid (lipid I). Transferase activity was inhibited by tunicamycin andmore » UMP, but it was unaffected by UDP. The reaction was reversible, and the synthesis of UDP-(/sup 3/H)GlcNAc from UMP and (/sup 3/H)GlcNAc-PP-lipid was also sensitive to tunicamycin. The simultaneous addition of UDP-(/sup 14/C)ManNAcUA and UDP-(/sup 3/H)GlcNAc to cell envelope preparations resulted in the synthesis of a more polar lipid (lipid II) that contained both labeled sugars in equimolar amounts. Synthesis of lipid II was dependent on prior synthesis of lipid I. Accordingly, (/sup 3/H)GlcNAc-PP-lipid that had been synthesized in vivo served as an acceptor in vitro of ManNAcUA residues from UDP-ManNAcUA. Lipid II has been tentatively identified as ManNAcUA-GlcNAc-pyrophosphorylundecaprenol.« less

The Biosynthesis of Capuramycin-type Antibiotics

PubMed Central

Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D.; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J.; Spork, Anatol P.; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S.; Van Lanen, Steven G.

2015-01-01

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5′-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5′-aldehyde transaldolase were uncovered, suggesting that C–C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5′-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. PMID:25855790

Photodetachment of Zwitterions: Probing Intramolecular Coulomb Repulsion and Attraction in the Gas Phase Using Mono- Decarboxylated Pyridinium Dicarboxylates. Implications on the Mechanism of Orotidine 5' -Monophosphate Decarboxylase

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

Wang, Xue B.; Dacres, J E.; Yang, Xin

2003-11-11

Negative ion photoelectron spectra resulting from the decarboxylation of nine zwitterionic pyridinium dicarboxylates (D(x,y)) are reported. Structural assignments are made on the basis of analogy to the spectra of related species, labeling experiments with C-13- or H-2-containing substrates, independent syntheses, and comparison to density functional theory and ab initio (B3LYP and CCSD(T), respectively) results. In some cases, an acid-catalyzed isomerization of the D(xy)-CO2 ions was found to take place. Adiabatic detachment energies of the resulting zwitterionic ions were measured and are well reproduced by theory. The relative stabilities of the D(x,Y)- CO2 decarboxylation products are largely determined by their intramolecularmore » electrostatic interactions, which are directly probed by the photoelectron spectra and were analyzed in terms of the resulting Coulombic forces. Expulsion of carbon dioxide from the D(x,y) ions was also used as an electrostatic model to probe the mechanism of the enzyme-catalyzed conversion of orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate (UMP). It was found that the loss of CO2 from these zwitterions; and from oxygen-protonated OMP is retarded by the presence of an additional anionic group. This suggests that the formation of a zwitterion intermediate in the enzyme-catalyzed transformation of OMP to UMP may have less of an energetic impact than commonly thought and could be a''red herring''. If so, the electrostatic stress mechanism proposed by Larsen et al. and Pai, Guo, and co-workers maybe followed.« less

Attachment, Symptom Severity, and Depression in Medically Unexplained Musculoskeletal Pain and Osteoarthritis: A Cross-Sectional Study

PubMed Central

Schroeter, Corinna; Ehrenthal, Johannes C.; Giulini, Martina; Neubauer, Eva; Gantz, Simone; Amelung, Dorothee; Balke, Doreen; Schiltenwolf, Marcus

2015-01-01

Background Attachment insecurity relates to the onset and course of chronic pain via dysfunctional reactions to pain. However, few studies have investigated the proportion of insecure attachment styles in different pain conditions, and results regarding associations between attachment, pain severity, and disability in chronic pain are inconsistent. This study aims to clarify the relationships between insecure attachment and occurrence or severity of chronic pain with and without clearly defined organic cause. To detect potential differences in the importance of global and romantic attachment representations, we included both concepts in our study. Methods 85 patients with medically unexplained musculoskeletal pain (UMP) and 89 patients with joint pain from osteoarthritis (OA) completed self-report measures of global and romantic attachment, pain intensity, physical functioning, and depression. Results Patients reporting global insecure attachment representations were more likely to suffer from medically unexplained musculoskeletal pain (OR 3.4), compared to securely attached patients. Romantic attachment did not differ between pain conditions. Pain intensity was associated with romantic attachment anxiety, and this relationship was more pronounced in the OA group compared to the UMP group. Both global and romantic attachment anxiety predicted depression, accounting for 15% and 17% of the variance, respectively. Disability was independent from attachment patterns. Conclusions Our results indicate that global insecure attachment is associated with the experience of medically unexplained musculoskeletal pain, but not with osteoarthritis. In contrast, insecure attachment patterns seem to be linked to pain intensity and pain-related depression in unexplained musculoskeletal pain and in osteoarthritis. These findings suggest that relationship-informed focused treatment strategies may alleviate pain severity and psychological distress in chronic pain independent of underlying pathology. PMID:25807172

The General Base in the Thymidylate Synthase Catalyzed Proton Abstraction

PubMed Central

Ghosh, Ananda K.; Islam, Zahidul; Krueger, Jonathan; Abeysinghe, Don Thelma; Kohen, Amnon

2015-01-01

The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2′-deoxythymidine-5′-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2′-deoxyuridine-5′-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic –OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton. PMID:25912171

EXEIS - Expert Screening and Optimal Extraction/Injection Pumping Systems for Short-Term Plume Immobilization

DTIC Science & Technology

1990-05-01

i at time t, (L). hL lower limit on head at pump i, (L). i xviii hU upper limit on head at -’ump i, (L).i (h j ,TT) d head at observation well j which...constraints: L U hL U h .. (9) h. ih. i ...I hi,t 1 S(hj Q........................(10) J ho ,TT - (h ,TT ) d . . . . .. . . . . . .O where: I = total number...at pump i at time period t, (L); = hi, 0 -s i,t hU = upper limit on head at pump i, (L); 1 (hTT ) d = head at each observation well j which is down

Chapter 2: Commercial and Industrial Lighting Evaluation Protocol. The Uniform Methods Project: Methods for Determining Energy Efficiency Savings for Specific Measures

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

Kurnik, Charles W; Gowans, Dakers; Telarico, Chad

The Commercial and Industrial Lighting Evaluation Protocol (the protocol) describes methods to account for gross energy savings resulting from the programmatic installation of efficient lighting equipment in large populations of commercial, industrial, and other nonresidential facilities. This protocol does not address savings resulting from changes in codes and standards, or from education and training activities. A separate Uniform Methods Project (UMP) protocol, Chapter 3: Commercial and Industrial Lighting Controls Evaluation Protocol, addresses methods for evaluating savings resulting from lighting control measures such as adding time clocks, tuning energy management system commands, and adding occupancy sensors.

Uridylylation of the PII protein from Herbaspirillum seropedicae.

PubMed

Benelli, E M; Buck, M; de Souza, E M; Yates, M G; Pedrosa, F O

2001-04-01

The PII protein is apparently involved in the control of NifA activity in Herbaspirillum seropedicae. To evaluate the probable role of PII in signal transduction, uridylylation assays were conducted with purified H. seropedicae PII and Escherichia coli GlnD, or a cell-free extract of H. seropedicae as sources of uridylylating activity. The results showed that alpha-ketoglutarate and ATP stimulate uridylylation whereas glutamine inhibits uridylylation. Deuridylylation of PII-UMP was dependent on glutamine and inhibited by ATP and alpha-ketoglutarate. PII uridylylation and (or) deuridylylation in response to these effectors suggests that PII is a nitrogen level signal transducer in H. seropedicae.

On the Observation of Discrete Fluorine NMR Spectra for Uridine 5′-β,γ-Fluoromethylenetriphosphate Diastereomers at Basic pH

PubMed Central

2015-01-01

Jakeman et al. recently reported the inability to distinguish the diastereomers of uridine 5′-β,γ-fluoromethylenetriphosphate (β,γ-CHF-UTP, 1) by 19F NMR under conditions we previously prescribed for the resolution of the corresponding β,γ-CHF-dGTP spectra, stating further that 1 decomposed under these basic conditions. Here we show that the 19F NMR spectra of 1 (∼1:1 diastereomer mixture prepared by coupling of UMP-morpholidate with fluoromethylenebis(phosphonic acid)) in D2O at pH 10 are indeed readily distinguishable. 1 in this solution was stable for 24 h at rt. PMID:24819695

An efficient parallel algorithm for the calculation of unrestricted canonical MP2 energies.

PubMed

Baker, Jon; Wolinski, Krzysztof

2011-11-30

We present details of our efficient implementation of full accuracy unrestricted open-shell second-order canonical Møller-Plesset (MP2) energies, both serial and parallel. The algorithm is based on our previous restricted closed-shell MP2 code using the Saebo-Almlöf direct integral transformation. Depending on system details, UMP2 energies take from less than 1.5 to about 3.0 times as long as a closed-shell RMP2 energy on a similar system using the same algorithm. Several examples are given including timings for some large stable radicals with 90+ atoms and over 3600 basis functions. Copyright © 2011 Wiley Periodicals, Inc.

Aspartate Carbamyltransferase : Site of End-Product Inhibition of the Orotate Pathway in Intact Cells of Cucurbita pepo.

PubMed

Lovatt, C J; Cheng, A H

1984-07-01

Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH(14)CO(3), but not [(14)C]carbamylaspartate or [(14)C]orotic acid, into uridine nucleotides (SigmaUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH(14)CO(3) into SigmaUMP by 80% but did not inhibit the incorporation of NaH(14)CO(3) into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH(14)CO(3) into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH(14)CO(3) into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides.

The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

PubMed

Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J; Spork, Anatol P; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S; Van Lanen, Steven G

2015-05-29

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Multifunctional phosphate-based inorganic-organic hybrid nanoparticles.

PubMed

Heck, Joachim G; Napp, Joanna; Simonato, Sara; Möllmer, Jens; Lange, Marcus; Reichardt, Holger M; Staudt, Reiner; Alves, Frauke; Feldmann, Claus

2015-06-17

Phosphate-based inorganic-organic hybrid nanoparticles (IOH-NPs) with the general composition [M](2+)[Rfunction(O)PO3](2-) (M = ZrO, Mg2O; R = functional organic group) show multipurpose and multifunctional properties. If [Rfunction(O)PO3](2-) is a fluorescent dye anion ([RdyeOPO3](2-)), the IOH-NPs show blue, green, red, and near-infrared fluorescence. This is shown for [ZrO](2+)[PUP](2-), [ZrO](2+)[MFP](2-), [ZrO](2+)[RRP](2-), and [ZrO](2+)[DUT](2-) (PUP = phenylumbelliferon phosphate, MFP = methylfluorescein phosphate, RRP = resorufin phosphate, DUT = Dyomics-647 uridine triphosphate). With pharmaceutical agents as functional anions ([RdrugOPO3](2-)), drug transport and release of anti-inflammatory ([ZrO](2+)[BMP](2-)) and antitumor agents ([ZrO](2+)[FdUMP](2-)) with an up to 80% load of active drug is possible (BMP = betamethason phosphate, FdUMP = 5'-fluoro-2'-deoxyuridine 5'-monophosphate). A combination of fluorescent dye and drug anions is possible as well and shown for [ZrO](2+)[BMP](2-)0.996[DUT](2-)0.004. Merging of functional anions, in general, results in [ZrO](2+)([RdrugOPO3]1-x[RdyeOPO3]x)(2-) nanoparticles and is highly relevant for theranostics. Amine-based functional anions in [MgO](2+)[RaminePO3](2-) IOH-NPs, finally, show CO2 sorption (up to 180 mg g(-1)) and can be used for CO2/N2 separation (selectivity up to α = 23). This includes aminomethyl phosphonate [AMP](2-), 1-aminoethyl phosphonate [1AEP](2-), 2-aminoethyl phosphonate [2AEP](2-), aminopropyl phosphonate [APP](2-), and aminobutyl phosphonate [ABP](2-). All [M](2+)[Rfunction(O)PO3](2-) IOH-NPs are prepared via noncomplex synthesis in water, which facilitates practical handling and which is optimal for biomedical application. In sum, all IOH-NPs have very similar chemical compositions but can address a variety of different functions, including fluorescence, drug delivery, and CO2 sorption.

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

PubMed Central

Beitlich, Thorsten; Lorenz, Thorsten; Reinstein, Jochen

2013-01-01

The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology. PMID:24205218

Hydrogen bonding in the neutron structure of the mononucleotide 5'-UMP disodium salt

NASA Astrophysics Data System (ADS)

Chitra, R.; Ranjan-Choudhury, R.; Ramanadham, M.

Disodium uridine 5'-monophosphate heptahydrate (5'-UMPNa2), Na2[C9H11N2O9P].7H2O, crystallises in space group C2221 with a=22.985, b=8.911 and c=19.494Å. A neutron beam of λ=1.216Å was used; Z=8 and V=3992.75Å3. Data consisted of 1785 unique reflections. Na ions were connected to the main molecule through water molecules and sugar oxygens. One of the Na ions occupied a special position, and the other at a general position was partially disordered. The uracil base was planar, and had anti conformation about the glycosidic bond. The sugar had C(2') endo conformation and was gauche-gauche.

Profile of nucleosides and nucleotides in donkey's milk.

PubMed

Vincenzetti, Silvia; Pucciarelli, Stefania; Nucci, Chiara; Polzonetti, Valeria; Cammertoni, Natalina; Polidori, Paolo

2014-01-01

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkey's milk may be considered a good replacer for cow's milk in feeding children with severe Ig-E mediated cow's milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.

Some considerations about Gaussian basis sets for electric property calculations

NASA Astrophysics Data System (ADS)

Arruda, Priscilla M.; Canal Neto, A.; Jorge, F. E.

Recently, segmented contracted basis sets of double, triple, and quadruple zeta valence quality plus polarization functions (XZP, X = D, T, and Q, respectively) for the atoms from H to Ar were reported. In this work, with the objective of having a better description of polarizabilities, the QZP set was augmented with diffuse (s and p symmetries) and polarization (p, d, f, and g symmetries) functions that were chosen to maximize the mean dipole polarizability at the UHF and UMP2 levels, respectively. At the HF and B3LYP levels of theory, electric dipole moment and static polarizability for a sample of molecules were evaluated. Comparison with experimental data and results obtained with a similar size basis set, whose diffuse functions were optimized for the ground state energy of the anion, was done.

Structural Dynamics of Picornaviral RdRP Complexes. Implications for the Design of Antivirals

NASA Astrophysics Data System (ADS)

Verdaguer, Núria; Ferrer-Orta, Cristina; Domingo, Esteban

Genome replication in picornavirus is catalyzed by a virally encoded RNA dependent RNA polymerase, termed 3D. These viruses also use a small protein primer, named VPg to initiate RNA replication. Polymerase 3D also catalyzes the covalent linkage of UMP to a N-terminal tyrosine on VPg. Seven different crystal structures of foot-and-mouth disease virus (FMDV) 3D catalytic complexes have enhanced our understanding of template and primer recognition, VPg uridylylation and rNTP binding and catalysis. In addition, the biochemical and structural analyses of six different FMDV 3D ribavirin resistant mutants provided evidences of three different mechanisms of resistance to this mutagenic nucleoside analogue. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds.

Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.

PubMed

Brooks, A I; Halterman, M W; Chadwick, C A; Davidson, B L; Haak-Frendscho, M; Radel, C; Porter, C; Federoff, H J

1998-04-30

To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 microl over 10 min. of either HSVlac (1 x 10(5) b.f.u), AdLac (1 x 10(5) p.f.u), Tfx-10 or Tfx-20 (2.6 microg DNA in 2.0 microl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 microg/microl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of beta-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect beta-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of beta-gal in HSVlac infected animals (p = 0.0029); a 29% increase in Adlac infected animals (p = 0.01); a 56% increase in Tfx-10 transduced animals (p = 0.04); a 24% increase in Tfx-20 transduced animals (p = 0.01); and a 69% increase in naked DNA gene transfer (p = 0.05). Total beta-galactosidase activity was greatest in HSVlac infected mice followed by Adlac > Tfx-20 > Tfx-10 = naked DNA.

PREFACE: 3rd International Conference of Mechanical Engineering Research (ICMER 2015)

NASA Astrophysics Data System (ADS)

Mamat, Riazalman; Rahman, Mustafizur; Mohd. Zuki Nik Mohamed, Nik; Che Ghani, Saiful Anwar; Harun, Wan Sharuzi Wan

2015-12-01

The 3rd ICMER2015 is the continuity of the NCMER2010. The year 2010 represents a significant milestone in the history for Faculty of Mechanical Engineering, Universiti Malaysia Pahang (UMP) Malaysia with the organization of the first and second national level conferences (1st and 2nd NCMER) at UMP on May 26-27 and Dec 3-4 2010. The Faculty then changed the name from National Conference on Mechanical Engineering Research (NCMER) to International Conference on Mechanical Engineering Research (ICMER) in 2011 and this year, 2015 is our 3rd ICMER. These proceedings contain the selected scientific manuscripts submitted to the conference. It is with great pleasure to welcome you to the "International Conference on Mechanical Engineering Research (ICMER2015)" that is held at Zenith Hotel, Kuantan, Malaysia. The call for papers attracted submissions of over two hundred abstracts from twelve different countries including Japan, Iran, China, Kuwait, Indonesia, Norway, Philippines, Morocco, Germany, UAE and more. The scientific papers published in these proceedings have been revised and approved by the technical committee of the 3rd ICMER2015. All of the papers exhibit clear, concise, and precise expositions that appeal to a broad international readership interested in mechanical engineering, combustion, metallurgy, materials science as well as in manufacturing and biomechanics. The reports present original ideas or results of general significance supported by clear reasoning and compelling evidence, and employ methods, theories and practices relevant to the research. The authors clearly state the questions and the significance of their research to theory and practice, describe how the research contributes to new knowledge, and provide tables and figures that meaningfully add to the narrative. In this edition of ICMER representatives attending are from academia, industry, governmental and private sectors. The plenary and invited speakers will present, discuss, promote and disseminate research in all fields of mechanical engineering. Topics cover synthesis, applications, and fundamental studies of the topics related to mechanical engineering. In addition, booths for industries to showcase their state-of-the-art products are also provided. The organizing committee of the conference thanks all the participants for their fruitful work and personal contribution to the development of these conference proceedings.

Effects of Specific Multi-Nutrient Enriched Diets on Cerebral Metabolism, Cognition and Neuropathology in AβPPswe-PS1dE9 Mice

PubMed Central

Jansen, Diane; Zerbi, Valerio; Arnoldussen, Ilse A. C.; Wiesmann, Maximilian; Rijpma, Anne; Fang, Xiaotian T.; Dederen, Pieter J.; Mutsaers, Martina P. C.; Broersen, Laus M.; Lütjohann, Dieter; Miller, Malgorzata; Joosten, Leo A. B.; Heerschap, Arend; Kiliaan, Amanda J.

2013-01-01

Recent studies have focused on the use of multi-nutrient dietary interventions in search of alternatives for the treatment and prevention of Alzheimer's disease (AD). In this study we investigated to which extent long-term consumption of two specific multi-nutrient diets can modulate AD-related etiopathogenic mechanisms and behavior in 11-12-month-old AβPPswe-PS1dE9 mice. Starting from 2 months of age, male AβPP-PS1 mice and wild-type littermates were fed either a control diet, the DHA+EPA+UMP (DEU) diet enriched with uridine monophosphate (UMP) and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), or the Fortasyn® Connect (FC) diet enriched with the DEU diet plus phospholipids, choline, folic acid, vitamins and antioxidants. We performed behavioral testing, proton magnetic resonance spectroscopy, immunohistochemistry, biochemical analyses and quantitative real-time PCR to gain a better understanding of the potential mechanisms by which these multi-nutrient diets exert protective properties against AD. Our results show that both diets were equally effective in changing brain fatty acid and cholesterol profiles. However, the diets differentially affected AD-related pathologies and behavioral measures, suggesting that the effectiveness of specific nutrients may depend on the dietary context in which they are provided. The FC diet was more effective than the DEU diet in counteracting neurodegenerative aspects of AD and enhancing processes involved in neuronal maintenance and repair. Both diets elevated interleukin-1β mRNA levels in AβPP-PS1 and wild-type mice. The FC diet additionally restored neurogenesis in AβPP-PS1 mice, decreased hippocampal levels of unbound choline-containing compounds in wild-type and AβPP-PS1 animals, suggesting diminished membrane turnover, and decreased anxiety-related behavior in the open field behavior. In conclusion, the current data indicate that specific multi-nutrient diets can influence AD-related etiopathogenic processes. Intervention with the FC diet might be of interest for several other neurodegenerative and neurological disorders. PMID:24086523

Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability .

PubMed

Huang, Xiao; Gibson, Lydia M; Bell, Brittnaie J; Lovelace, Leslie L; Peña, Maria Marjorette O; Berger, Franklin G; Berger, Sondra H; Lebioda, Lukasz

2010-03-23

Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of approximately 27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K(m,app) values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F.FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K(m,app) value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K(m,app) values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

Analytic energy gradients in combined second order Møller-Plesset perturbation theory and conductorlike polarizable continuum model calculation.

PubMed

Si, Dejun; Li, Hui

2011-10-14

The analytic energy gradients in combined second order Møller-Plesset perturbation theory and conductorlike polarizable continuum model calculations are derived and implemented for spin-restricted closed shell (RMP2), Z-averaged spin-restricted open shell (ZAPT2), and spin-unrestricted open shell (UMP2) cases. Using these methods, the geometries of the S(0) ground state and the T(1) state of three nucleobase pairs (guanine-cytosine, adenine-thymine, and adenine-uracil) in the gas phase and aqueous solution phase are optimized. It is found that in both the gas phase and the aqueous solution phase the hydrogen bonds in the T(1) state pairs are weakened by ~1 kcal/mol as compared to those in the S(0) state pairs. © 2011 American Institute of Physics

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique

PubMed Central

Zhang, Wei; Tan, Shenglan; Paintsil, Elijah; Dutschman, Ginger E.; Gullen, Elizabeth A.; Chu, Edward; Cheng, Yung-Chi

2011-01-01

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC-1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies. PMID:21620803

Chapter 1: Introduction. The Uniform Methods Project: Methods for Determining Energy-Efficiency Savings for Specific Measures

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

Li, Michael; Haeri, Hossein; Reynolds, Arlis

This chapter provides a set of model protocols for determining energy and demand savings that result from specific energy efficiency measures implemented through state and utility efficiency programs. The methods described here are approaches that are or are among the most commonly used and accepted in the energy efficiency industry for certain measures or programs. As such, they draw from the existing body of research and best practices for energy efficiency program evaluation, measurement, and verification (EM&V). These protocols were developed as part of the Uniform Methods Project (UMP), funded by the U.S. Department of Energy (DOE). The principal objectivemore » for the project was to establish easy-to-follow protocols based on commonly accepted methods for a core set of widely deployed energy efficiency measures.« less

cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)β(1).

PubMed

Beste, Kerstin Y; Seifert, Roland

2013-02-01

Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate are second messengers that regulate multiple physiological functions. The existence of additional cyclic nucleotides in mammalian cells was postulated many years ago, but technical problems hampered development of the field. Using highly specific and sensitive mass spectrometry methods, soluble guanylyl cyclase has recently been shown to catalyze the formation of several cyclic nucleotides in vitro. This minireview discusses the broad substrate-specificity of soluble guanylyl cyclase and the possible second messenger roles of cyclic nucleotides other than adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. We hope that this article stimulates productive and critical research in an area that has been neglected for many years.

Understanding behavioural intention to play online game: The case of VocBlast

NASA Astrophysics Data System (ADS)

Ali, Z.

2018-04-01

Research has shown that mobile learning enables its users to learn at any time and place. The current study investigates the use of VocBlast; an app that integrates technical and engineering vocabulary, in terms of understanding the behavioural intention of its players. The study employs 129 engineering and technical students from Universiti Malaysia Pahang (UMP). Online survey was used to collect their opinions; in particular male and female students’ opinions on the use of the app in the future. The results of the study indicated that there was no significant difference pertaining to their behavioural intention using VocBlast in the course of time. The study implies that more time needs to be given to the students in playing VocBlast as it is believed that playing the game repetitively would promote positive perceptions among its players.

After Action Report - Kazakhstan NSDD July 2015

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

Fox, Caterina; Eppich, Gary; Kips, Ruth

On Monday 20 July, Caterina Fox, Ruth Kips and Kim Knight were invited to participate in Kazakhstan's nuclear material inventory management working group meeting coordinated by Alexander Vasilliev as nuclear forensics subject matter experts. The meeting included participants from Kazakhstan's nuclear regulatory agency (CAESC, the Committee on Atomic and Energetic Supervision and Control) and 3 institutes 1. Institute of Nuclear Physics, INP (Almaty), 2. National Nuclear Center, NNC (Kurchatov), and 3. Ulba Metallurgical Plant, UMP (Oskemen). CAESC requested attendance of an MC&A expert, an IT Specialist, and a Physical Security Specialist from each site. The general meeting concerned considerations formore » creating unified or compatible systems for nuclear material inventory management. NSDD representatives provided an overview of nuclear forensics and presented considerations for developments of inventory management that might be synergistic with future consideration of development of a National Nuclear Forensics Library to support nuclear forensics investigations.« less

Sedimentological Investigations of the Martian Surface using the Mars 2001 Robotic Arm Camera and MECA Optical Microscope

NASA Technical Reports Server (NTRS)

Rice, J. W., Jr.; Smith, P. H.; Marshall, J. R.

1999-01-01

The first microscopic sedimentological studies of the Martian surface will commence with the landing of the Mars Polar Lander (MPL) December 3, 1999. The Robotic Arm Camera (RAC) has a resolution of 25 um/p which will permit detailed micromorphological analysis of surface and subsurface materials. The Robotic Ann will be able to dig up to 50 cm below the surface. The walls of the trench will also be inspected by RAC to look for evidence of stratigraphic and / or sedimentological relationships. The 2001 Mars Lander will build upon and expand the sedimentological research begun by the RAC on MPL. This will be accomplished by: (1) Macroscopic (dm to cm): Descent Imager, Pancam, RAC; (2) Microscopic (mm to um RAC, MECA Optical Microscope (Figure 2), AFM This paper will focus on investigations that can be conducted by the RAC and MECA Optical Microscope.

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

NASA Technical Reports Server (NTRS)

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

1997-01-01

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

Characterization of the Polyoxin Biosynthetic Gene Cluster from Streptomyces cacaoi and Engineered Production of Polyoxin H*S⃞

PubMed Central

Chen, Wenqing; Huang, Tingting; He, Xinyi; Meng, Qingqing; You, Delin; Bai, Linquan; Li, Jialiang; Wu, Mingxuan; Li, Rui; Xie, Zhoujie; Zhou, Huchen; Zhou, Xiufen; Tan, Huarong; Deng, Zixin

2009-01-01

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics. PMID:19233844

High-level QM/MM calculations support the concerted mechanism for Michael addition and covalent complex formation in thymidylate synthase.

PubMed

Kaiyawet, Nopporn; Lonsdale, Richard; Rungrotmongkol, Thanyada; Mulholland, Adrian J; Hannongbua, Supot

2015-02-10

Thymidylate synthase (TS) is a promising cancer target, due to its crucial function in thymine synthesis. It performs the reductive methylation of 2'-deoxyuridine-5'-phosphate (dUMP) to thymidine-5'-phosphate (dTMP), using N-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent conformational activation, this complex has been proposed to react via nucleophilic attack (Michael addition) by Cys146, followed by methylene-bridge formation to generate the ternary covalent intermediate. Herein, QM/MM (B3LYP-D/6-31+G(d)-CHARMM27) methods are used to model the formation of the ternary covalent intermediate. A two-dimensional potential energy surface reveals that the methylene-bridged intermediate is formed via a concerted mechanism, as indicated by a single transition state on the minimum energy pathway and the absence of a stable enolate intermediate. A range of different QM methods (B3LYP, MP2 and SCS-MP2, and different basis sets) are tested for the calculation of the activation energy barrier for the formation of the methylene-bridged intermediate. We test convergence of the QM/MM results with respect to size of the QM region. Inclusion of Arg166, which interacts with the nucleophilic thiolate, in the QM region is important for reliable results; the MM model apparently does not reproduce energies for distortion of the guanidinium side chain correctly. The spin component scaled-Møller-Plessett perturbation theory (SCS-MP2) approach was shown to be in best agreement (within 1.1 kcal/mol) while the results obtained with MP2 and B3LYP also yielded acceptable values (deviating by less than 3 kcal/mol) compared with the barrier derived from experiment. Our results indicate that using a dispersion-corrected DFT method, or a QM method with an accurate treatment of electron correlation, increases the agreement between the calculated and experimental activation energy barriers, compared with the semiempirical AM1 method. These calculations provide important insight into the reaction mechanism of TS and may be useful in the design of new TS inhibitors.

Structure-Activity Relationships of Orotidine-5′-Monophosphate Decarboxylase Inhibitors as Anticancer Agents

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

Bello, A.; Konforte, D; Poduch, E

2009-01-01

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro,more » 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.« less

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

PubMed Central

Begley, Darren W.; Edwards, Thomas E.; Raymond, Amy C.; Smith, Eric R.; Hartley, Robert C.; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D.; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J.

2011-01-01

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

Reaction of atomic hydrogen with formic acid.

PubMed

Cao, Qian; Berski, Slawomir; Latajka, Zdzislaw; Räsänen, Markku; Khriachtchev, Leonid

2014-04-07

We study the reaction of atomic hydrogen with formic acid and characterize the radical products using IR spectroscopy in a Kr matrix and quantum chemical calculations. The reaction first leads to the formation of an intermediate radical trans-H2COOH, which converts to the more stable radical trans-cis-HC(OH)2via hydrogen atom tunneling on a timescale of hours at 4.3 K. These open-shell species are observed for the first time as well as a reaction between atomic hydrogen and formic acid. The structural assignment is aided by extensive deuteration experiments and ab initio calculations at the UMP2 and UCCSD(T) levels of theory. The simplest geminal diol radical trans-cis-HC(OH)2 identified in the present work as the final product of the reaction should be very reactive, and further reaction channels are of particular interest. These reactions and species may constitute new channels for the initiation and propagation of more complex organic species in the interstellar clouds.

Reversible Sialylation: Synthesis of CMP-NeuAc from 5′-CMP using α2,3-sialyl O-glycan, glycolipid and macromolecule based donors allow for the synthesis of diverse sialylated products

PubMed Central

Chandrasekaran, E.V.; Xue, Jun; Xia, Jie; Locke, Robert D.; Matta, Khushi L.; Neelamegham, Sriram

2008-01-01

Sialyltransferases transfer sialic acid from CMP-NeuAc to an acceptor molecule. Trans-sialidases of parasites transfer α2,3 linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here, we report another type of sialylation termed reverse sialylation catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcα2,3Galβ1,3GalNAcα-unit of O-glycans, 3-sialyl globo unit of glycolipids and sialylated macromolecules to 5′-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I forming α2,6 sialylated compounds. ST3Gal-II also catalyzed the conversion of 5′-UMP to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions or energy. The direct sialylation using CMP-NeuAc as well as the formation of CMP-NeuAc from 5′-CMP had a wide optimum range (pH 5.2–7.2 and 4.8–6.4 respectively) whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (the activity level 50% at pH 5.2 & 6.8 and 25% at pH 4.8 & 7.2). Several properties distinguish forward/conventional vs. reverse sialylation: i. Sodium citrate inhibited forward sialylation but not reverse sialylation. ii. 5′-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5′-CMP to CMP-NeuAc. iii. The mucin core 2 compound 3-O-sulfoα2,3Galβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5′-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products. PMID:18067323

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

PubMed Central

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-01-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase. PMID:12429023

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

PubMed

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-03-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

Backbone ¹H, ¹³C, ¹⁵N NMR assignments of yeast OMP synthase in unliganded form and in complex with orotidine 5'-monophosphate.

PubMed

Hansen, Michael Riis; Harris, Richard; Barr, Eric W; Cheng, Hong; Girvin, Mark E; Grubmeyer, Charles

2014-04-01

The type I phosphoribosyltransferase OMP synthase (EC 2.4.2.10) is involved in de novo synthesis of pyrimidine nucleotides forming the UMP precursor orotidine 5'-monophosphate (OMP). The homodimeric enzyme has a Rossman α/β core topped by a base-enclosing "hood" domain and a flexible domain-swapped catalytic loop. High-resolution X-ray structures of the homologous Salmonella typhimurium and yeast enzymes show that a general compacting of the core as well as movement of the hood and a major disorder-to-order transition of the loop occur upon binding of ligands MgPRPP and orotate. Here we present backbone NMR assignments for the unliganded yeast enzyme (49 kDa) and its complex with product OMP. We were able to assign 212-213 of the 225 non-proline backbone (15)N and amide proton resonances. Significant difference in chemical shifts of the amide cross peaks occur in regions of the structure that undergo movement upon ligand occupancy in the S. typhimurium enzyme.

Fe(II)-dependent, uridine-5'-monophosphate α-ketoglutarate dioxygenases in the synthesis of 5'-modified nucleosides.

PubMed

Yang, Zhaoyong; Unrine, Jason; Nonaka, Koichi; Van Lanen, Steven G

2012-01-01

Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. The functional assignment of LipL and the homologous enzymes-including bioinformatic analysis, iron detection and quantification, and assay development for biochemical characterization-is presented herein. Copyright © 2012 Elsevier Inc. All rights reserved.

Structure-activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase.

PubMed

Bello, Angelica M; Poduch, Ewa; Liu, Yan; Wei, Lianhu; Crandall, Ian; Wang, Xiaoyang; Dyanand, Christopher; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2008-02-14

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

A Nutrient Combination that Can Affect Synapse Formation

PubMed Central

Wurtman, Richard J.

2014-01-01

Brain neurons form synapses throughout the life span. This process is initiated by neuronal depolarization, however the numbers of synapses thus formed depend on brain levels of three key nutrients—uridine, the omega-3 fatty acid DHA, and choline. Given together, these nutrients accelerate formation of synaptic membrane, the major component of synapses. In infants, when synaptogenesis is maximal, relatively large amounts of all three nutrients are provided in bioavailable forms (e.g., uridine in the UMP of mothers’ milk and infant formulas). However, in adults the uridine in foods, mostly present at RNA, is not bioavailable, and no food has ever been compelling demonstrated to elevate plasma uridine levels. Moreover, the quantities of DHA and choline in regular foods can be insufficient for raising their blood levels enough to promote optimal synaptogenesis. In Alzheimer’s disease (AD) the need for extra quantities of the three nutrients is enhanced, both because their basal plasma levels may be subnormal (reflecting impaired hepatic synthesis), and because especially high brain levels are needed for correcting the disease-related deficiencies in synaptic membrane and synapses. PMID:24763080

Ultra-mini PNL (UMP): Material, indications, technique, advantages and results.

PubMed

Desai, Janak D

2017-01-01

Stone disease has afflicted mankind since centuries; records from ancient civilisations of India and Egypt have shown stones in human bodies. The scientific mind of humans has always made smart endeavours to remove the kidney stones. From large instruments made like the beaks of different animals and birds in 600 BC (Indian civilisation) to extremely sophisticated and miniaturised endoscopic intruments of today the human race has travelled a long way. The theme has always been to remove the stones with minimal morbidity and mortality and with minimum pain to the patient. The article takes you through the journey of instruments used in 600 BC until today. The story of instrumentation is a symbiosis of the medical minds along with engineering advances. The story of miniaturisation could not have moved further without the development of lasers, fiberoptics and sophisticated cameras. As the field stands today, we remove more complex stones by larger endoscopic intervention and smaller stones by miniaturised instruments. The article discusses all the merits and shortcomings of various techniques: from open surgery to standard PCNL to Mini PCNL to Ultra- Mini PCNL to Micro-PCNL.

Confronting the Gaia and NLTE spectroscopic parallaxes for the FGK stars

NASA Astrophysics Data System (ADS)

Sitnova, Tatyana; Mashonkina, Lyudmila; Pakhomov, Yury

2018-04-01

The understanding of the chemical evolution of the Galaxy relies on the stellar chemical composition. Accurate atmospheric parameters is a prerequisite of determination of accurate chemical abundances. For late type stars with known distance, surface gravity (log g) can be calculated from well-known relation between stellar mass, T eff, and absolute bolometric magnitude. This method weakly depends on model atmospheres, and provides reliable log g. However, accurate distances are available for limited number of stars. Another way to determine log g for cool stars is based on ionisation equilibrium, i.e. consistent abundances from lines of neutral and ionised species. In this study we determine atmospheric parameters moving step-by-step from well-studied nearby dwarfs to ultra-metal poor (UMP) giants. In each sample, we select stars with the most reliable T eff based on photometry and the distance-based log g, and compare with spectroscopic gravity calculated taking into account deviations from local thermodinamic equilibrium (LTE). After that, we apply spectroscopic method of log g determination to other stars of the sample with unknown distances.

Evaluation of umami taste in mushroom extracts by chemical analysis, sensory evaluation, and an electronic tongue system.

PubMed

Phat, Chanvorleak; Moon, BoKyung; Lee, Chan

2016-02-01

Seventeen edible mushrooms commercially available in Korea were analysed for their umami taste compounds (5'-nucleotides: AMP, GMP, IMP, UMP, XMP; free amino acids: aspartic, glutamic acid) and subjected to human sensory evaluation and electronic tongue measurements. Amanita virgineoides featured the highest total 5'-nucleotide content (36.9 ± 1.50 mg/g), while monosodium glutamate-like components (42.4 ± 6.90 mg/g) were highest in Agaricus bisporus. The equivalent umami concentration (EUC) ranged from 1.51 ± 0.42 to 3890 ± 833 mg MSG/g dry weight; most mushrooms exhibited a high umami taste. Pleurotus ostreatus scored the highest in the human sensory evaluation, while Flammulina velutipes obtained the maximum score in the electronic tongue measurement. The EUC and the sensory score from the electronic tongue test were highly correlated, and also showed significant correlation with the human sensory evaluation score. These results suggest that the electronic tongue is suitable to determine the characteristic umami taste of mushrooms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

PubMed

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

Synthesis, characterization and properties of uridine 5'-( -D-apio-D-furanosyl pyrophosphate).

PubMed

Kindel, P K; Watson, R R

1973-06-01

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

PubMed Central

Kindel, Paul K.; Watson, Ronald R.

1973-01-01

1. A method was developed for synthesizing UDP-apiose [uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5′-(α-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5′-(α-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [3H]UDP-[U-14C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the 3H/14C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100°C for 15min; (b) degraded at pH8.0 and 100°C for 3min; (c) used as a substrate in the enzymic synthesis of [14C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [3H]UDP-[U-14C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-14C]apiose and phosphate formed on alkaline degradation of UDP-[U-14C]apiose was α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-14C]apiose and phosphate formed on acid hydrolysis of α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-14C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-14C]apiose to α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80°C, at pH8.0 and 25°C and at pH8.0 and 4°C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-14C]-apiose to d-[U-14C]apiose and UDP at pH3.0 and 40°C was 4.67min. After 20 days at pH6.2–6.6 and 4°C, 17% of the starting UDP-[U-14C]apiose was degraded to α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-14C]apiose and UDP. After 120 days at pH6.4 and −20°C 2% of the starting UDP-[U-14C]apiose was degraded and 4% was hydrolysed. PMID:4723773

Depopulation of highly excited singlet states of DNA model compounds: quantum yields of 193 and 245 nm photoproducts of pyrimidine monomers and dinucleoside monophosphates.

PubMed

Gurzadyan, G G; Görner, H

1996-02-01

Formation of uracil and orotic acid photodimers, uridine and 5'-UMP photohydrates, TpT photodimers and (6-4)photoproducts, dCpT photohydrates and (6-4)photoproducts and UpU, CpC and CpU photohydrates were studied in neutral deoxygenated aqueous solution at room temperature upon irradiation at either 193 or 254 nm. The photoproducts were identified and quantified and the contribution from photoionization to substrate decomposition, using lambda irr = 193 nm, was separated. The ratio of the quantum yields of respective stable products, eta = phi 193/phi 254, is indicative of the yield of internal conversion from the second to the first excited singlet state, S2-->S1. For the observed photodimers eta decreases from 0.94 for uracil to 0.7 for TpT and further to 0.55 for orotic acid. For the (6-4)photoproducts of TpT and dCpT eta = 0.5-0.8 and for the photohydrates in the cases of UpU, CpC, CpU and dCpT eta ranges from 0.55 to 1.

Estudio ab initio del mecanismo de la reacción HSO + O3

NASA Astrophysics Data System (ADS)

Nebot Gil, I.

La reacción entre el radical HSO y el ozono ha sido ampliamente estudiada desde el punto de vista experimental debido a la importancia que tiene el radical HSO en la oxidación de los compuestos de azufre reductores y a que puede contribuir a la producción de H2SO4 [1-4]. Se realizaron diversos estudios teóricos sobre la cinética de la reacción entre el radical HSO y el ozono. La reacción del HSO con el ozono presenta tres canales diferentes : HSO + O3 &rightarrow &HSO2 + O2 &rightarrow &HS + 2 O2 &rightarrow &SO + OH + O2 La controversia existente entre los grupos experimentales sobre cuál de las tres vías es la predominante, se ha resuelto mediante un estudio teórico de todas ellas utilizando métodos ab initio. La estructura de todos los reactivos, productos, intermedios y estados de transición ha sido optimizada a nivel ab initio utilizando los métodos UMP2 /6-31G** y QCISD/6-31G**.

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide

PubMed Central

Burton, K.

1967-01-01

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0° by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO3NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient. PMID:6048808

The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

PubMed Central

Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Yuan, Zheng; Zhang, Dabing

2016-01-01

Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise. PMID:27635142

Allosteric inhibitors of Coxsackie virus A24 RNA polymerase.

PubMed

Schein, Catherine H; Rowold, Diane; Choi, Kyung H

2016-02-15

Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3D(pol)) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3D(pol)), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3D(pol). Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10-20μM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed Central

Vanstapel, F; Blanckaert, N

1988-01-01

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

Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite.

PubMed

Cho, J M; Kimball, A P

1982-08-15

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.

Improving the Organoleptic Properties of a Craft Mezcal Beverage by Increasing Fatty Acid Ethyl Ester Contents through ATF1 Expression in an Engineered Kluyveromyces marxianus UMPe-1 Yeast.

PubMed

Campos-García, Jesús; Vargas, Alejandra; Farías-Rosales, Lorena; Miranda, Ana L; Meza-Carmen, Víctor; Díaz-Pérez, Alma L

2018-05-02

Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide.

PubMed

Burton, K

1967-08-01

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0 degrees by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO(3)NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.

Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

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

Krungkrai, Sudaratana R.; Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871; Tokuoka, Keiji

Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 asmore » a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})« less

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue.

PubMed Central

Wild, K.; Bohner, T.; Folkers, G.; Schulz, G. E.

1997-01-01

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments. PMID:9336833

Structure and inhibition of orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum.

PubMed

Langley, David B; Shojaei, Maryam; Chan, Camilla; Lok, Hiu Chuen; Mackay, Joel P; Traut, Thomas W; Guss, J Mitchell; Christopherson, Richard I

2008-03-25

Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis-Menten kinetics with K m = 350 +/- 60 nM and V max = 2.70 +/- 0.10 micromol/min/mg protein. Inhibition patterns for nucleoside 5'-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5'-monophosphate ( K i = 3.6 +/- 0.7 nM) > xanthosine 5'-monophosphate (XMP, K i = 4.4 +/- 0.7 nM) > 6-azauridine 5'-monophosphate (AzaUMP, K i = 12 +/- 3 nM) > allopurinol-3-riboside 5'-monophosphate ( K i = 240 +/- 20 nM). XMP is an approximately 150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of the enzyme. Both the phosphate-binding loop and the betaalpha5-loop have conformational flexibility, which may be associated with substrate capture and product release along the reaction pathway.

The fate of H atom adducts to 3'-uridine monophosphate.

PubMed

Wang, Ran; Zhang, Ru Bo; Eriksson, Leif A

2010-07-29

The stabilities of the adducts deriving from H free radical addition to the O2, O4, and C5 positions of 3'-uridine monophosphate (3'UMP) are studied by the hybrid density functional B3LYP approach. Upon H atom addition at the O2 position, a concerted low-barrier proton-transfer process will initially occur, followed by the potential ruptures of the N-glycosidic or beta-phosphate bonds. The rupture barriers are strongly influenced by the rotational configuration of the phosphate group at the 3' terminal, and are influenced by bulk solvation effects. The O4-H adduct has the highest thermal stability, as the localization of the unpaired electron does not enable cleavage of either the C1'-N1 or the C3'-O(P) bonds. For the most stable adduct, with H atom added to the C5 position, the rate-controlled step is the H2'a abstraction by the C6 radical site, after which the subsequent strand rupture reactions proceed with low barriers. The main unpaired electron densities are presented for the transient species. Combined with previous results, it is concluded that the H atom adducts are more facile to drive the strand scission rather than N-glycosidic bond ruptures within the nucleic acid bases.

Histone-poly(A) hybrid molecules as tools to block nuclear pores.

PubMed

Cremer, G; Wojtech, E; Kalbas, M; Agutter, P S; Prochnow, D

1995-04-01

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

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

PubMed

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

2013-11-15

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

Capacity building toward evidence-based medicine among healthcare professionals at the university of medicine and pharmacy, ho chi minh city, and its related institutes.

PubMed

Nga, LE Thi Quynh; Goto, Aya; Trung, Tran The; Vinh, Nguyen Quang; Khue, Nguyen Thy

2014-02-01

Research capacity development enhances a country's ownership of activities aimed at strengthening its health system. In Vietnam, continuing medical education (CME) is attracting increasing attention with the establishment of legal and policy frameworks. During 2010-2013, the Japan International Cooperation Agency funded a research capacity building project targeting physicians in Ho Chi Minh City. The project had been developed in four previous courses that were conducted in collaboration with Fukushima Medical University and Ho Chi Minh City University of Medicine and Pharmacy (UMP). The project succeeded in obtaining accreditation as the city's CME course. A total of 262 physicians attended three courses that have a divided set of research competencies. Following the Kirkpatrick Model for evaluating the effectiveness of training programs, we confirmed the participants' positive reaction to the courses (Level 1 evaluation), their perceived increase in knowledge and confidence in research skills (Level 2 evaluation), and application of learned knowledge in their practice (Level 3 evaluation). Presented here is a step-by-step scaling-up model of health research capacity building. Strategies for the further expansion include: further capacity building of instructors; responding to clinicians' specific needs; building a recruiting system with authorization; and improving the Level 3 training evaluation.

The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

PubMed

Doucet, Nicolas; Jayasundera, Thusitha B; Simonović, Miljan; Loria, J Patrick

2010-08-15

Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme. 2010 Wiley-Liss, Inc.

Assessing self-efficacy and college readiness level among new undergraduate students in computer science using metacognitive awareness inventory (MAI)

NASA Astrophysics Data System (ADS)

Othman, Wan Nor Afiqah Wan; Abdullah, Aziman

2018-04-01

This preliminary study was conducted to address the issue of academic planning skills among new university student. Due to lack of proper measurement mechanism for awareness and readiness among students, this study proposes Metacognitive Awareness Inventory (MAI) to assess the connection between student self-efficacy and college readiness. Qualitative and quantitative approach were used by provide an online self-assessment for new student of Faculty of Computer Systems & Software Engineering (FSKKP) and analyse the data respectively. The possible relationships between MAI and College Readiness Item (CRI) in self-assessment has been evaluated. The sample size of 368 respondents from UMP are responding to the online self-assessment. The initial finding shows most student (71%) of the respondent lack of skills in planning. We manage to use Pearson Product-moment correlation coefficient to find the significant relationship between MAI and CRI. Thus, we found that College Readiness provide sufficient evidence that there is a significant correlation with most of MAI items. The findings also indicated not much difference was found between gender in terms of self-efficacy level. This paper suggests the MAI and CRI is a reliable and valid scale to respond the planning skills issues among new university students.

Organization of Nucleotides in Different Environments and the Formation of Pre-Polymers

NASA Astrophysics Data System (ADS)

Himbert, Sebastian; Chapman, Mindy; Deamer, David W.; Rheinstädter, Maikel C.

2016-08-01

RNA is a linear polymer of nucleotides linked by a ribose-phosphate backbone. Polymerization of nucleotides occurs in a condensation reaction in which phosphodiester bonds are formed. However, in the absence of enzymes and metabolism there has been no obvious way for RNA-like molecules to be produced and then encapsulated in cellular compartments. We investigated 5‧-adenosine monophosphate (AMP) and 5‧-uridine monophosphate (UMP) molecules confined in multi-lamellar phospholipid bilayers, nanoscopic films, ammonium chloride salt crystals and Montmorillonite clay, previously proposed to promote polymerization. X-ray diffraction was used to determine whether such conditions imposed a degree of order on the nucleotides. Two nucleotide signals were observed in all matrices, one corresponding to a nearest neighbour distance of 4.6 Å attributed to nucleotides that form a disordered, glassy structure. A second, smaller distance of 3.4 Å agrees well with the distance between stacked base pairs in the RNA backbone, and was assigned to the formation of pre-polymers, i.e., the organization of nucleotides into stacks of about 10 monomers. Such ordering can provide conditions that promote the nonenzymatic polymerization of RNA strands under prebiotic conditions. Experiments were modeled by Monte-Carlo simulations, which provide details of the molecular structure of these pre-polymers.

Characterization of Streptococcus pneumoniae thymidylate kinase: steady-state kinetics of the forward reaction and isothermal titration calorimetry.

PubMed Central

Petit, Chantal M; Koretke, Kristin K

2002-01-01

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis. The tmk gene from the bacterial pathogen Streptococcus pneumoniae was identified. The gene, encoding a 212-amino-acid polypeptide (23352 Da), was cloned and overexpressed in Escherichia coli with an N-terminal hexahistidine tag. The enzyme was purified to homogeneity, and characterized in the forward reaction. The pH profile of TMK indicates that its activity is optimal at pH 8.5. The substrate specificity of the enzyme was examined; it was found that not only ATP, but also dATP and to a lesser extent CTP, could act as phosphate donors, and dTMP and dUMP could serve as phosphate acceptors. Furthermore, AZT-MP (3'-azido-3'-deoxythymidine 5'-monophosphate) was shown not to be a substrate for S. pneumoniae TMK. Steady-state kinetics and inhibition studies with adenosine 5'-[beta-thio]diphosphate and dTDP in addition to isothermal titration calorimetry were performed. The data showed that binding follows an ordered pathway, in which ATP binds first with a K(m) of 235 +/- 46 microM and a K(d) of 116 +/- 3 microM, and dTMP binds secondly with a K(m) of 66 +/- 12 microM and a K(d) of 53 +/- 2 microM. PMID:11964185

UV irradiation of biomarkers adsorbed on minerals under Martian-like conditions: Hints for life detection on Mars

NASA Astrophysics Data System (ADS)

Fornaro, Teresa; Boosman, Arjen; Brucato, John R.; ten Kate, Inge Loes; Siljeström, Sandra; Poggiali, Giovanni; Steele, Andrew; Hazen, Robert M.

2018-10-01

Laboratory simulations of Martian conditions are essential to develop quantitative models for the survival of organic biomarkers for future Mars exploration missions. In this work, we report the results of ultraviolet (UV) irradiation processing of biomarkers adsorbed on minerals under Martian-like conditions. Specifically, we prepared Mars soil analogues by doping forsterite, lizardite, antigorite, labradorite, natrolite, apatite and hematite minerals with organic compounds considered as potential biomarkers of extant terrestrial life such as the nucleotides adenosine monophosphate (AMP) and uridine monophosphate (UMP). We characterized such Mars soil analogues by means of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) and Confocal Raman Imaging Spectroscopy (CRIS), in order to get insights into the specific molecule-mineral interactions and explore the capabilities of different techniques to reveal diagnostic features of these biomarkers. Then, we performed irradiation experiments in the mid-UV spectral region under simulated Martian conditions and under terrestrial ambient conditions for comparison, monitoring the degradation process through DRIFTS. We observed that degradation under Martian-like conditions occurs much slower than in terrestrial ambient conditions. The minerals labradorite and natrolite mainly promote photodegradation of nucleotides, hematite and forsterite exhibit an intermediate degrading effect, while apatite, lizardite and antigorite do not show any significant catalytic effect on the degradation of the target organic species.

Deficiency of uridine monophosphate synthase (DUMPS) and X-chromosome deletion in fetal mummification in cattle.

PubMed

Ghanem, Mohamed Elshabrawy; Nakao, Toshihiko; Nishibori, Masahide

2006-01-01

Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

PubMed

Kandeel, Mahmoud; Kitade, Yukio

2018-02-01

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

Distinct hydroxy-radical-induced damage of 3'-uridine monophosphate in RNA: a theoretical study.

PubMed

Zhang, Ru bo; Eriksson, Leif A

2009-01-01

RNA strand scission and base release in 3'-uridine monophosphate (UMP), induced by OH radical addition to uracil, is studied at the DFT B3LYP/6-31+G(d,p) level in the gas phase and in solution. In particular, the mechanism of hydrogen-atom transfer subsequent to radical formation, from C2' on the sugar to the C6 site on the base, is explored. The barriers of (C2'-)H2'(a) abstraction by the C6 radical site range from 11.2 to 20.0 kcal mol(-1) in the gas phase and 14.1 to 21.0 kcal mol(-1) in aqueous solution, indicating that the local surrounding governs the hydrogen-abstraction reaction in a stereoselective way. The calculated N1-C1' (N1-glycosidic bond) and beta-phosphate bond strengths show that homolytic and heterolytic bond-breaking processes are largely favored in each case, respectively. The barrier for beta-phosphate bond rupture is approximately 3.2-4.0 kcal mol(-1) and is preferred by 8-12 kcal mol(-1) over N1-glycosidic bond cleavage in both the gas phase and solution. The beta-phosphate bond-rupture reactions are exothermal in the gas phase and solution, whereas N1-C1' bond-rupture reactions require both solvation and thermal corrections at 298 K to be energetically favored. The presence of the ribose 2'-OH group and its formation of low-barrier hydrogen bonds with oxygen atoms of the 3'-phosphate linkage are highly important for hydrogen transfer and the subsequent bond-breakage reactions.

Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

PubMed

Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

2011-11-01

Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR

PubMed Central

Bonner, Eric R.; D’Elia, John N.; Billips, Benjamin K.; Switzer, Robert L.

2001-01-01

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5′-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4–5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure. PMID:11726695

Comparative analysis of positive and negative attitudes toward statistics

NASA Astrophysics Data System (ADS)

Ghulami, Hassan Rahnaward; Ab Hamid, Mohd Rashid; Zakaria, Roslinazairimah

2015-02-01

Many statistics lecturers and statistics education researchers are interested to know the perception of their students' attitudes toward statistics during the statistics course. In statistics course, positive attitude toward statistics is a vital because it will be encourage students to get interested in the statistics course and in order to master the core content of the subject matters under study. Although, students who have negative attitudes toward statistics they will feel depressed especially in the given group assignment, at risk for failure, are often highly emotional, and could not move forward. Therefore, this study investigates the students' attitude towards learning statistics. Six latent constructs have been the measurement of students' attitudes toward learning statistic such as affect, cognitive competence, value, difficulty, interest, and effort. The questionnaire was adopted and adapted from the reliable and validate instrument of Survey of Attitudes towards Statistics (SATS). This study is conducted among engineering undergraduate engineering students in the university Malaysia Pahang (UMP). The respondents consist of students who were taking the applied statistics course from different faculties. From the analysis, it is found that the questionnaire is acceptable and the relationships among the constructs has been proposed and investigated. In this case, students show full effort to master the statistics course, feel statistics course enjoyable, have confidence that they have intellectual capacity, and they have more positive attitudes then negative attitudes towards statistics learning. In conclusion in terms of affect, cognitive competence, value, interest and effort construct the positive attitude towards statistics was mostly exhibited. While negative attitudes mostly exhibited by difficulty construct.

Unraveling the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress by ¹H NMR-based metabolomics.

PubMed

Ye, Yangfang; Wang, Xin; Zhang, Limin; Lu, Zhenmei; Yan, Xiaojun

2012-07-01

Nicotine can cause oxidative damage to organisms; however, some bacteria, for example Pseudomonas sp. HF-1, are resistant to such oxidative stress. In the present study, we analyzed the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress using ¹H NMR spectroscopy coupled with multivariate data analysis. We found that the dominant metabolites in Pseudomonas sp. HF-1 were eight aliphatic organic acids, six amino acids, three sugars and 11 nucleotides. After 18 h of cultivation, 1 g/L nicotine caused significant elevation of sugar (glucose, trehalose and maltose), succinate and nucleic acid metabolites (cytidine, 5'-CMP, guanine 2',3'-cyclic phosphate and adenosine 2',3'-cyclic phosphate), but decrease of glutamate, putrescine, pyrimidine, 2-propanol, diethyl ether and acetamide levels. Similar metabolomic changes were induced by 2 g/L nicotine, except that no significant change in trehalose, 5'-UMP levels and diethyl ether were found. However, 3 g/L nicotine led to a significant elevation in the two sugars (trehalose and maltose) levels and decrease in the levels of glutamate, putrescine, pyrimidine and 2-propanol. Our findings indicated that nicotine resulted in the enhanced nucleotide biosynthesis, decreased glucose catabolism, elevated succinate accumulation, severe disturbance in osmoregulation and complex antioxidant strategy. And a further increase of nicotine level was a critical threshold value that triggered the change of metabolic flow in Pseudomonas sp. HF-1. These findings revealed the comprehensive insights into the metabolic response of nicotine-degrading bacteria to nicotine-induced oxidative toxicity.

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

PubMed

Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

2014-06-01

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

RNA sequencing reveals significant miRNAs in Atypical endometrial hyperplasia.

PubMed

Tang, Shiqian; Dai, Yinmei

2018-06-01

In this paper, we aimed to investigate the miRNAs that played a regulatory role in the development of atypical endometrial hyperplasia (AEH). RNA sequencing was performed for endometrial tissues from 3 AEH patients and 3 endometrial normal hyperplasia patients. RNA sequencing data were processed and differentially expressed (DE) miRNAs were identified between AEH and controls. The target genes for DE miRNAs were identified and mapped to the protein-protein interaction (PPI) network. The miRNA related functions were predicted and miRNA-disease gene network was constructed. Total 18 DE miRNAs were overlapped in three sample groups, among which hsa-miR-577, hsa-miR-182-5p and hsa-miR-183-5p were top three miRNAs that targeting largest number of genes. Function analysis showed that the 18 overlapped miRNAs mainly related with cancer and signaling transduction related pathways. PPI network showed that total 12 genes were among top 20 genes based on three network topological features including BCL2, UMPS, MAPK13, PRKCB, CREB1, IGF1, SP1, SMAD3, IGF1R, NOTCH2, WNT5A, TK2. Top 10 miRNAs in miRNA-disease gene network were identified such as hsa-miR-577 (degree = 17), hsa-miR-182-5p (degree = 16) and hsa-miR-3609 (degree = 13). hsa-miR-577 and hsa-miR-182-5p may play regulatory role in AEH through AMPK signal pathway and Wnt signaling pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Cyclic Nucleotide Monophosphates in Plants and Plant Signaling.

PubMed

Marondedze, Claudius; Wong, Aloysius; Thomas, Ludivine; Irving, Helen; Gehring, Chris

2017-01-01

Cyclic nucleotide monophosphates (cNMPs) and the enzymes that can generate them are of increasing interest in the plant sciences. Arguably, the major recent advance came with the release of the complete Arabidopsis thaliana genome that has enabled the systematic search for adenylate (ACs) or guanylate cyclases (GCs) and did eventually lead to the discovery of a number of GCs in higher plants. Many of these proteins have complex domain architectures with AC or GC centers moonlighting within cytosolic kinase domains. Recent reports indicated the presence of not just the canonical cNMPs (i.e., cAMP and cGMP), but also the noncanonical cCMP, cUMP, cIMP, and cdTMP in plant tissues, and this raises several questions. Firstly, what are the functions of these cNMPs, and, secondly, which enzymes can convert the substrate triphosphates into the respective noncanonical cNMPs? The first question is addressed here by comparing the reactive oxygen species (ROS) response of cAMP and cGMP to that elicited by the noncanonical cCMP or cIMP. The results show that particularly cIMP can induce significant ROS production. To answer, at least in part, the second question, we have evaluated homology models of experimentally confirmed plant GCs probing the substrate specificity by molecular docking simulations to determine if they can conceivably catalytically convert substrates other than ATP or GTP. In summary, molecular modeling and substrate docking simulations can contribute to the evaluation of cyclases for noncanonical cyclic mononucleotides and thereby further our understanding of the molecular mechanism that underlie cNMP-dependent signaling in planta.

Joint conference of iMEC 2015 (2nd International Manufacturing Engineering Conference & APCOMS 2015 (3rd Asia-Pacific Conference on Manufacturing Systems)

NASA Astrophysics Data System (ADS)

2016-02-01

The iMEC 2015 is the second International Manufacturing Engineering Conference organized by the Faculty of Manufacturing, Universiti Malaysia Pahang (UMP), held from 12-14th November 2015 in Kuala Lumpur, Malaysia, with a theme "Materials, Manufacturing and Systems for Tomorrow". For the first time, iMEC is organized together with 3rd Asia- Pacific Conference on Manufacturing System (APCOMS 2015) which owned by Fakulti Teknologi Industri, Institut Teknologi Bandung (ITB), Indonesia. This is an extended collaboration between UMP and ITB to intensify knowledge sharing and experiences between higher learning institutions. This conference (iMEC & APCOMS 2015) is a platform for knowledge exchange and the growth of ideas, particularly in manufacturing engineering. The conference aims to bring researchers, academics, scientists, students, engineers and practitioners from around the world together to present their latest findings, ideas, developments and applications related to manufacturing engineering and other related research areas. With rapid advancements in manufacturing engineering, iMEC is an appropriate medium for the associated community to keep pace with the changes. In 2015, the conference theme is “Materials, Manufacturing and Systems for Tomorrow” which reflects the acceleration of knowledge and technology in global manufacturing. The papers in these proceedings are examples of the work presented at the conference. They represent the tip of the iceberg, as the conference attracted over 200 abstracts from Malaysia, Indonesia, Japan, United Kingdom, Australia, India, Bangladesh, South Africa, Turkey and Morocco and 151 full papers were accepted in these proceedings. The conference was run in four parallel sessions with 160 presenters sharing their latest finding in the areas of manufacturing process, systems, advanced materials and automation. The first keynote presentation was given by Prof. B. S. Murthy (IIT, Madras) on "Nanomaterials with Exceptional Properties Synthesized through Top Down Approach”, which was warmly welcomed by an eager and highly motivated audience. The second keynote speaker was Prof. Ir. Dradjad Irianto (ITB, Indonesia) on “Collaborative Manufacturing for Small-medium Enterprises”. The organizers are very grateful to them for supporting the conference and sharing their latest research results with the conference participants. The conference organizers would like to express our sincere gratitude and thanks to the honorary chairman Prof. Dato, Dr. Daing Nasir Ibrahim and Prof. Dr. Wan Azhar Wan Yusoff and organizing committee members of iMEC2015, Assoc. Prof. Dr. A.K. Prasada Rao (chairman), Dr. Ing. Mohd Azmir Mohd Azhari (co-chairman), and all committee members Assoc. Prof. Dr. Ahmad Razlan Yusoff, Assoc. Prof. Dr. Ismed Iskandar, Dr Noraini Mohd Razali, Dr Muhammed Nafis Osman Zahid, Dr Noor Mazni Ismail, Dr Izwan Ismail, Dr Zamzuri Hamedon, Dr Faiz Mohd Turan, Ms Suraya Sulaiman and secretariat Miss Zuryaty, Mrs Wan Norshihah, Mr. Mohd Khairulnazri and Mr Rafilah and also a million thanks to the steering committee from APCOMS Prof. Dr. Kadarsyah Suryadi, Prof. Dr. Bermawi, P. Iskandar, Prof. Dr. Abdul Hakim Halim, Prof. Dr. Dradjad Irianto, Dr. TMA Ari Samadhi, Ir. I Made Dana Tangkas and APCOMS organizing committee Prof. Dr. Abdul Hakim Halim (chair), Dr. Rachmawati Wangsaputra (co-chair) and other committee members Dr. Iwan Inrawan Wiratmadja, Dr. Anas Ma'ruf, Dr. Sukoyo and Dr. Wisnu Aribowo. The editorial board are indebted to all of the reviewers who were willing to spend their precious time in reviewing the papers. Thanks also to all who contributed direct or indirectly in making this conference a success, especially Mr. Mohd Khairulnazri in helping the editors complete the proceedings. Dr. Zamzuri Hamedon Editor in-chief iMEC & APCOMS 2015 Associate Editors 1. Assoc. Prof. Dr. A.K. Prasada Rao 2. Assoc. Prof. Dr. Ahmad Razlan Yusoff 3. Dr. Ing. Mohd Azmir Mohd Azhari 4. Dr. Muhammed Nafis Osman Zahid 5. Dr. Faiz Mohd Turan

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes*

PubMed Central

Lopata, Anna; Leveles, Ibolya; Bendes, Ábris Ádám; Viskolcz, Béla; Vértessy, Beáta G.; Jójárt, Balázs; Tóth, Judit

2016-01-01

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent. PMID:27815500

Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidiness

PubMed Central

Ali, Juma A. M.; Creek, Darren J.; Burgess, Karl; Allison, Harriet C.; Field, Mark C.; Mäser, Pascal; De Koning, Harry P.

2016-01-01

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2′deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2′deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity through incorporation into RNA. 5F-2′dUrd and 5F-2′dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase as 5F-dUMP. We present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. PMID:23188714

NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU)

PubMed Central

Schein, Catherine H.; Oezguen, Numan; van der Heden van Noort, Gerbrand J.; Filippov, Dmitri V.; Paul, Aniko; Kumar, Eric; Braun, Werner

2010-01-01

Picornaviruses have a 22–24 amino acid peptide, VPg, bound covalently at the 5’ end of their RNA, that is essential for replication. VPgs are uridylylated at a conserved Tyrosine to form VPgpU, the primer of RNA synthesis by the viral polymerase. This first complete structure for any uridylylated VPg, of poliovirus type 1 (PV1)-VPgpU, shows that conserved amino acids in VPg stabilize the bound UMP, with the uridine atoms involved in base pairing and chain elongation projected outward. Comparing this structure to PV1-VPg and partial structures of VPg/VPgpU from other picornaviruses suggests that enteroviral polymerases require a more stable VPg structure than does the distantly related aphthovirus, foot and mouth disease virus (FMDV). The glutamine residue at the C-terminus of PV1-VPgpU lies in back of the uridine base and may stabilize its position during chain elongation and/or contribute to base specificity. Under in vivo-like conditions with the authentic cre(2C) hairpin RNA and Mg++, 5-methylUTP cannot compete with UTP for VPg uridylyation in an in vitro uridylyation assay, but both nucleotides are equally incorporated by PV1-polymerase with Mn++ and a poly-A RNA template. This indicates the 5 position is recognized under in vivo conditions. The compact VPgpU structure docks within the active site cavity of the PV-polymerase, close to the position seen for the fragment of FMDV-VPgpU with its polymerase. This structure could aid in design of novel enterovirus inhibitors, and stabilization upon uridylylation may also be pertinent for post-translational uridylylation reactions that underlie other biological processes. PMID:20441784

Ab initio study of the structural properties of ascorbic acid (vitamin C)

NASA Astrophysics Data System (ADS)

Allen, Reeshemah N.; Shukla, M. K.; Reed, Demarcio; Leszczynski, Jerzy

Geometries of the neutral and ionic tautomeric species of ascorbic acid were optimized at the density functional theory (DFT) level using the B3LYP functional. The radical species were evaluated using the unrestricted B3LYP method. Single-point energy calculations were also performed using the Møller-Plesset (MP2) and unrestricted MP2 (UMP2) methods for the closed-shell and open-shell systems, respectively. The effects of aqueous solution were evaluated using the conducting polarized continuum model (CPCM) and polarized continuum model (PCM). The geometries of most stable radicals in the respective groups were also optimized in the water solution using the CPCM model at the UB3LYP level. All calculation were performed using the 6-311++G(d,p) basis set. The nature of stationary points on the gas phase potential energy surfaces (PESs) was evaluated using vibrational frequency calculations; all geometries characterize local minima. The species obtained by the deprotonation of the O3 site is the most stable monoanion of ascorbic acid. For the radical species, the structure obtained by the dehydrogenation of the O3 site is the most stable monoradical. Among the radical anions, the species obtained by the deprotonation of the O3 site and subsequent dehydrogenation of the O2 site is the most stable in the gas phase and in an aqueous medium. The computed isotropic hyperfine coupling constants of this species were found to be in good agreement with the experimental data. Our investigation also supports the earlier findings that the oxidized species of ascorbic acid in water solution by the OH? radical is radical anion of the AAO?3O-2 form. The spin densities and molecular electrostatic potentials are also discussed.

Dissociative electron attachment to the gas-phase nucleobase hypoxanthine

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

Dawley, M. Michele; Tanzer, Katrin; Denifl, Stephan, E-mail: Stephan.Denifl@uibk.ac.at, E-mail: Sylwia.Ptasinska.1@nd.edu

We present high-resolution measurements of the dissociative electron attachment (DEA) to isolated gas-phase hypoxanthine (C{sub 5}H{sub 4}N{sub 4}O, Hyp), a tRNA purine base. The anion mass spectra and individual ion efficiency curves from Hyp were measured as a function of electron energy below 9 eV. The mass spectra at 1 and 6 eV exhibit the highest anion yields, indicating possible common precursor ions that decay into the detectable anionic fragments. The (Hyp − H) anion (C{sub 5}H{sub 3}N{sub 4}O{sup −}) exhibits a sharp resonant peak at 1 eV, which we tentatively assign to a dipole-bound state of the keto-N1H,N9H tautomermore » in which dehydrogenation occurs at either the N1 or N9 position based upon our quantum chemical computations (B3LYP/6-311+G(d,p) and U(MP2-aug-cc-pVDZ+)) and prior studies with adenine. This closed-shell dehydrogenated anion is the dominant fragment formed upon electron attachment, as with other nucleobases. Seven other anions were also observed including (Hyp − NH){sup −}, C{sub 4}H{sub 3}N{sub 4}{sup −}/C{sub 4}HN{sub 3}O{sup −}, C{sub 4}H{sub 2}N{sub 3}{sup −}, C{sub 3}NO{sup −}/HC(HCN)CN{sup −}, OCN{sup −}, CN{sup −}, and O{sup −}. Most of these anions exhibit broad but weak resonances between 4 and 8 eV similar to many analogous anions from adenine. The DEA to Hyp involves significant fragmentation, which is relevant to understanding radiation damage of biomolecules.« less

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: implications for the nature of the first genetic material

NASA Technical Reports Server (NTRS)

Stribling, R.; Miller, S. L.

1991-01-01

Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system. Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5'-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template. Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3 x 10(-6) M), as well as the difficulty of prebiotic activation of phosphates.

New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies*

PubMed Central

Liu, Yao; Rodrigues, João P. G. L. M.; Bonvin, Alexandre M. J. J.; Zaal, Esther A.; Berkers, Celia R.; Heger, Michal; Gawarecka, Katarzyna; Swiezewska, Ewa; Breukink, Eefjan; Egmond, Maarten R.

2016-01-01

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, short-chain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDP-MurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide. PMID:27226570

Molecularly Tuning the Radicaloid N-H···O═C Hydrogen Bond.

PubMed

Lu, Norman; Chung, Wei-Cheng; Ley, Rebecca M; Lin, Kwan-Yu; Francisco, Joseph S; Negishi, Ei-Ichi

2016-03-03

Substituent effects on the open shell N-H···O═C hydrogen-bond has never been reported. This study examines how 12 functional groups composed of electron donating groups (EDG), halogen atoms and electron withdrawing groups (EWG) affect the N-H···O═C hydrogen-bond properties in a six-membered cyclic model system of O═C(Y)-CH═C(X)N-H. It is found that group effects on this open shell H-bonding system are significant and have predictive trends when X = H and Y is varied. When Y is an EDG, the N-H···O═C hydrogen-bond is strengthened; and when Y is an EWG, the bond is weakened; whereas the variation in electronic properties of X group do not exhibit a significant impact upon the hydrogen bond strength. The structural impact of the stronger N-H···O═C hydrogen-bond are (1) shorter H and O distance, r(H···O) and (2) a longer N-H bond length, r(NH). The stronger N-H···O═C hydrogen-bond also acts to pull the H and O in toward one another which has an effect on the bond angles. Our findings show that there is a linear relationship between hydrogen-bond angle and N-H···O═C hydrogen-bond energy in this unusual H-bonding system. In addition, there is a linear correlation of the r(H···O) and the hydrogen bond energy. A short r(H···O) distance corresponds to a large hydrogen bond energy when Y is varied. The observed trends and findings have been validated using three different methods (UB3LYP, M06-2X, and UMP2) with two different basis sets.

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

PubMed

Djordjevic, Michael A; Chen, Han Cai; Natera, Siria; Van Noorden, Giel; Menzel, Christian; Taylor, Scott; Renard, Clotilde; Geiger, Otto; Weiller, Georg F

2003-06-01

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation*

PubMed Central

Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M.

2014-01-01

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation. PMID:24563465

Integrated RNA-seq and sRNA-seq analysis reveals miRNA effects on secondary metabolism in Solanum tuberosum L.

PubMed

Qiao, Yan; Zhang, Jinjin; Zhang, Jinwen; Wang, Zhiwei; Ran, An; Guo, Haixia; Wang, Di; Zhang, Junlian

2017-02-01

Light is a major environmental factor that affects metabolic pathways and stimulates the production of secondary metabolites in potato. However, adaptive changes in potato metabolic pathways and physiological functions triggered by light are partly explained by gene expression changes. Regulation of secondary metabolic pathways in potato has been extensively studied at transcriptional level, but little is known about the mechanisms of post-transcriptional regulation by miRNAs. To identify light-responsive miRNAs/mRNAs and construct putative metabolism pathways regulated by the miRNA-mRNA pairs, an integrated omics (sRNAome and transcriptome) analysis was performed to potato under light stimulus. A total of 31 and 48 miRNAs were identified to be differentially expressed in the leaves and tubers, respectively. Among the DEGs, 1353 genes in the leaves and 1841 genes in the tubers were upregulated, while 1595 genes in the leaves and 897 genes in the tubers were downregulated by light. Mapman enrichment analyses showed that genes related to MVA pathway, alkaloids-like, phenylpropanoids, flavonoids, and carotenoids metabolism were significantly upregulated, while genes associated with major CHO metabolism were repressed in the leaves and tubers. Integrated miRNA and mRNA profiles revealed that light-responsive miRNAs are important regulators in alkaloids metabolism, UMP salvage, lipid biosynthesis, and cellulose catabolism. Moreover, several miRNAs may participate in glycoalkaloids metabolism via JA signaling pathway, UDP-glucose biosynthesis and hydroxylation reaction. This study provides a global view of miRNA and mRNA expression profiles in potato response to light, our results suggest that miRNAs might play important roles in secondary metabolic pathways, especially in glycoalkaloid biosynthesis. The findings will enlighten us on the genetic regulation of secondary metabolite pathways and pave the way for future application of genetically engineered potato.

Kinetics and mechanism for platination of thione-containing nucleotides and oligonucleotides: evaluation of the salt dependence.

PubMed

Kjellström, Johan; Elmroth, Sofi K C

2003-01-01

Reactions of cis-[PtCl(NH(3))(CyNH(2))(OH(2))](+) (Cy=cyclohexyl) with thione-containing single-stranded oligonucleotides d(T(8)XT(8)) and d(XT(16)) (X=(s6)I or (s4)U) and the mononucleotides 4-thiouridine ((s4)UMP) and 6-mercaptoinosine ((s6)IMP) have been studied in aqueous solution at pH 4.1. The reaction kinetics was followed using HPLC methodology as a function of ionic strength in the interval 5.0 mM

Structure and Activity Analyses of Escherichia coli K-12 NagD Provide Insight into the Evolution of Biochemical Function in the Haloakanoic Acid Dehlogenase Superfamily

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

Tremblay,L.; Dunaway-Mariano, D.; Allen, K.

2006-01-01

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia colimore » K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 Angstroms with R{sub work} = 19.8% and R{sub free} = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with kcat/Km = 3.12 x 10{sup 4} and 1.28 x 10{sup 4} {micro}M{sup -1} s{sup -1} for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k{sub cat}/K{sub m}) are low (1 x 10{sup 4} M{sup -1} s{sup -1}) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.« less

Toxic effects of two brominated flame retardants BDE-47 and BDE-183 on the survival and protein expression of the tubificid Monopylephorus limosus.

PubMed

Chiu, K H; Lin, C-R; Huang, H-W; Shiea, J; Liu, L L

2012-10-01

The toxic effects of two brominated diphenyl ethers (BDE), BDE-47, and BDE-183, on a benthic oligochaete tubificid, Monopylephorus limosus were studied under laboratory conditions. Investigated responses included survival, growth, and protein expression profiles, at BDE concentrations of 1, 10, 100, and 700 ng/g on a dry soil weight basis, with isooctane as the carrier solvent. Body weight losses among treatments were insignificant after 8 weeks of exposure. The 8-wk LC(50) of BDE-47 and -183 were 2311 and 169 ng/g, respectively. By applying multivariate analysis techniques, protein expression patterns were compared and correlated with stressful sources of long-term culture, carrier solvent, BDE-47 and -183. The treatment of 8-wk 100 ng/g BDE-47 was most closely clustered to the 10 ng/g BDE-183 treatment, based on the 40 examined protein spots. This indicated that BDE-183 was more potent to M. limosus, than was BDE-47. The 2-wk and 8-wk controls clustered into different groups indicating the occurrence of physiological changes due to long-term laboratory culture. Additionally, solvent effect was shown by grouping the isooctane carrier to different clusters. With further characterization by principle component analysis, it was found that the separation was mainly contributed by the 2nd principal-component. And, the primarily inhibitory variation was at spots 2 (UMP-CMP kinase) and 40 (plasma retinol-binding protein precursor) in the 8-wk groups. On the contrary, protein spots 16 (cell division control protein 2 homolog) and 24 (mitochondrial DNA mismatch repair protein) showed stimulatory variation. In all, the observed proteomic responses suggest that BDEs disrupted metabolic function in M. limosus and multivariate analysis tool offers significant potential for the assessment of various stress sources at biochemical level. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural Defects in the Regulatory Particle-Core Particle Interface of the Proteasome Induce a Novel Proteasome Stress Response*

PubMed Central

Park, Soyeon; Kim, Woong; Tian, Geng; Gygi, Steven P.; Finley, Daniel

2011-01-01

Proteasomes consist of a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP), an α7β7β7α7 structure. The RP recognizes substrates and translocates them into the CP for degradation. At the RP-CP interface, a heterohexameric Rpt ring joins to a heteroheptameric CP α ring. Rpt C termini insert individually into the α ring pockets to form a salt bridge with a pocket lysine residue. We report that substitutions of α pocket lysine residues produce an unexpected block to CP assembly, arising from a late stage defect in β ring assembly. Substitutions α5K66A and α6K62A resulted in abundant incorporation of immature CP β subunits, associated with a complete β ring, into proteasome holoenzymes. Incorporation of immature CP into the proteasome depended on a proteasome-associated protein, Ecm29. Using ump1 mutants, we identified Ecm29 as a potent negative regulator of RP assembly and confirmed our previous findings that proper RP assembly requires the CP. Ecm29 was enriched on proteasomes of pocket lysine mutants, as well as those of rpt4-Δ1 and rpt6-Δ1 mutants, in which the C-terminal residue, thought to contact the pocket lysine, is deleted. In both rpt6-Δ1 and α6K62A proteasomes, Ecm29 suppressed opening of the CP substrate translocation channel, which is gated through interactions between Rpt C termini and the α pockets. The ubiquitin ligase Hul5 was recruited to these proteasomes together with Ecm29. Proteasome remodeling through the addition of Ecm29 and Hul5 suggests a new layer of the proteasome stress response and may be a common response to structurally aberrant proteasomes or deficient proteasome function. PMID:21878652

THE STAR FORMATION HISTORIES OF z {approx} 2 DUST-OBSCURED GALAXIES AND SUBMILLIMETER-SELECTED GALAXIES

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

Bussmann, R. S.; Dey, Arjun; Jannuzi, B. T.

The Spitzer Space Telescope has identified a population of ultraluminous infrared galaxies (ULIRGs) at z {approx} 2 that may play an important role in the evolution of massive galaxies. We measure the stellar masses (M{sub *}) of two populations of Spitzer-selected ULIRGs that have extremely red R - [24] colors (dust-obscured galaxies, or DOGs) and compare our results with submillimeter-selected galaxies (SMGs). One set of 39 DOGs has a local maximum in their mid-infrared (mid-IR) spectral energy distribution (SED) at rest frame 1.6 {mu}m associated with stellar emission ({sup b}ump DOGs{sup )}, while the other set of 51 DOGs havemore » power-law mid-IR SEDs that are typical of obscured active galactic nuclei ({sup p}ower-law DOGs{sup )}. We measure M{sub *} by applying Charlot and Bruzual stellar population synthesis models to broadband photometry in the rest-frame ultraviolet, optical, and near-infrared of each of these populations. Assuming a simple stellar population and a Chabrier initial mass function, we find that power-law DOGs and bump DOGs are on average a factor of 2 and 1.5 more massive than SMGs, respectively (median and inter-quartile M{sub *} values for SMGs, bump DOGs, and power-law DOGs are log(M{sub *}/M{sub Sun }) = 10.42{sup +0.42}{sub -0.36}, 10.62{sup +0.36}{sub -0.32}, and 10.71{sup +0.40}{sub -0.34}, respectively). More realistic star formation histories drawn from two competing theories for the nature of ULIRGs at z {approx} 2 (major merger versus smooth accretion) can increase these mass estimates by up to 0.5 dex. A comparison of our stellar masses with the instantaneous star formation rate (SFR) in these z {approx} 2 ULIRGs provides a preliminary indication supporting high SFRs for a given M{sub *}, a situation that arises more naturally in major mergers than in smooth accretion-powered systems.« less

Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults

PubMed Central

Skibola, Christine F.; Smith, Martyn T.; Kane, Eleanor; Roman, Eve; Rollinson, Sara; Cartwright, Raymond A.; Morgan, Gareth

1999-01-01

Reduction of 5,10-methylenetetrahydrofolate (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofolate (methylTHF), the primary methyl donor for methionine synthesis, is catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR). A common 677 C → T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of methylTHF and increases the pool of methyleneTHF. Recently, another polymorphism in MTHFR (1298 A → C) has been identified that also results in diminished enzyme activity. We tested whether carriers of these variant alleles are protected from adult acute leukemia. We analyzed DNA from a case–control study in the United Kingdom of 308 adult acute leukemia patients and 491 age- and sex-matched controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. The MTHFR 677TT genotype was lower among 71 acute lymphocytic leukemia (ALL) cases compared with 114 controls, conferring a 4.3-fold decrease in risk of ALL [odds ratio (OR = 0.23; 95% CI = 0.06–0.81]. We observed a 3-fold reduction in risk of ALL in individuals with the MTHFR 1298AC polymorphism (OR = 0.33; 95% CI = 0.15–0.73) and a 14-fold decreased risk of ALL in those with the MTHFR 1298CC variant allele (OR = 0.07; 95% CI = 0.00–1.77). In acute myeloid leukemia, no significant difference in MTHFR 677 and 1298 genotype frequencies was observed between 237 cases and 377 controls. Individuals with the MTHFR 677TT, 1298AC, and 1298CC genotypes have a decreased risk of adult ALL, but not acute myeloid leukemia, which suggests that folate inadequacy may play a key role in the development of ALL. PMID:10536004

Substrate-induced fit of the ATP binding site of cytidine monophosphate kinase from Escherichia coli: time-resolved fluorescence of 3'-anthraniloyl-2'-deoxy-ADP and molecular modeling.

PubMed

Li de La Sierra, I M; Gallay, J; Vincent, M; Bertrand, T; Briozzo, P; Bârzu, O; Gilles, A M

2000-12-26

The conformation and dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichia coli (CMPK(coli)), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the enzyme. The spectroscopic properties of the bound fluorescent nucleotide change strongly with respect to those in aqueous solution. These changes (red shift of the absorption and excitation spectra, large increase of the excited state lifetime) are compared to those observed in different solvents. These data, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected from the aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP. The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completely blocked by CMP binding. The energy-minimized models of CMPK(coli) complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes and, respectively) were compared to the crystal structure of UMP/CMP kinase from Dictyostelium discoideum complexed with substrates (PDB code ). The key residues for ATP/ADP binding to CMPK(coli) were identified as R157 and I209, their side chains sandwiching the adenine ring. Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins. In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LID conformation remained unchanged, by contrast to the fluorescence data. These results provide the first experimental arguments about an ATP-mediated induced-fit of the LID in CMPK(coli) modulated by CMP, leading to a closed conformation of the active site, protected from water.

Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

PubMed Central

Breda, Ardala; Martinelli, Leonardo K. B.; Bizarro, Cristiano V.; Rosado, Leonardo A.; Borges, Caroline B.; Santos, Diógenes S.; Basso, Luiz A.

2012-01-01

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme. PMID:22745722

Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

PubMed

Krokan, Hans E; Sætrom, Pål; Aas, Per Arne; Pettersen, Henrik Sahlin; Kavli, Bodil; Slupphaug, Geir

2014-07-01

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Differential release of cell-signaling metabolites by male and female bovine embryos cultured in vitro.

PubMed

Gómez, E; Carrocera, S; Martin, D; Herrero, P; Canela, N; Muñoz, M

2018-07-01

Male and female early bovine embryos show dimorphic transcription that impacts metabolism. Individual release of metabolites was examined in a 24h single culture medium from Day-6 male and female morulae that developed to Day-7 expanded blastocysts. Embryos were produced in vitro, fertilized with a single bull and cultured in SOFaaci+6  g/L BSA. The embryonic sex was identified (amelogenin gene amplification). Embryos (N = 10 males and N = 10 females) and N = 6 blank samples (i.e. SOFaaci+6  g/L BSA incubated with no embryos) were collected from 3 replicates. Metabolome was analyzed by UHPLC-TOF-MS in spent culture medium. After tentative identification, N = 13 metabolites significantly (P < 0.05; ANOVA) differed in their concentrations between male and female embryos, although N = 10 of these metabolites showed heterogeneity (Levene's test; P > 0.05). LysoPC(15:0) was the only metabolite found at higher concentration in females (fold change [FC] male to female = 0.766). FC of metabolites more abundant in male culture medium (N = 12) varied from 1.069 to 1.604. Chemical taxonomy grouped metabolites as amino-acids and related compounds (DL-2 aminooctanoic acid, arginine, 5-hydroxy-l-tryptophan, and palmitoylglycine); lipids (2-hexenoylcarnitine; Lauroyl diethanolamide; 5,6 dihydroxyprostaglandin F1a; LysoPC(15:0); DG(14:0/14:1(9Z)/0:0) and triterpenoid); endogenous amine ((S)-N-Methylsalsolinol/(R)-N-Methylsalsolinol); n-acyl-alpha-hexosamine (N-acetyl-alpha-d-galactosamine 1-phosphate); and dUMP, a product of pyrimidine metabolism. Among the compounds originally contained in CM, female embryos significantly depleted more arginine than males and blank controls (P < 0.001). Male and female embryos induce different concentrations of metabolites with potential signaling effects. The increased abundance of metabolites released from males is consistent with the higher metabolic activity attributed to such blastocysts. Copyright © 2018 Elsevier Inc. All rights reserved.

The [C{sub 6}H{sub 10}]{sup {sm{underscore}bullet}+} hypersurface: The parent radical cation Diels-Alder reaction

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

Hoffmann, M.; Schaefer, H.F. III

1999-07-21

Various possible reaction pathways between ethene and butadiene radical cation (cis- and trans-), have been investigated at different levels of theory up to UCCSD(T)/DZP/UMP2(fc)/DZP and with density functional theory at B3LYP/DZP. A stepwise addition involving open chain intermediates and leading to the Diels-Alder product, the cyclohexene radical cation, was found to have a total activation barrier {Delta}G{sup 298{ne}} = 6.3 kcal mol{sup {minus}1} and a change in free Gibbs energy, {Delta}G{sup 298}, of {minus}33.5 kcal mol{sup {minus}1}. On the E{degree} potential energy surface, all transition states are lower in energy than separated ethene and butadiene, the exothermicity {Delta}E = -45.6more » kcal mol{sup {minus}1}. A more direct path could be characterized as stepwise with one intermediate only at the SCF level but not at electron-correlated levels and hence might actually be a concerted strongly asynchronous addition with a very small or no activation barrier (UCCSD(T)/DZP/UHF/6-31G* gives a {Delta}G{sup 298{ne}} of 0.8 kcal mol{sup {minus}1}). The critical step for another alternative, the cyclobutanation-vinylcyclobutane/cyclohexene rearrangement, is a 1,3-alkyl shift which involves a barrier ({Delta}G{sup 298{ne}}) only 1.7 kcal mol{sup {minus}1} higher than that of stop use addition for both cis-, and trans-butadiene radical cation. However, from the (ethene and trans-butadiene) reactions, ring expansion of the vinylcyclobutane radical cation intermediate, to a methylene cyclopentane radical cation, requires an activation only 1.3 kcal mol{sup {minus}1} larger than for (trans-butadiene radical). While cis/trans isomerization of free butadiene radical cation requires a high activation (24.9 kcal mol{sup {minus}1}), a reaction sequence involving addition of ethene (to stepwise give an open chain intermediate and vinyl cyclobutane radical cation) has a barrier of only 3.5 kcal mol{sup {minus}1} ({Delta}G{sup 298{ne}}). This sequence also makes ethene and butadiene radical cations to exchange terminal methylene groups.« less

Direct dynamics simulation of dioxetane formation and decomposition via the singlet .O-O-CH2-CH2. biradical: Non-RRKM dynamics

NASA Astrophysics Data System (ADS)

Sun, Rui; Park, Kyoyeon; de Jong, Wibe A.; Lischka, Hans; Windus, Theresa L.; Hase, William L.

2012-07-01

Electronic structure calculations and direct chemical dynamics simulations are used to study the formation and decomposition of dioxetane on its ground state singlet potential energy surface. The stationary points for 1O2 + C2H4, the singlet .O-O-CH2-CH2. biradical, the transition state (TS) connecting this biradical with dioxetane, and the two transition states and gauche .O-CH2-CH2-O. biradical connecting dioxetane with the formaldehyde product molecules are investigated at different levels of electronic structure theory including UB3LYP, UMP2, MRMP2, and CASSCF and a range of basis sets. The UB3LYP/6-31G* method was found to give representative energies for the reactive system and was used as a model for the simulations. UB3LYP/6-31G* direct dynamics trajectories were initiated at the TS connecting the .O-O-CH2-CH2. biradical and dioxetane by sampling the TS's vibrational energy levels, and rotational and reaction coordinate energies, with Boltzmann distributions at 300, 1000, and 1500 K. This corresponds to the transition state theory model for trajectories that pass the TS. The trajectories were directed randomly towards both the biradical and dioxetane. A small fraction of the trajectories directed towards the biradical recrossed the TS and formed dioxetane. The remainder formed 1O2 + C2H4 and of these ˜ 40% went directly from the TS to 1O2 + C2H4 without getting trapped and forming an intermediate in the .O-O-CH2-CH2. biradical potential energy minimum, a non-statistical result. The dioxetane molecules which are formed dissociate to two formaldehyde molecules with a rate constant two orders of magnitude smaller than that predicted by Rice-Ramsperger-Kassel-Marcus theory. The reaction dynamics from dioxetane to the formaldehyde molecules do not follow the intrinsic reaction coordinate or involve trapping in the gauche .O-CH2-CH2-O. biradical potential energy minimum. Important non-statistical dynamics are exhibited for this reactive system.

Map-based cloning and functional analysis of YGL8, which controls leaf colour in rice (Oryza sativa).

PubMed

Zhu, Xiaoyan; Guo, Shuang; Wang, Zhongwei; Du, Qing; Xing, Yadi; Zhang, Tianquan; Shen, Wenqiang; Sang, Xianchun; Ling, Yinghua; He, Guanghua

2016-06-13

As the indispensable part of plant, leaf blade mainly functions as the production workshops where organic substance is produced by photosynthesis. Leaf colour mutation is a genetic phenomenon that has a high frequency and is easily identified. The mutations always exhibit negative impact on the development of plants in any of the different stages of growth. Up to now, numerous genes involved in leaf colour mutations have been cloned. In this study, a yellow-green leaf mutant, yellow-green leaf 8 (ygl8), with stable genetic phenotype, has been screened out in the progeny of an excellent indica restorer line Jinhui 10 with seeds treated by EMS. The levels of Chl a, Chl b and total chlorophyll were significantly lower in ygl8 than those in the WT throughout the whole growth period, while no clear change was noted in the Chl a/b ratio. Transmission electron microscopy demonstrated that the lamellae were clearly intumescent and intricately stacked in ygl8. Furthermore, compared with those of the WT, the stomatal conductance, intercellular CO2 concentration, photosynthetic rate and transpiration rate of ylg8 were all significantly lower. Map-based cloning results showed that Loc_Os01g73450, encoding a chloroplast-targeted UMP kinase, corresponded to Ygl8 and played an important role in regulating leaf colour in rice (Oryza sativa). Complementation of ygl8 with the WT DNA sequence of Loc_Os01g73450 led to restoration of the normal phenotype, and transgenic RNA interference plants showed a yellow-green colour. Analysis of the spatial and temporal expression of Ygl8 indicated that it was highly expressed in leaf blades and weakly expressed in other tissues. qRT-PCR also showed that the expression levels of the major Photosystem I core subunits plastome-encoded PsaA, PsaB and PsbC were significantly reduced in ygl8. The expression levels of nuclear-encoded gene involved in Chl biosynthesis HEMC, HEME, and PORA were also decreased when compared with the wild-type. Independent of Chl biosynthesis and photosystem, YGL8 may affect the structure and function of chloroplasts grana lamellae by regulating plastid genome encoded thylakoid membrane constitutive gene expression and indirectly influences Chl biosynthesis.

Primary retention following nuclear recoil in β-decay: Proposed synthesis of a metastable rare gas oxide ((38)ArO4) from ((38)ClO4(-)) and the evolution of chemical bonding over the nuclear transmutation reaction path.

PubMed

Timm, Matthew J; Matta, Chérif F

2014-12-01

Argon tetroxide (ArO4) is the last member of the N=50 e(-) isoelectronic and isosteric series of ions: SiO4(4-), PO4(3-), SO4(2-), and ClO4(-). A high level computational study demonstrated that while ArO4 is kinetically stable it has a considerable positive enthalpy of formation (of ~298kcal/mol) (Lindh et al., 1999. J. Phys. Chem. A 103, pp. 8295-8302) confirming earlier predictions by Pyykkö (1990. Phys. Scr. 33, pp. 52-53). ArO4 can be expected to be difficult to synthesize by traditional chemistry due to its metastability and has not yet been synthesized at the time of writing. A computational investigation of the changes in the chemical bonding of chlorate (ClO4(-)) when the central chlorine atom undergoes a nuclear transmutation from the unstable artificial chlorine isotope (38)Cl to the stable rare argon isotope (38)Ar through β-decay, hence potentially leading to the formation of ArO4, is reported. A mathematical model is presented that allows for the prediction of yields following the recoil of a nucleus upon ejecting a β-electron. It is demonstrated that below a critical angle between the ejected β-electron and that of the accompanying antineutrino their respective linear momentums can cancel to such an extent as imparting a recoil to the daughter atom insufficient for breaking the Ar-O bond. As a result, a primary retention yield of ~1% of ArO4 is predicted following the nuclear disintegration. The study is conducted at the quadratic configuration interaction with single and double excitations [QCISD/6-311+G(3df)] level of theory followed by an analysis of the electron density by the quantum theory of atoms in molecules (QTAIM). Crossed potential energy surfaces (PES) were used to construct a PES from the metastable ArO4 ground singlet state to the Ar-O bond dissociation product ArO3+O((3)P) from which the predicted barrier to dissociation is ca. 22kcal/mol and the exothermic reaction energy is ca. 28kcal/mol [(U)MP2/6-311+G(d)]. Copyright © 2014 Elsevier Ltd. All rights reserved.

Core Proteomic Analysis of Unique Metabolic Pathways of Salmonella enterica for the Identification of Potential Drug Targets.

PubMed

Uddin, Reaz; Sufian, Muhammad

2016-01-01

Infections caused by Salmonella enterica, a Gram-negative facultative anaerobic bacteria belonging to the family of Enterobacteriaceae, are major threats to the health of humans and animals. The recent availability of complete genome data of pathogenic strains of the S. enterica gives new avenues for the identification of drug targets and drug candidates. We have used the genomic and metabolic pathway data to identify pathways and proteins essential to the pathogen and absent from the host. We took the whole proteome sequence data of 42 strains of S. enterica and Homo sapiens along with KEGG-annotated metabolic pathway data, clustered proteins sequences using CD-HIT, identified essential genes using DEG database and discarded S. enterica homologs of human proteins in unique metabolic pathways (UMPs) and characterized hypothetical proteins with SVM-prot and InterProScan. Through this core proteomic analysis we have identified enzymes essential to the pathogen. The identification of 73 enzymes common in 42 strains of S. enterica is the real strength of the current study. We proposed all 73 unexplored enzymes as potential drug targets against the infections caused by the S. enterica. The study is comprehensive around S. enterica and simultaneously considered every possible pathogenic strain of S. enterica. This comprehensiveness turned the current study significant since, to the best of our knowledge it is the first subtractive core proteomic analysis of the unique metabolic pathways applied to any pathogen for the identification of drug targets. We applied extensive computational methods to shortlist few potential drug targets considering the druggability criteria e.g. Non-homologous to the human host, essential to the pathogen and playing significant role in essential metabolic pathways of the pathogen (i.e. S. enterica). In the current study, the subtractive proteomics through a novel approach was applied i.e. by considering only proteins of the unique metabolic pathways of the pathogens and mining the proteomic data of all completely sequenced strains of the pathogen, thus improving the quality and application of the results. We believe that the sharing of the knowledge from this study would eventually lead to bring about novel and unique therapeutic regimens against the infections caused by the S. enterica.

Hydraulic Properties of Diatomaceous Structure on Physical Properties and Wire-line Logging - An example of off Sanriku, North Japan -

NASA Astrophysics Data System (ADS)

Ojima, T.; Saito, S.

2013-12-01

During Ocean Drilling Program (ODP) Leg. 186, two sites (Site 1150 and Site 1151) were drilled on the continental slope of the deep-sea forearc basin of northern Japan. Diatomaceous sediments were recovered Site 1150 (39° 10.9' N, 143° 19.9' E) and Site 1151 (38° 45.1' N, 143° 20.0' E), and the depth of each site is 1181.60 mbsf and 1113.60 mbsf, respectively. This area is under the influence of the Oyashio current and is one of the highly bio-productive regions of the North Pacific Ocean (Motoyama et al., 2004). The combination of high productivity and active tectonic deformation that often caused high rate accumulating of fossil and organic rich sediments. The onboard results of porosity measurements show high value (50-70 %) down to 1000 mbsf, and obviously higher than nearby subduction trench, Nankai Trough (Taylor and Fisher, 1993). There is a possibility that diatomaceous shell keep a frame structure from effective stress and load pressure. On another drilling site result, DSDP (Deep Sea Drilling Project) Leg. 19 located 60 km to the north of ODP sites, was reported high value of porosity, but recognized only shallow range (>500 mbsf) (Shephard and Bryant, 1980). We focused on the relationships between physical property, microstructure, and logging data at deep range(~1000 mbsf). We picked 14 samples to observe microstructure using SEM and measure permeability using flow-ump approach (1.5-4.5 MPa). Logging data were collected using wireline logging (Sacks and Suyehiro, 2003). Based on these results, it is expected that microstructure and logging can be integrated into a general model of core-log correlation. We observed many pores in and around diatom fossils using SEM even in the sample from deeper than 1000 mbsf, and measured pore size distribution and permeability at each depth. Generally, porosity decreases by effective stress and load pressure, and permeability also decrease with down hole. In this site, we recognized down hole decreases of pore space and preservations of diatom shell by using SEM. However, measured permeability displayed subtle patterns in downhole. These results can be considered that intact diatom shells don't always contribute to permeability, while fractured fabrics play good correlation with consolidation. Furthermore, correlations between water content ratio, permeability, and ithostratigraphic variance can be considered as a good index for hydraulic sedimental conditions. In this presentation, We show results of data integration of lithostratigraphy, measurements values, logging data, and physical properties.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

The concentration of prebiotic organic building blocks may have promoted the formation of biopolymers in the environment of the early Earth. We therefore studied the adsorption of RNA monomers AMP, GMP, CMP, and UMP, and DNA monomers dGMP, dCMP, and TMP, on minerals that were abundant in the early Earth environment as the result of aqueous or hydrothermal alteration of the primitive oceanic crust. We focused our study on swelling clays, i.e. nontronite and montmorillonite, and non-swelling phyllosilicates, i.e. pyrophyllite, chlorite, lizardite and chrysotile suspended in an aqueous saline solution analog to seawater. In this reference study, adsorption experiments were carried out under standard conditions of pressure and temperature and controlled pH. Under such conditions, this work is also relevant to the preservation of nucleic acids in Fe-Mg-rich terrestrial and Martian soils. We compared the adsorption of the different monomers on individual minerals, as well as the adsorption of single monomers on the whole suite of minerals. We found that DNA monomers adsorb much more strongly than RNA monomers, and that any monomer containing the G nucleobase adsorbed more strongly than one containing the C nucleobase. At high surface loadings (greater than about 1 mM monomer in aqueous solution) we also found a dramatic increase in the slope of adsorption isotherm on the swelling clays, leading to large increases in the amounts adsorbed. Data were processed in order to understand the adsorption mechanism of nucleotides onto mineral surfaces. We infer that all nucleotides behave as homologous molecules in regard to their adsorption onto the studied mineral surfaces. At low to moderate surface loadings, their adsorption is best explained by a single mechanism common to the suite of minerals of the present study. At pH 7, adsorption certainly proceeds by ligand exchange between the phosphate group and the hydroxyls of the broken edges of phyllosilicates leading to the saturation of lateral surfaces. Below pH 4, swelling clays also adsorb nucleotides through cation exchange on basal surfaces, whereas non-swelling phyllosilicates do not. However, at high surface loadings an additional mechanism stabilizing adsorption occurs. Given the proposed adsorption mechanisms, the possibility of a favorable polymerization at the mineral-water interface is discussed. We propose that Fe-Mg rich phyllosilicates tightly bind nucleotides, under ambient conditions, and concentrate them up to 1000 times the solution concentration upon saturation. Nontronites have the most diverse and favorable adsorption behaviors and could have helped to the concentration and polymerization of nucleotides under primitive Earth-like conditions.

The Hypothesis that the Genetic Code Originated in Coupled Synthesis of Proteins and the Evolutionary Predecessors of Nucleic Acids in Primitive Cells

PubMed Central

Francis, Brian R.

2015-01-01

Although analysis of the genetic code has allowed explanations for its evolution to be proposed, little evidence exists in biochemistry and molecular biology to offer an explanation for the origin of the genetic code. In particular, two features of biology make the origin of the genetic code difficult to understand. First, nucleic acids are highly complicated polymers requiring numerous enzymes for biosynthesis. Secondly, proteins have a simple backbone with a set of 20 different amino acid side chains synthesized by a highly complicated ribosomal process in which mRNA sequences are read in triplets. Apparently, both nucleic acid and protein syntheses have extensive evolutionary histories. Supporting these processes is a complex metabolism and at the hub of metabolism are the carboxylic acid cycles. This paper advances the hypothesis that the earliest predecessor of the nucleic acids was a β-linked polyester made from malic acid, a highly conserved metabolite in the carboxylic acid cycles. In the β-linked polyester, the side chains are carboxylic acid groups capable of forming interstrand double hydrogen bonds. Evolution of the nucleic acids involved changes to the backbone and side chain of poly(β-d-malic acid). Conversion of the side chain carboxylic acid into a carboxamide or a longer side chain bearing a carboxamide group, allowed information polymers to form amide pairs between polyester chains. Aminoacylation of the hydroxyl groups of malic acid and its derivatives with simple amino acids such as glycine and alanine allowed coupling of polyester synthesis and protein synthesis. Use of polypeptides containing glycine and l-alanine for activation of two different monomers with either glycine or l-alanine allowed simple coded autocatalytic synthesis of polyesters and polypeptides and established the first genetic code. A primitive cell capable of supporting electron transport, thioester synthesis, reduction reactions, and synthesis of polyesters and polypeptides is proposed. The cell consists of an iron-sulfide particle enclosed by tholin, a heterogeneous organic material that is produced by Miller-Urey type experiments that simulate conditions on the early Earth. As the synthesis of nucleic acids evolved from β-linked polyesters, the singlet coding system for replication evolved into a four nucleotide/four amino acid process (AMP = aspartic acid, GMP = glycine, UMP = valine, CMP = alanine) and then into the triplet ribosomal process that permitted multiple copies of protein to be synthesized independent of replication. This hypothesis reconciles the “genetics first” and “metabolism first” approaches to the origin of life and explains why there are four bases in the genetic alphabet. PMID:25679748

Interaction of ribonucleotides with oxide and silicate minerals under varying environmental conditions

NASA Astrophysics Data System (ADS)

Feuillie, C.; Sverjensky, D. A.; Hazen, R. M.

2013-12-01

Large quantities of nucleic acids are found in natural environments, released after the death of an organism and subsequent cell lysis [1]. Nucleic acids are known to adsorb on mineral surfaces [2, 3, 4], which protect them from degradation, whether enzymatic [5, 6] or UV-mediated [7]. It may then contribute to the extracellular genetic pool available in soils to microorganisms for horizontal gene transfers [8]. In order to better understand the behaviour of extracellular nucleic acids in soils, we have investigated the interactions between nucleotides, 5'-GMP, 5'-CMP, 5'-AMP and 5'-UMP, and α-alumina as a model compound for Al in six-fold coordination in soil minerals. We carried out batch adsorption experiments over a wide range of pH, ionic strength and surface loading. Alumina adsorbs high amounts of nucleotides > 2 μmol/m2. In similar environmental conditions, swelling clays such as nontronite and montmorillonite adsorb less than 0.1 μmol/m2 if the total surface area is taken under consideration. However, if only the edges of clay particles are considered, the amount of nucleotides adsorbed reaches values between 1.2 and 2 μmol/m2 [9], similar to the alumina and consistent with ';oxide-like' surface sites on the edges of the clay particles. Surface complexation modeling enabled us to predict the speciation of the surface species on the alumina, as well as the stoichiometry and thermodynamic equilibrium constants for the adsorption of nucleotides. We used the extended triple-layer model (ETLM), that takes into account the electrical work linked to the desorption of chemisorbed water molecules during the formation of inner-sphere complexes. Two surface species are thought to form on the surface of corundum: a monodentate inner-sphere complex, dominant at pH < 7.5, and a bidentate outer-sphere complex, dominant at higher pH. Both complexes involve interactions between the negatively charged phosphate group and the positively charged surface of alumina. Our results provide a better understanding of how nucleic acids attach to mineral surfaces under varying environmental conditions in soil environments. Moreover, the predicted configuration of nucleotide surface species, bound via the phosphate group, could have implications for the abiotic formation and concentration of nucleic acids in the context of the origin of life. References : [1] Lorenz and Wackernagel (1987), Applied and environmental microbial., 2948-2952 [2] Ferris (2005), Reviews in mineralogy & geochemistry 59, 187-210 [3] Cleaves H.J. et al. (2011), Chemosphere 83, 1560-1567 [4] Arora & Kamaluddin (2009), Astrobiology 9, 165-171 [5] Cai et al. (2006), Environ. Sci. Technol. 40 (9), 2971-2976 [6] Franchi and Gallori (2005),Gene 346, 205-214 [7] Scappini et al. (2004), International Journal of Astrobiology 3(1), 17-19 [8] Levy-Booth et al. (2007), Soil Biol. Biochem. 39, 2977-2991. [9] Feuillie et al. (2013), Geochimica et Cosmochimica Acta (in press)

Isomerization and fragmentation reactions of gaseous phenylarsane radical cations and phenylarsanyl cations. A study by tandem mass spectrometry and theoretical calculations.

PubMed

Letzel, Matthias; Kirchhoff, Dirk; Grützmacher, Hans-Friedrich; Stein, Daniel; Grützmacher, Hansjörg

2006-04-28

The unimolecular reactions of radical cations and cations derived from phenylarsane, C6H5AsH2 (1) and dideutero phenylarsane, C6H5AsD2 (1-d2), were investigated by methods of tandem mass spectrometry and theoretical calculations. The mass spectrometric experiments reveal that the molecular ion of phenylarsane, 1*+, exhibits different reactivity at low and high internal excess energy. Only at low internal energy the observed fragmentations are as expected, that is the molecular ion 1*+ decomposes almost exclusively by loss of an H atom. The deuterated derivative 1-d2 with an AsD2 group eliminates selectively a D atom under these conditions. The resulting phenylarsenium ion [C6H5AsH]+, 2+, decomposes rather easily by loss of the As atom to give the benzene radical cation [C6H6]*+ and is therefore of low abundance in the 70 eV EI mass spectrum. At high internal excess energy, the ion 1*+ decomposes very differently either by elimination of an H2 molecule, or by release of the As atom, or by loss of an AsH fragment. Final products of these reactions are either the benzoarsenium ion 4*+, or the benzonium ion [C6H7]+, or the benzene radical cation, [C6H6]*+. As key-steps, these fragmentations contain reductive eliminations from the central As atom under H-H or C-H bond formation. Labeling experiments show that H/D exchange reactions precede these fragmentations and, specifically, that complete positional exchange of the H atoms in 1*+ occurs. Computations at the UMP2/6-311+G(d)//UHF/6-311+G(d) level agree best with the experimental results and suggest: (i) 1*+ rearranges (activation enthalpy of 93 kJ mol(-1)) to a distinctly more stable (DeltaH(r)(298) = -64 kJ mol(-1)) isomer 1 sigma*+ with a structure best represented as a distonic radical cation sigma complex between AsH and benzene. (ii) The six H atoms of the benzene moiety of 1 sigma*+ become equivalent by a fast ring walk of the AsH group. (iii) A reversible isomerization 1+<==>1 sigma*+ scrambles eventually all H atoms over all positions in 1*+. The distonic radical cation 1*+ is predisposed for the elimination of an As atom or an AsH fragment. The calculations are in accordance with the experimentally preferred reactions when the As atom and the AsH fragment are generated in the quartet and triplet state, respectively. Alternatively, 1*(+) undergoes a reductive elimination of H2 from the AsH2 group via a remarkably stable complex of the phenylarsandiyl radical cation, [C6H5As]*+ and an H2 molecule.

Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis.

PubMed

Richard, John P; Amyes, Tina L; Reyes, Archie C

2018-04-17

The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme-substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5'-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5'-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 10 23 -fold rate acceleration for this enzyme, which had " jolted many biochemists' assumptions about the catalytic potential of enzymes." Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid p K a that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the p K a = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein-ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO 2 by H + that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO 2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.

[System of telesonography with synchronous teleconsultations and asynchronous telediagnoses (Togo)].

PubMed

Adambounou, K; Farin, F; Boucher, A; Adjenou, K V; Gbeassor, M; N'dakena, K; Vincent, N; Arbeille, P

2012-01-01

Ultrasonography is an important nonirradiating diagnostic medical imaging procedure, frequently used, especially in urgent circumstances. This relatively inexpensive noninvasive examination makes it possible to diagnose disorders in various parts of the human body, by examining, for example, the abdomen and pelvis, the cardiovascular system, and the muscles and joints. Ultrasound is also an operator-dependent examination, in that the quality of the result depends on precision in the manipulation of the probe. Unfortunately, many small medical centers and isolated sites do not have an appropriate well-trained sonographer to perform initial evaluations, and an untrained operator cannot capture the appropriate echographic views required for a safe diagnosis of current patients, even with realtime vocal guidance (personal data). The lack of experienced physicians or qualified technicians means that diagnostic ultrasound is not always accessible to patients for rapid examination worldwide, especially in Africa, Amazonia or near the North or South Poles. This situation has led to the development of a new concept of telemedicine: telesonography, with a remote ultrasound diagnosis either in real time (synchronous) or delayed (asynchronous; store-and-forward). These systems of real-time telesonography and data transmission require expensive and complex technology with sophisticated equipment not available in many developing countries. The purpose of this study is to design a low-cost real-time system of telesonography for teleconsultations with experts and a delayed telediagnostic mode between isolated peripheral hospitals and a University Hospital center (UHC). An IP camera and an internet video server were installed in a geographically isolated site equipped with an ultrasound machine and an operator with basic training in its use. Synchronous teleconsultation (second-opinion diagnosis) is possible via internet with a UHC expert. If no ultrasound operator is available at the isolated center, volume capture-and-store software is used. Later on, the UHC expert uses Echo-Cnes 3D software to reconstruct the organs scanned. The expert can then navigate within the reconstructed volume and display any plane. Volume capture is performed by tilting (± 40°) to both sides vertically to the skin. To locate the probe on the organ acoustic window, the novice operator uses acoustic window mapping designed by our laboratory (UMPS-Tours). The system was tested between the Tsévié regional hospital in Togo (40 km from Lomé, Togo, and 4500 km from Tours, France) and the UHC at Lomé and the Trousseau UHC in Tours. With an average internet connection of 2 Mbps, the quality of transmission of the background video and ultrasound sequence videos from Tsévié towards Lomé was satisfactory (16 images/s) with a maximal transmission delay of 1.5 s (almost in real time). A video conference between the Trousseau UHC in Tours, the UHC Campus in Lomé and the Tsévié Hospital was possible and the bandwidth allowed the Lomé experts (radiologists) to perform real-time telesonography with very satisfactory results (ultrasound diagnoses obtained) for abdominal (n = 5), pelvic (n = 3), obstetric (n = 2), prostate (n = 2) and mammary (n = 2) ultrasound, both normal and pathological. Because the doctors at Tsévié had minimal experience with ultrasound, complete ultrasound diagnoses were obtained by combining remote voice instruction for image capture and full diagnosis by Echo-Cnes. Asynchronous telediagnosis was also performed with Tsévié operators who lacked ultrasound expertise but could perform the required tilt movements (after 3 training sessions). The expert at Trousseau UHC performed real-time telesonography with the Tsévié Hospital for two cases requiring abdominal images and another viewing of the prostate. He also performed asynchronous reconstruction of the abdominal organs with Echo-Cnes. A demonstration seminar of our platform was organized successfully for 2 days during the 9(th) Congress of the French-speaking Black Africa Society of Radiology (SRANF in French) held from 4 till 6 May 2011 at the hotel EDA OBA. During this seminar, 4 ultrasound teleconsultations were performed from the hotel by eminent African radiologists. This preliminary study, although limited in the number of patients, allowed us to assess the technical features of our telesonography system. Togo, a developing country with a very modest infrastructure for information and communication, was an ideal site for a first test of this platform. Our system of remote ultrasound requires the local patient center to be equipped simply with a 2D ultrasound machine. The cost is quite low, in comparison to the asynchronous techniques requiring 3D devices. The high cost of 3D or 4D ultrasound machines and their fragility make it difficult to install them at the isolated sites and was a serious obstacle in the development of this system. If the center already has a 2D device and a computer, the cost to equip it with the remaining communications materials is 1,500 €. The experience in Togo clearly highlighted the possibility of teletraining and complete teleradiology with our system. The next stage of this work will seek to validate the results of this preliminary experience on a larger sample with more precise assessment criteria in 2012. The results will allow the widespread dissemination and routine use of this system in developing countries.