The 2'-5'-oligoadenylate synthetase 3 enzyme potently synthesizes the 2'-5'-oligoadenylates required for RNase L activation.

UNLABELLED: The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS. IMPORTANCE: We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.

A role of erythrocytes in adenosine monophosphate initiation of hypometabolism in mammals.

Biochemical and mechanistic aspects into how various hypometabolic states are initiated in mammals are poorly understood. Here, we show how a state of hypometabolism is initiated by 5'-AMP uptake by erythrocytes. Wild type, ecto-5'-nucleotidase-deficient, and adenosine receptor-deficient mice undergo 5'-AMP-induced hypometabolism in a similar fashion. Injection of 5'-AMP leads to two distinct declining phases of oxygen consumption (VO(2)). The phase I response displays a rapid and steep decline in VO(2) that is independent of body temperature (T(b)) and ambient temperature (T(a)). It is followed by a phase II decline that is linked to T(b) and moderated by T(a). Altering the dosages of 5'-AMP from 0.25- to 2-fold does not change the phase I response. For mice, a T(a) of 15 degrees C is effective for induction of DH with the appropriate dose of 5'-AMP. Erythrocyte uptake of 5'-AMP leads to utilization of ATP to synthesize ADP. This is accompanied by increased glucose but decreased lactate levels, suggesting that glycolysis has slowed. Reduction in glycolysis is known to stimulate erythrocytes to increase intracellular levels of 2,3-bisphosphoglycerate, a potent allosteric inhibitor of hemoglobin's affinity for oxygen. Our studies showed that both 2,3-bisphosphoglycerate and deoxyhemoglobin levels rose following 5'-AMP administration and is in parallel with the phase I decline in VO(2). In summary, our investigations reveal that 5'-AMP mediated hypometabolism is probably triggered by reduced oxygen transport by erythrocytes initiated by uptake of 5'-AMP.

Aptamer-modified micellar electrokinetic chromatography for the enantioseparation of nucleotides.

In this paper, a new aptamer-based capillary electrophoresis (CE) method, which was able to separate the enantiomers of an anionic target (adenosine monophosphate, AMP) displaying the same electrophoretic mobility as that of the oligonucleotidic chiral selector, is reported. The design of the aptamer-modified micellar electrokinetic chromatography (MEKC) mode consisted of nonionic micelles which acted as a pseudostationary phase and a hydrophobic cholesteryl group-tagged aptamer (Chol-Apt) which partitioned into the uncharged micellar phase. Under partial-filling format and suppressed electroosmotic flow conditions, the strong mobility alteration of Chol-Apt permitted AMP enantiomers to pass through the micelle-anchored aptamer zone and promoted the target enantioseparation. The influence of several electrophoretic parameters (such as concentration and nature of the nonionic surfactant, preincubation of the Chol-Apt and surfactant, capillary temperature, and applied voltage) on the AMP enantiomer migration was investigated in order to define the utilization conditions of the aptamer-modified MEKC mode. The chiral resolution, in a single run, of three adenine nucleotides, i.e., AMP, ADP (adenosine diphosphate), and ATP (adenosine triphosphate), was further accomplished using such methodology. This approach demonstrates the possibility to extend the CE applicability of aptamer chiral selectors to potentially any target, without restriction on its charge-to-mass ratio.

Adenosine-3',5'-phosphate: identification as acrasin in a species of cellular slime mold.

Acrasin, the chemotactic agent responsible for aggregation in thecellular slime molds, has been identified for one species, Dictyostelium discoideum, as adenosine-3', 5'-phosphate.

A sensitive method for the separation and quantification of low-level adenine nucleotides using porous graphitic carbon-based liquid chromatography and tandem mass spectrometry.

A liquid chromatography coupled to heated electrospray ionization/tandem mass spectrometry (LC-HESI-MS/MS) method was developed for the simultaneous quantitative analysis of low nanomolar level adenine nucleotides AMP, ADP, ATP, cyclic AMP (cAMP), and the nucleoside adenosine. For analyte retention and separation, reverse phase chromatography using porous graphitic carbon (PGC) was employed as it provided full resolution. The erratic chromatographic behaviour characteristic of PGC, including deterioration of analyte resolution and increased peak tailing (leading to decreased sensitivity), was mitigated by incorporating acidic equilibration within runs using a quaternary gradient. Analyte resolution and chromatographic sensitivity were still lost after a period of column inactivity; hence a pre-conditioning protocol was implemented between batches to regenerate the column. These column regeneration measures also allowed elution of AMP, ADP and ATP in the sequence of mono- to tri- nucleotides, differing from conventional reverse phase elution where analytes elute with decreasing polarity. This nucleotide elution sequence has the advantage of overcoming potential mis-annotation and inaccurate quantification of smaller nucleotides caused by in-source fragmentation of ATP. The method was validated in granulosa cell conditioned media, with the LLOQs ranging between 10-50nM for most analytes. To verify the method using biological samples, nucleotide secretion was measured in granulosa cell conditioned media under various treatments known to alter their levels. Moreover, the method was applied to cumulus-oocyte complex cell lysates to examine its linearity in a complex matrix.

Chromatographic analysis of dopamine metabolism in a Parkinsonian model.

The present study examined the metabolism of released dopamine from rat striatum upon chronic rotenone exposure. The sample separation was carried out by two-dimensional, reversed-phase and ion pair reversed-phase chromatography using on-line solid phase extraction enrichment. Reduced dopamine content and decreased extracellular level of [(3)H] and endogenous dopamine evoked by electrical stimulation indicated the injury of dopaminergic pathway. Sensitivity of dopaminergic neurons were increased to oxidative stress with enhanced release of dopamine and formation of oxidized metabolite dopamine quinone (DAQ). Utilizing multidimensional detection, EC at -100 mV reduction potential, the method has been applied for identification of DAQ and aminochrome (DAC).

Separation of nucleotides with an aqueous mobile phase using pH- and temperature-responsive polymer modified packing materials.

A new method for the qualitative analysis of adenosine nucleotides (AMP, ADP, and ATP) and synthetic oligonucleotides has been proposed, utilizing a pH- and temperature-responsive polymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm) as the stationary phase of HPLC. In the chromatographic system using the copolymer with ionizable groups of modified packing materials, we investigated how to separate adenosine nucleotides and oligonucleotides by temperature. The properties of the surface of the copolymer-grafted stationary phase altered from hydrophilic to hydrophobic and from charged to non-charged due to changes in the temperature and in the pH, respectively. In addition, it is possible to exhibit and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena result from changes in the charge and hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface with the controlling temperature. A pH- and temperature-responsive chromatography would be greatly useful for biopolymer and nucleotide separation and purification.

Quantitative separation of nucleotides on mercurated dextran.

Dextran gels of varying porosites (Sephadex G series) were chemically modified so as to contain covalently bound monofunctional mercury. Mercurated Sephadex of the porosity G-25 was then used to fractionate mixtures of mono- and dinucleotides into the constituent components. Separation is based on the affinity of the nitrogen binding sites of the purine and pyrimidine derivatives for organomercurial Hg+. Thus, a mixture composed of the four mononucleotides Cyd-5'-P, Ado-3'-P, Guo-2'(3')-P, dThd-5'-P and of the four dinucleotides Cyd-P-Cyd, Ado-P-Ado, Guo-P-Urd, and Urd-P-Urd could be separated into eight well-resolved fractions by using a combination Tris-bicarbonate/carbonate buffer system of increasing pH as an eluent. Complete separation was also achieved when a mixture of Ado 3:5'-P, Ado 5'-P, Ado-5'-PP, and Ado-5'-PPP was fractionated on mercurated Sephadex G-25. Again, Tris-bicarbonate/carbonate buffer served as an eluent. Lastly, fractionation can also be performed at a constant pH by offering other suitable ligands, for instance Cl-, that will compete with nucleotides for monofunctional Hg+. The fractionation behavior of mercurated Sephadex G-25 can be fully understood on the basis of the complexing properties of monofunctional Hg+. This has been shown by calculating the net retention volume ratios of several nucleotides with the help of the known interaction parameters of corresponding nucleosides with CH3 HgOH and by comparing the predicted ratios with the experimentally measured ones. Finally, the acid-base properties of mercurated Sephadex G-25 as well as its affinity for chloride and iodide ions have been determined. The data agree quite well with those known for CH3 HgOH.

Presence of epsilon-adenosine tetraphosphate in chromaffin granules after transport of epsilon-ATP.

Adenosine 5'-tetraphosphate (Ap4) is a natural constituent of chromaffin granules with concentration values of 2.2 +/- 0.1 nmol/mg of protein and a ratio 245 +/- 40 times lower with respect to ATP (n = 4). The granular transport of epsilon-ATP resulted in a time- and concentration-dependent production of epsilon-adenosine tetraphosphate (epsilon-Ap4) at the intragranular level. The epsilon-Ap4 formation followed a hyperbolic saturation kinetic at low epsilon-ATP concentrations with K(m) value of 0.4 microM epsilon-ATP intragranular (1.15 pmol/mg of granular protein). Intragranular concentrations of epsilon-ATP higher than 500 pmol/mg of protein (approximately to 175 microM intragranular) resulted in a non-saturable production of epsilon-Ap4.

Adenosine 5'-tetraphosphate (Ap(4)), a new agonist on rat midbrain synaptic terminal P2 receptors.

The aim of this study was to see whether the compound adenosine 5'-tetraphosphate (Ap(4)) is active in the central nervous system by examining its effect on isolated rat brain synaptic terminals. Ap(4) proved to be more resistant to ecto-enzymatic hydrolysis than adenosine triphosphate (ATP), showing only 2% hydrolysis after a 2-min incubation, compared to 75% for ATP. In addition, Ap(4) was able to produce concentration-dependent increases in intracellular Ca(2+) when applied extracellularly. This action was dependent upon the presence of extracellular calcium. Ap(4) acts through ionotropic ATP receptors (P2X receptors) and not through diadenosine polyphosphate receptors, since ATP abolished the response elicited by Ap(4) whereas Ap(5)A did not. Ap(4), ATP and ATP-gamma-S were of similar potency (EC(50) approximately 20 microM) while 2MeSATP, alpha,beta-meATP and ADP-beta-S possessed slightly lower potency (EC(50) approximately 50 microM). The P2-purinoceptor antagonists suramin and PPADS blocked the Ap(4) effect. The IC(50) values for these compounds were 35.5 and 7.8 microM respectively. Diinosine polyphosphates and inosine tetraphosphate inhibited the response elicited by Ap(4) with IC(50) values that varied between approximately 40 and 50 microM. These results show that Ap(4) is as good an agonist as ATP on synaptosomal P2X receptors, being more resistant to extracellular hydrolysis by ecto-nucleotidases.

Polymeric liquid membrane electrodes incorporated with macrocyclic hexaamines for screening adenine nucleotides.

Lipophilic macrocyclic hexaamines supported by a poly(vinyl chloride) PVC matrix were used for the construction of liquid membrane electrodes sensitive toward adenine nucleotide polyanions. The membrane potential strongly depended on the pH of the sample solution. This phenomenon occurs due to the ability of the ionophore to accept protons. Therefore, the optimum pH was determined based on potential pH profile. The potential measurements were carried out at pH 6.0 in the presence of 10(-2) M 2-[N-morpholino] ethanesulfonic acid (MES) buffer. The potential response of these electrodes toward ATP(-4) and/or HATP(-3) was close to the Nernstian slope. The selectivities against ADP(-3), AMP(-2), HPO(4)(-2), and monovalent inorganic anions were estimated using the matched potential method. Chloride ions slightly affected potential response of the electrodes toward ATP(-4)/HATP(-3). The influence of ionophore chemical structure on the selectivity and the sensitivity of these electrodes is briefly discussed.

Amphetamine-induced release of diadenosine polyphosphates--Ap4A and Ap5A--from caudate putamen of conscious rat.

The release of diadenosine polyphosphates--diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A)--was measured by intracerebral push-pull perfusion in conscious rats after systemic amphetamine injection. Samples were collected from the caudate putamen, and nucleotide compounds were analyzed by HPLC. The presence of Ap4A and Ap5A was demonstrated by their retention times and phosphodiesterase digestion. Dinucleotides were not detectable before amphetamine injection (5 mg/kg). The maximal levels were reached 20 min after the injection with values of 12.9 +/- 0.9 and 11.5 +/- 0.9 pmol/fraction for Ap4A and Ap5A, respectively. A slow and progressive decrease in their concentration followed. This study shows for the first time the amphetamine-induced release of diadenosine polyphosphates in conscious rats, and a role for Ap4A and Ap5A in the central nervous system is therefore suggested.

Determination of ATP impurity in adenine dinucleotides.

Adenine dinucleotides (ApnA) are extracellular signal molecules that are released from blood platelets, following stress, into the vascular system. The most abundant and best-characterized ApnA (Ap4A) interacts with a unique receptor on bovine aortic endothelial cells (BAEC) where it induces nitric oxide. Ap4A also interacts with P2 purinoceptors on BAEC to modulate Ca2+ mobilization and prostacyclin release; this behavior can be equally well explained by Ap4A being either a partial agonist to these receptors, or an antagonist in the presence of ATP contamination. To discern between these two possibilities, we have investigated the presence of such contaminants in ApnA preparations. The studies herein indicate that ApnAs (n = 3-6) contain ATP impurities; thus, when characterizing the ApnA interaction with ATP-binding sites, investigators must assure that the response elicited is not partly due to an ATP impurity. We here provide a means for detecting and estimating ATP impurities within Ap4A preparations while also eliminating them; the level of this contamination is estimated to be as low as 0.2%. We applied our method to distinguish the true effect of Ap4A at P2 purinoceptors; our findings are consistent with Ap4A acting as a partial agonist to these receptors. We also applied our method to characterizing the ApnA interaction with luciferase, and found that decontaminated ApnA (n = 4-6) are weak substrates for luciferase.

Measurement of red blood cell adenosine nucleotides by high-performance liquid chromatography.

A new method for measuring ATP in stored red blood cells using high-performance liquid chromatography was compared with an established enzymatic method. The new method is based upon isocratic reverse phase chromatography using a polyvinyl alcohol gel stationary phase. The chromatograms produce quantitative results for ADP, AMP, and other nucleotides, and can be used to determine adenylate energy charge. The correlation coefficient between the two methods was 0.91, and mean ATP was 3.0 mumol/g Hb for both methods. Tests of hypothesis for mean and variance were not significant. The method is recommended as a means to study the relationships between poststorage red blood cell ATP, adenylate energy charge, total adenylates, and posttransfusion erythrocyte survival.

[Identification of the isoadenylate synthetase in the messenger RNA translation products of antiviral proteins]. / Identifikatsiia izoadenilatsintetazy v produktakh transliatsii informatsionnykh RNK antivirusnykh belkov.

A further study of conditions for induction, determinations of activity and products of mRNAs translation of AVP having nonspecific antiviral activity from L-929 cells treated with poly(I) . poly(C) was carried out. Under conditions of superinduction of interferon the yield of AVP mRNA is reduced unlike that of interferon mRNA. Manifestations of the antiviral effect of AVP mRNA translation products do not seem to require the stages of transcription as dihydrorifampicin exerts no influence on this process. The inhibiting effect of actinomycin D is more likely to be associated with its effect on the energy of cells and synthesis of macroergic compounds of ATP type. Active AVP mRNAs from L-929 cells like interferon mRNA consist mainly of poly-A-deficient mRNAs. The sedimentation rate of AVP mRNAs differs from that of interferon mRNA and is about 10S. AVP mRNA translation products in ovocytes of Xenopus laevis, in contrast to control mRNAs, contain isoadenylate synthetase responsible for synthesis of 2'5'-oligoisoadenylate, a nonspecific translation inhibitor the mechanism of action of which consists in activation of cellular endogenous nuclease. The results suggest that the mode of development of the antiviral condition by activation of isoadenylate synthetase is similar in the cells treated with interferon and poly(I) . poly(C).

Origin of salt additive effect on solute partitioning in aqueous polyethylene glycol-8000-sodium sulfate two-phase system.

Partitioning of a homologous series of dinitrophenylted (DNP-) amino acids with aliphatic side chains was examined in aqueous polyethylene glycol (PEG)-8000-sodium sulfate two-phase systems (ATPS) with the additives NaSCN, NaClO4, and NaH2PO4 at concentrations varied from 0.025M up to 0.54M. The differences between the relative hydrophobicities and electrostatic properties of the two phases in all ATPS were estimated. Partitioning of adenine, adenosine mono-, di- and tri-phosphates was also examined in all ATPSs, including those with NaCl additive. Partition coefficients for these compounds and for nonionic organic compounds previously reported [L.A. Ferreira, P. Parpot, J.A. Teixeira, L.M. Mikheeva, B.Y. Zaslavsky, J. Chromatogr. A 1220 (2012) 14.] were analyzed in terms of linear solvent regression relationship. The results obtained suggest that the effects of the salts additives are related to their influence on the water structure.

Facile preparation of organic-silica hybrid monolith for capillary hydrophilic liquid chromatography based on "thiol-ene" click chemistry.

In this work, a one-step approach to facile preparation of organic-inorganic hybrid monoliths was successfully developed. After vinyl-end organic monomers and azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethoxysilane (TMOS) and 3-mercaptopropyltrimethoxysilane (MPTMS), the homogeneous mixture was introduced into a fused-silica capillary for simultaneous polycondensation and "thiol-ene" click reaction to form the organic-silica hybrid monoliths. By employing this strategy, two types of organic-silica hybrid monoliths with positively charged quaternary ammonium and amide groups were prepared, respectively. The functional groups were successfully introduced onto the monoliths during the sol-gel process with "thiol-ene" click reaction, which was demonstrated by ζ-potential assessment, energy dispersive X-ray spectroscopy (EDX), and Fourier transform infrared (FT-IR) spectroscopy. The porous structure of the prepared monolithic columns was examined by scanning electron microscopy (SEM), nitrogen adsorption-desorption measurement, and mercury intrusion porosimetry. These results indicate the prepared organic-silica hybrid monoliths possess homogeneous column bed, large specific surface area, good mechanical stability, and excellent permeability. The prepared monolithic columns were then applied for anion-exchange/hydrophilic interaction liquid chromatography. Different types of analytes, including benzoic acids, inorganic ions, nucleosides, and nucleotides, were well separated with high column efficiency around 80,000-130,000 plates/m. Taken together, we present a facile and universal strategy to prepare organic-silica hybrid monoliths with a variety of organic monomers using one-step approach.

In situ sol-gel preparation of porous alumina monoliths for chromatographic separations of adenosine phosphates.

A method enabling the in situ preparation of porous alumina monoliths within 100 µm i.d. fused silica capillaries has been developed. These monoliths were prepared using the sol-gel process from a mixture consisting of an inorganic aluminum salt, a porogen, an epoxide, and a solvent. We investigated the effects of varying the preparation conditions on the physical characteristics of the monoliths with respect to their potential application in chromatographic separations. The best columns were obtained from a mixture of aluminum chloride hexahydrate, N,N-dimethylformamide, water, ethanol and propylene oxide. Adenosine phosphates were then separated in the optimized column with retention increasing according to number of phosphate functionalities.

Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings.

A novel core-shell composite (SiO(2)-nLPD), consisting of micrometer-sized silica spheres as a core and nanometer titania particles as a surface coating, was prepared by liquid phase deposition (LPD). Here, we show the resulting core-shell composite to have better efficient and selective enrichment for mono- and multi-phosphopeptides than commercially available TiO(2) spheres without any enhancer. The material exhibited favorable characteristics for HPLC, which include narrow pore size distribution, high surface area and pore volume. We also show that the core-shell composite can efficiently separate adenosine phosphate compounds due to the Lewis acid-base interaction between titania and phosphate group when used as HPLC packings. After coating the silica sphere with titania by LPD, the silanol of silica spheres will be shielded and that the stationary phase, C(18) bonded SiO(2)-3LPD, could be used under extreme pH condition.