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.

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.

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.

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.

Ribonucleotide content of mononuclear cells from normal subjects and patients with chronic lymphocytic leukemia: increased nicotinamide adenine dinucleotide concentration in chronic lymphocytic leukemia lymphocytes.

The ribonucleotide content of lymphocytes obtained from normal subjects and patients with chronic lymphocytic leukemia (CLL) was determined by means of high-performance liquid chromatography. The levels of normal B- and T-cells were compared to each other as well as those of their CLL counterparts. Unfractionated CLL lymphocytes, predominantly B-cells, had significantly lower levels of adenosine-5'-triphosphate, cytidine-5'-triphosphate, uridine-5'-triphosphate, cytidine-5'-diphosphate, and guanosine-5'-phosphate, while the concentration of nicotinamide-adenine dinucleotide was significantly higher than in normal unfractionated lymphocytes which consisted mainly of T-cells. For enriched populations: (a) CLL B-cells had much lower adenosine-5'-triphosphate (3439 versus 5689) (pmol/1 X 10(7) cells), cytidine-5'-triphosphate (107 versus 313), guanosine-5'-triphosphate (462 versus 978), and uridine-5'-triphosphate (633 versus 1214) than normal B-cells; (b) CLL T-enriched subpopulations had significantly lower ribonucleoside triphosphates, adenosine-5'-triphosphate (3217 versus 5468), cytidine-5'-triphosphate (119 versus 209), guanosine-5'-triphosphate (422 versus 826), and uridine-5'-triphosphate (504 versus 969) than normal T-cells. The lower ribonucleoside triphosphate levels found in unfractionated CLL lymphocytes, therefore, are the result of differences between the CLL and normal B-cells as well as between CLL and normal T-cells. These findings establish a framework for studying the reasons underlying the decreased ribonucleoside triphosphate levels in unfractionated CLL lymphocytes. T-helper and T-suppressor lymphocytes showed similar ribonucleotide patterns. Nucleoside and base levels were significantly higher in normal monocytes than in normal lymphocytes. The only compound found to be increased in the CLL B-lymphocytes when compared to their normal counterparts was nicotinamide-adenine dinucleotide. The level in CLL lymphocytes was 404 versus 209 pmol/10(7) cells for normal B-lymphocytes. No correlation was found between any ribonucleotide levels and the expression of 5'-nucleotidase activity.

Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a mechanism of self-potentiation.

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

Precipitation of nucleotides by calcium phosphate.

Earlier it has been shown that nucleic acids of high molecular weight can be be introduced into cells by coprecipitation with calcium phosphate. We have studied the requirements for calcium phosphate coprecipitation of shorter nucleotides. The degree of coprecipitation of dodecanucleotides lacking terminal phosphate varied between 25 and 72%. Tetramers with a 5'-monophosphate were coprecipitated to 29-87% by calcium phosphate. A high content of guanosine residues and an increased number of terminal phosphate groups increased the degree of coprecipitation of nucleotides. The trinucleotide pppA2'p5'A2'p5'A was effectively precipitated by calcium phosphate but the monophosphate and the core structure were not.

Conformation affinity of cross-linked polyacrylic gels towards nucleobases, nucleosides and nucleotides.

By radical copolymerisation of monofunctional acrylic derivatives with 1,4-tetramethylene dimethacrylate, cross-linked polyacrylic gels which show a high affinity towards nucleic acid residues have been synthesised. Using these polyacrlic gels, mixtures of nucleobases, nucleosides and nucleotides can be chromatographically separated to differing extents, in some cases quantitatively. The elution of nucleobases and nucleosides from the gel in the order Cyt, Gua, Thy, Ade (or dC, dG, dT, dA and C, G, U, A, respectively) shows that Ade (dA, A) is retarded to the highest degree from the gel matrix, Cyt (dC, C) to the lowest degree. Further, the results of the separations prove that the affinity of the polyacrylic gels is much stronger towards deoxyribonucleosides than towards ribonucleosides and nucleobases. The affinity of the polyacrylic gels towards nucleobases and nucleosides depends mainly upon their tertiary structure whereas the functional side groups of the polyacrylate matrix do not contribute significantly to the interaction of the gels.

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.

Metabolic activation of 2,6-diaminopurine and 2,6-diaminopurine-2'-deoxyriboside to antitumor agents.

2,6-Diaminopurine (DAP) and 2,6-diaminopurine 2'-deoxyriboside (DAPdR) are analogs of adenine and deoxyadenosine, respectively. It was the purpose of this study to compare these analogs under identical conditions in order to define their inhibitory properties and the underlying mechanism in L1210 mouse leukemia cells. In a 5-day cell growth experiment, DAP exerted a significantly stronger antiproliferative effect than DAPdR. Correspondingly, colony formation of L1210 cells in soft agarose was inhibited by DAP to a greater extent than by DAPdR. A differential distribution of L1210 cells in the cell cycle resulted from an exposure to DAP and DAPdR. While DAPdR arrested cells in the G1/G0 phase of the cell cycle, DAP appeared to lead to an accumulation of G2/M cells. The diaminopurines were combined with modulatory agents to test the antiproliferative action of the combinations. Deoxycytidine partially rescued the cells from the growth inhibitory action of DAPdR without affecting the growth of DAP-treated cells. When adenine was used, the antiproliferative effect of DAPdR was slightly enhanced while the effect of DAP was completely abolished. 8-Aminoguanosine, a specific inhibitor of purine nucleoside phosphorylase, synergistically potentiated the cytostatic effect of DAPdR. However, this inhibitor did not alter DAP effects. At the biochemical level, the target of DAPdR was ribonucleotide reductase which was in line with a drastic expansion of the dGTP pool in DAPdR-treated cells. In cells exposed to DAP, high levels of DAP riboside triphosphate were measured; concomitantly, the ATP level dropped markedly. Enzymological studies revealed that DAPdR is an excellent substrate of adenosine deaminase giving rise to the formation of deoxyguanosine. DAP was found to be activated in the purine nucleoside phosphorylase reaction and in a phosphoribosyl-pyrophosphate-dependent reaction. The data from this comparative study suggest that DAPdR and DAP possess different toxicity mechanisms. DAPdR and DAP possess different toxicity mechanisms. DAPdR acts as a precursor of deoxyguanosine, and DAP is metabolically activated to DAP-containing ribonucleotide analogs. These different metabolic routes seem to account for the different effects of DAP and DAPdR at the cellular level.

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.

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.

In vitro processing of the adenosine analog formycin A to the mono-, di-, and triphosphate by a soluble multienzyme system from mouse liver.

The adenosine kinase activity present in a soluble preparation from rat liver was investigated using formycin A (FoA), a fluorescent analog of adenosine as the phosphoryl acceptor and ATP as the donor. Reversed-phase high-performance liquid chromatography (h.p.l.c.) was used to separate substrate from product, and the progress of the phosphorylation reaction was followed by monitoring fluorometrically the amount of formycin 5'-monophosphate (FoMP), and the AMP analog, that was formed. The results showed that while FoMP was formed during the reaction indicating that an adenosine kinase activity was present, both formycin 5'-di- and triphosphate (FoDP and FoTP respectively), the corresponding analogs of ADP and ATP, were also formed, suggesting than an adenylate kinase activity was present. This result was confirmed with FoMP as the substrate and showing the formation of FoDP and FoTP. Other experiments carried out with FoMP as the substrate revealed the formation of FoA. Taken together, these results indicated that a 5'-nucleotidase activity as well as an adenylate kinase was present. Using this analog and h.p.l.c., it has been possible to demonstrate for the first time in an in vitro system the complete salvage of a nucleoside to the triphosphate level.

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.

Rapid separation and quantitation of 3',5'-cyclic nucleotides and 5'-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography.

A simple, rapid high-performance liquid-chromatography system for the fractionation and direct quantitation of substrates and products in crude phosphodiesterase reaction mixtures is described. Phosphate buffers and a pellicular anion exchange resin are used at ambient temperature. The method is sensitive, measuring picomoles of products with ultraviolet detection and femtomoles with isotopic measurement, and offers several advantages over the more popular batch sorption and manual methods for measuring phosphodiesterase activity. The time required for analysis, less than 8 min for single substrate reaction mixtures, is a fraction of that required with other chromatographic systems, and precision is +/- 5%. Results of studies with an activatable form of phosphodiesterase demonstrate the accuracy, precision and utility of the procedure for biochemical analyses.

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.

High-pressure liquid-chromatographic assay of nucleotide-pool concentrations during polysaccharide biosynthesis in four ascomycetes.

High-resolution liquid-chromatographic methods developed for analyzing nucleotide pools at the nanogram level in four representative species of ascomycetes (Penicillium citrinum, Aspergillus niger, Fusarium moniliforme, and Cladosporium herbarum) were used to study polysaccharide biosynthesis. Nucleotides extracted from the mycelial mat were preseparated from interfering polysaccharides, glycoproteins, and nucleic acids on a column of Biogel P-2. Resolution of 18 nucleotides from each fungal species was accomplished on AS-Pellionex-SAX, pellicular anion-exchanger by using a high-pressure liquid chromatograph. Nucleotides were identified by comparing peak retention-times, by differential u.v. absorption with two detectors in series at selected wavelengths, and by acid or enzymic hydrolysis with product identification by liquid chromatography. Pyrimidine bases exceeded purines by at least three fold, and uridine nucleotides often constituted 60-80 mole percent of the total nucleotides; extractable cytidine nucleotides were negligible. Uridine 5'-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl disphosphate) is the preponderant nucleotide throughout the growth cycles of all four species, amounting to 30-60% of all nucleotides present. For all four fungal species, a burst of nucleotide formation was observed after the first 48h (15-30 mumol/g tissue), with fluctuations that eventually fell to 0.1 mumol/g on the tenth day.

Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA.

Study of optimal operational conditions for RNA enzymatic hydrolysis to obtain 5'-ribonucleotides has been carried out. RNA from brewer's yeasts, obtained by ammonium extraction, was hydrolysed by a partially purified 5'-phosphodiesterase from barley rootlets. Temperature of 60 degrees C and pH 7 have been determined as the best operational conditions. Low RNA initial concentration (approximately 0.1%) and reaction time (approximately 1 h) have been identified as necessary to obtain a good yield of 5'-ribonucleotides.

[Separation and analysis of ribonucleotide mixtures by anion exchange high performance liquid chromatography]. / Razdelenie i analiz smesei ribonukleotidov metodom vysokoéffektivnoi zhidkostnoi khromatografii na anionoobmennikakh.

Ribonucleotides in the artificial mixture or from natural extracts were separated by HPLC on a column with strong anion-exchanger in gradient of pH and concentration of phosphate-chloride buffer system. Peaks were detected by UV-absorbance at 254 nm and detector sensitivity of 0,02 optical units for a full scale, whereas their identification was based on comparison of retention times with those of pure standards. An electronic integrator calibrated for each nucleotide was used for quantitative analysis. The conditions for separating 13 ribonucleotides (including IMP) during 40-minute HPLC-analysis were specified. Some problems pertinent to nucleotide extractions from biological samples are discussed.