Mass transfer process and separation mechanism of four 5'-ribonucleotides on a strong acid cation exchange resin.

5'-ribonucleotides including adenosine 5'-monophosphate (AMP), cytidine 5'-monophsphate (CMP), guanosine 5'-monophosphate (GMP) and uridine 5'-monophosphate (UMP) have been widely used in the food and pharmaceutical industries. This work focused on the assessment of mass transfer process and separation mechanism of four 5'-ribonucleotides and counter-ion Na+ on the strong cation exchange resin NH-1. The intraparticle diffusion was determined as the rate-limiting step for the mass transfer of AMP, CMP, GMP, and Na+ on the resin NH-1 through the Boyd model. Meanwhile, a homogeneous surface diffusion model (HSDM) combing ion exchange and physical adsorption was proposed and tested against adsorption kinetic data in the batch adsorption systems. The fixed-bed film-surface diffusion model based on the HSDM was then developed and successfully predicted the concentration profiles of 5'-ribonucleotides and the change of pH at the outlet of the fixed-bed in the dynamic adsorption and separation process. Finally, the separation mechanism of 5'-ribonucleotides was presented combining model prediction and experimental results. The separation of UMP, GMP and CMP were mainly based on their differences in isoelectric points, while that of AMP and CMP were lied with the discrepancy of their physical adsorption binding capacity with the resin NH-1.

A new ribonucleotide from Cordyceps militaris.

One new ribonucleotide, 5'-(3''-deoxy-ß-D-ribofuranosyl)-3'-deoxyadenosine (1), and 14 known compounds (2-15) were isolated from an ethanol extract of Cordyceps militaris. The chemical structures of these compounds were determined from 1D and 2D NMR (1H-1H COSY, HMBC, HMQC and NOESY) and HR-ESI-MS spectra, and results were compared with data from the literature. The effects of all isolated compounds were measured on NF-κB activation, with compound 2 exhibiting significant inhibitory activity against TNF-α-induced NF-κB reporter gene expression in HeLa cells from 3 to 100 µM.

A method for assessing efficiency of bacterial cell disruption and DNA release.

BACKGROUND: DNA-based testing is becoming the preferred method both for identifying microorganisms and for characterizing microbial communities. However, no single DNA extraction method exists that is suitable for all types of microorganisms because bacteria are variable in their susceptibility to lysis by available extraction procedures. To develop and test new DNA extraction procedures, it would be helpful to determine their efficiencies. While the amount of extracted DNA can readily be measured by different methods, calculation of true efficiency requires knowledge of the initial amount of DNA in the starting bacterial sample, which cannot be done with precision by any existing method. In the process of developing a new extraction procedure, we developed a method that can be used to determine the total amount of both DNA and RNA in bacteria. The amount of DNA can be calculated from the amount of purines released after mild acid and alkali treatment. The amount of RNA in the same extract can also be calculated from the amount of ribonucleoside monophosphates. The released purines and ribonucleoside monophosphates can be quantified by absorbance using HPLC, with reference to appropriate standards. RESULTS: The acid/HPLC method was used to measure the efficiency of commonly used bead-beating and chemical protocols for releasing DNA from a particularly hardy organism, Mycobacterium smegmatis as well as several other species (Bacillus subtilis vegetative cells and spores; Francisella philomiragia; Pseudomonas aeruginosa; Moraxella catarrhalis; Bacillus thuringiensis; Staphylococcus aureus). Surprisingly large differences in efficiency between methods were found. CONCLUSIONS: The acid/HPLC method is a new tool to determine DNA extraction efficiencies and should aid in the development of improved protocols for releasing DNA from a broad range of microorganisms.

Se-derivatized RNAs for X-ray crystallography.

Selenium derivatization of RNA is an important strategy for crystal structure determination and functional studies of noncoding RNAs and protein-RNA interactions. We describe here the synthesis of nucleoside 5'-(α-P-seleno)-triphosphate analogs (Se-NTPs) and their use in vitro transcription and purification of Se-derivatized RNA samples (phosphoroselenoate RNA, PSe-RNA).

Preparative separation of ribonucleoside monophosphates by ion-pair reverse-phase HPLC.

Structural and dynamic investigations of RNA by nuclear magnetic resonance (NMR) spectroscopy strongly benefit from isotopic-labeling strategies. Among these, nucleotide-specific and site-specific labeling methods can help tremendously in simplifying complex NMR data, while providing unique opportunities for structural investigation of larger RNAs. Such methods generally require separation of individual isotopically labeled ribonucleoside monophosphates prior to their conversion into nucleoside triphosphates and selective incorporation of these nucleoside triphosphates into the RNA. This chapter provides the experimental details for preparative separation of ribonucleoside monophosphates by ion-pair reverse-phase HPLC. It also describes a quick procedure for clean-up and quality control of the individual ribonucleoside monophosphates.

Applications of reversible covalent chemistry in analytical sample preparation.

Reversible covalent chemistry (RCC) adds another dimension to commonly used sample preparation techniques like solid-phase extraction (SPE), solid-phase microextraction (SPME), molecular imprinted polymers (MIPs) or immuno-affinity cleanup (IAC): chemical selectivity. By selecting analytes according to their covalent reactivity, sample complexity can be reduced significantly, resulting in enhanced analytical performance for low-abundance target analytes. This review gives a comprehensive overview of the applications of RCC in analytical sample preparation. The major reactions covered include reversible boronic ester formation, thiol-disulfide exchange and reversible hydrazone formation, targeting analyte groups like diols (sugars, glycoproteins and glycopeptides, catechols), thiols (cysteinyl-proteins and cysteinyl-peptides) and carbonyls (carbonylated proteins, mycotoxins). Their applications range from low abundance proteomics to reversible protein/peptide labelling to antibody chromatography to quantitative and qualitative food analysis. In discussing the potential of RCC, a special focus is on the conditions and restrictions of the utilized reaction chemistry.

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter.

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 min with a flow rate of 200 µL/min. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 7 0min using a flow rate of 100 µL/min.

Preparation of 5-amino-4-imidazole-N-succinocarboxamide ribotide, 5-amino-4-imidazole-N-succinocarboxamide riboside and succinyladenosine, compounds usable in diagnosis and research of adenylosuccinate lyase deficiency.

The enzyme adenylosuccinate lyase (ADSL) intervenes twice in the biosynthesis of adenine nucleotides. ADSL deficiency is an inherited metabolic disease characterized by various degrees of psychomotor retardation and accumulation of dephosphorylated enzyme substrates 5-amino-4-imidazole-N-succinocarboxamide riboside (SAICAr) and succinyladenosine (SAdo) in body fluids. Severity of symptoms seems to correlate with residual activity of mutant enzyme and with SAdo/SAICAr concentration ratio in cerebrospinal fluid. To better understand the pathogenetic mechanisms of the disease symptoms, studies of catalytic properties of mutant enzymes together with in vitro and in vivo experiments utilizing SAICAr and SAdo must be performed. Such studies require availability of both ADSL substrates, 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR) and succinyladenosine 5'-monophosphate (SAMP) and their dephosphorylated products in sufficient amounts and purity. Except for SAMP, none of these compounds is commercially available and they must therefore be synthesized. SAICAR was prepared by recombinant human ADSL-catalysed reaction of AICAR (5-aminoimidazole-4-carboxamide) with fumarate and isolated by thin-layer chromatography. SAICAr and SAdo were prepared by calf intestine alkaline phosphatase-catalysed dephosphorylation of SAICAR and SAMP and isolated on cation- and anion-exchange resin columns. The procedures described are easily scalable and provide high yields of sufficiently pure products for use in experiments related to studies of pathogenetic mechanisms in ADSL deficiency.

RNA aptamers selected against the receptor activator of NF-kappaB acquire general affinity to proteins of the tumor necrosis factor receptor family.

The receptor activator of NF-kappaB (RANK) is a member of the tumor necrosis factor (TNF) receptor family and acts to cause osteoclastgenesis through the interaction with its ligand, RANKL. We isolated RNA aptamers with high affinity to human RANK by SELEX. Sequence and mutational analysis revealed that the selected RNAs form a G-quartet conformation that is crucial for binding to RANK. When the aptamer binding to RANK was challenged by RANKL, there was no competition between the aptamer and RANKL. Instead, the formation of a ternary complex, aptamer-RANK-RANKL, was detected by a spin down assay and by BIAcore surface plasmon resonance analysis. Moreover, the selected aptamer efficiently bound to other TNF receptor family proteins, such as TRAIL-R2, CD30, NGFR as well as osteoprotegerin, a decoy receptor for RANK. These results suggest that the selected aptamer recognizes not the ligand-binding site, but rather a common structure conserved in the TNF receptor family proteins.

Functional characterization of 2'-O-methylation and pseudouridylation guide RNAs.

Pseudouridines and 2'-O-methylated nucleotides are ubiquitous constituents of stable cellular RNAs. In eukaryotes, posttranscriptional synthesis of most pseudouridines and 2'-O-methylated nucleotides is directed by sequence-specific guide RNAs (gRNAs). In recent years, an enormous number of novel putative modification gRNAs have been identified in a broad variety of organisms by using bioinformatics and large-scale cDNA-sequencing approaches. Understanding of the function of the novel modification gRNAs as well as the functional importance of modified nucleotides requires techniques that support the site-specific detection of 2'-O-methylated nucleotides and pseudouridines. Here, we describe rapid, reverse transcription-based methods to map 2'-O-methylated nucleotides and pseudouridines in any cellular RNA.

Capillary electrophoresis separation of the new anti-AIDS agents 9-(2-phosphonylmethoxyethyl)adenine and 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine in mixtures with some monoribonucleotides or the most common deoxynucleotides.

The present work describes an electrophoretic method for the separation and determination of the new antivirals, 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(2-phosphonylmethoxyethyl)-2,9-diaminopurine (PMEDAP) in model mixtures with some monoribonucleotide isomers (3'-AMP, 2'-CMP, 3'-CMP, 3'-GMP, 2'-GMP, 3'-UMP, 5'-GMP, and 5'-UMP) or with the most common deoxynucleotides (dCMP, dCDP, dCTP, dTMP, dTDP, dTTP, dGMP, dGDP, dGTP, dAMP, dADP, dATP). A fused-silica capillary tube, 75 microm ID, 67.8 cm total length (60.3 cm length to the detector), with detection at 210 nm was employed. A hydrodynamic injection for 10 s (1.5 psi vacuum) was utilized to introduce the sample, and 30 kV voltage was applied for the separation. The complete separation of PMEA and PMEDAP from the mononucleotide isomers and deoxynucleotide mixtures is possible in less that 10 min and 25 min, respectively, using 20 mM borate buffer, pH 9.9, with the addition of 10 mM beta-cyclodextrin. Efficiencies of more than 120 000 and resolution higher than 1.9 were reached for each of the compounds studied. This capillary electrophoretic procedure opens the possibility for future determination of PMEA and PMEDAP in cell pool samples.

Simple and rapid chromatographic method for the separation of major classes of ribosomal ribomononucleotides.

We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.

Regulation of T4 phage aerobic ribonucleotide reductase. Simultaneous assay of the four activities.

We have devised an assay procedure that permits simultaneous monitoring of the four activities of ribonucleotide reductase. Using this assay, we have compared the reduction of all four substrates by the T4 bacteriophage aerobic ribonucleotide reductase within different allosteric environments. Specifically, we compared the relative turnover rates by the enzyme when activated with "in vivo" concentrations of the known allosteric effectors versus activation by ATP alone. Consistent with the known allosteric properties of this enzyme, our results show that ATP does act as a general activator, although the rate of purine nucleotide reduction was approximately 5% of the rate for the pyrimidine nucleotides. However, addition of the allosteric effectors at their estimated physiological concentrations dramatically changed the relative rates of substrate reduction, creating a more "balanced" pool of products. Addition of the substrates at their respective in vivo concentrations further pushed rates of product formation toward a ratio similar to the base composition of the T4 genome. The similarity of the product profile produced under in vivo conditions to the genomic composition of T4 phage is discussed.

T-lymphocytes from AIDS patients are unable to synthesize ribonucleotides de novo in response to mitogenic stimulation. Impaired pyrimidine responses are already evident at early stages of HIV-1 infection.

Proliferative defects have been reported at the level of DNA synthesis, even in T-lymphocytes from asymptomatic human immunodeficiency virus type-1+ (HIV-1+) patients. Since purine and pyrimidine ribonucleotide availability is crucial for proliferation, we compared the ability of HIV-1- and HIV-1+ T-lymphocytes (> 95% CD4+ and CD8+) to activate de novo biosynthetic and salvage pathways following phytohemagglutinin stimulation using 14C-labeled precursors. The striking abnormality already detectable in asymptomatic patients' cells was the impaired ability of CTP, UDP-Glc, and UTP pools to expand over 72 h (44-70% of control), although ATP and GTP pools and responses were normal. In symptomatic patients, resting T-cells showed markedly reduced pyrimidine pools (53-74% of control) with no change following activation. Relatively normal ATP, GTP, and NAD pools masked the same impaired response of de novo synthesis to activation, with ATP and GTP being reduced by 50% at 48 h. Purine salvage was more active than the control in unstimulated HIV-1+ cells. This impaired de novo synthesis in HIV-1+ T-lymphocytes severely restricts the availability of ribonucleotides for vital growth-related activities such as membrane expansion and strand break repair as well as DNA and RNA synthesis. The data indicate that resting T-lymphocytes from symptomatic patients survive through enhanced salvage, but the stimulation induces metabolic cell death, and provide an explanation for the activation-associated lymphocyte death seen in HIV-1+ T-lymphocytes.

Enzymatic synthesis of ATP analogs and their purification by reverse-phase high-performance liquid chromatography.

The enzymatic syntheses of ATP analogs, such as tubercidin 5'-triphosphate, formycin A 5'-triphosphate, and etheno-ATP, from their respective mono- and diphosphate are described. The reaction products were purified by reverse-phase HPLC using a C-18 matrix and a volatile mobile phase at pH 7, with tributylamine as the ion-pairing agent. Each of the analogs required a buffer of somewhat different composition for the baseline separation of reaction product and reactants. The elutions were isocratic and allowed several successive runs without any intermediate equilibration of the column. After freeze-drying of the pooled fractions, the yield of the synthesized nucleoside triphosphate was approximately 70%. The described procedures are applicable either for analytical investigations or for semi-preparative purposes.

A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.

A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.

Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.

Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.