A new and improved method for 3′-end labelling DNA using [α-32P]ddATP

We have synthesised dideoxyadenosine-5′-[α-³²P]triphosphate ([α-³²P]ddATP) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5′-[α-³²P]triphosphate ([α-³²P]KTP) as a means to 3′-end label DNA. The [α-³²P]ddATP was found to be three to five times more efficient than [α-³²P]KTP. Blunt and 3′-protruding ends were labelled more efficiently with [α-³²P]ddATP using terminal transferase than were the 5′-ends with [γ-³²P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3′-end labelling of DNA to become a realistic alternative to 5′-end labelling. We have also compared [α-³²P]ddATP- and [α-³²P]KTP-labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.     

The effect of sample composition and vial type on Ĉerenkov counting in a liquid scintillation counter

The effect of sample vial type and sample composition on the ?erenkov count rate detected from ³²P and ³⁶Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate.     

Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism‐related micro‐organisms

Aims: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. Methods and Results: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10‐μl sample volume onto the sample support was 10⁶ particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 μl, the detection limit could be reduced to 10⁵ particles per ml for spores and to 5 × 10⁴ particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. Conclusions: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. Significance and Impact of the Study: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.     

A highly sensitive adenylate cyclase assay

A highly sensitive adenylate cyclase assay method has been developed which employs sequential chromatography on columns of Dowex cation exchange resin and aluminum oxide. With the use of [α-³²P]ATP as substrate, this method permits the nearly complete separation of cyclic [³²P]AMP formed from the substrate and other ³²P-containing compounds, i.e., ³²P in the assay blanks was barely detectable. In comparative studies, this method was found to be considerably more sensitive than previously reported methods. The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies.     

Rapid concentration of bacteria using submicron magnetic anion exchangers for improving PCR-based multiplex pathogen detection

Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detection is investigated in this study. Submicron (average size 500 nm) superparamagnetic anion-exchangers (SiMAG-DEAE) were used successfully to concentrate target bacteria from very dilute solutions. A mass-balance model predicted that for Escherichia coli, the extent of cell concentrating increases almost linearly with increasing sample/SiMAG volume ratio up to about 2000, accompanied by only a slight decrease in the capture efficiency (< 10%). Our experimental data generally support this analysis in that the SiMAG beads concentrated bacterial targets by two to three orders of magnitude using a sample/bead volume ratio of about 1000, and lowered the PCR detection limit to a level of 10² CFU/mL, from 10⁴ to 10⁵ CFU/mL without concentrating. Several target bacteria can be concentrated concurrently and detected via multiplex PCR, as illustrated using E. coli and Agrobacterium tumefaciens as model bacteria. Finally, concentration and detection of bacteria in fresh produce samples were demonstrated. The integration of submicron magnetic ion exchangers and PCR detection provides an appealing alternative to immunomagnetic separation/PCR in improving pathogen detection.     

Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity.

Conditions are described where 5-phosphoribosyl-α-1-pyrophosphate (PRPP) can be determined by thin-layer chromatographic methods commonly used for the determination of nucleoside triphosphate pools in ³²P-labeled bacteria. A two-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 μmol per gram dry weight bacteria) can be determined. In a uni-dimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 μmol per gram dry weight bacteria. This uni-dimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [γ-³²P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of ³²P labeling.The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase have been applied to the analysis of some mutants of Salmonella typhimurium and have provided results that agree well with the results obtained by conventional methods of PRPP analysis.     

Use of diacylclycerol kinase to quantitate picomole levels of 1,2-diacylglycerol.

An extremely sensitive and specific analytic procedure is described for quantitating 1,2-diacylglycerol (DAG). Escherichia coli DAG kinase (EC 2.7.1.-) catalyzed the formation of [³²P]phosphatidic acid by the transfer of ³²PO₄ from [γ-³²P]adenosine 5′-triphosphate to DAG in linear proportion to the quantity of added DAG from 10 to 1000 pmol. This technique allowed reliable detection of as little as 2 pmol of added DAG. To assess levels of DAG in tissue lipid extracts a miniaturized method for silicic acid column chromatography was developed to separate DAG from triglycerides and phospholipids. When these procedures were applied to erythrocytes, lysis in the presence of Ca²⁺ caused a 10.6-fold rise in cellular DAG confirming not only the results obtained in an earlier investigation (1), but also the utility of this technique in the analysis of exceedingly small quantities of cellular DAG.     

Lethal modifications of DNA via the transmutation of32P and33P incorporated in the genome of the S13 bacteriophage

Summary When circular single-stranded DNA of phage S13 is labelled with³²P or³³P, the transmutations very efficiently bring about a loss of phage infectiousness (efficiency = 1 for³²P and 0.73 for³³P). For both radionuclides, the lethal efficiencies as well as the lethal events are different. In the case of³²P, the lethal event is the loss of the circular integrity of the DNA molecule, occurring as a consequence of a systematic single strand-break caused by each³²P decay (100%). Conversely, in the case of³³P, the lethal events are either a single strand-break (40%) or a local stereochemical modification (33%). The same primary event, the substitution at each³³P decay of a phosphate by a sulfate molecule, leads to one of these lethal events in relation to the decay site. Moreover, neither the phage adsorption nor its genome injection into bacteria depends on the physical state of the genome, and thus lethality is revealed at only the genetic level.     

Fluorescence correlation spectroscopy. II. An experimental realization

This paper describes the first experimental application of fluorescence correlation spectroscopy, a new method for determining chemical kinetic constants and diffusion coefficients. These quantities are measured by observing the time behaviour of the tiny concentration fluctuations which occur spontaneously in the reaction system even when it is in equilibrium. The equilibrium of the system is not disturbed during the experiment. The diffusion coefficients and chemical rate constants which determine the average time behaviour of these spontaneous fluctuations are the same as those sought by more conventional methods including temperature-jump or other perturbation techniques. The experiment consists essentially in measuring the variation with time of the number of molecules of specified reactants in a defined open volume of solution. The concentration of a reactant is measured by its fluorescence; the sample volume is defined by a focused laser beam which excites the fluorescence. The fluorescent emission fluctuates in proportion with the changes in the number of fluorescent molecules as they diffuse into and out of the sample volume and as they are created or eliminated by the chemical reactions. The number of these reactant molecules must be small to permit detection of the concentration fluctuations. Hence the sample volume is small (10^?8 ml) and the concentration of the solutes is low (～ 10^?9 M). We have applied this technique to the study of two prototype systems: the simple example of pure diffusion of a single fluorescent species, rhodamine 6G, and the more interesting but more challenging example of the reaction of macromolecular DNA with the drug ethidium bromide to form a fluorescent complex. The increase of the fluorescence of the ethidium bromide upon formation of the complex permits the observation of the decay of concentration fluctuations via the chemical reaction and consequently the determination of chemical rate constants.     

Tamm Plasmon Polariton as Refractive Index Sensor Excited by Gyroid Metals/Porous Ta2O5 Photonic Crystal

In this paper, a novel refractive index sensor in terahertz region is proposed. The proposed structure is prism/(sample/porousTa₂O₅)¹⁵/sample/gyroid metal/substrate. The sensor is based on the Tamm plasmon polariton at the interface between porous one-dimensional photonic crystal and gyroidal metal. The gyroidal metal has been used as an alternative metal and its refraction index can be tuned by the gyroid parameters. The effects of the metal volume fraction and sample refractive index on the performance are studied to improve the ability of the sensor. The proposed sensor achieves high sensitivity of 6.7 THz/RIU, a high figure of merit 6*10³ RIU^?1, a high-quality factor of 3*10³, and a low detection limit of 9*10^?6 RIU. The proposed device can be a good candidate for fabricating gyroid metal and porous material-based biosensors, active optoelectronic and polaritonic devices.

     

Liquid scintillation counting of aqueous suspensions of microbiological specimens in a Triton X-100/toluene scintillant

The pulse height spectra and the relative efficiencies of aqueous suspensions of [³H]DNA T4D bacteriophages, of [³H]DNA Escherichia coli bacteria, and of [³²P]DNA T4D phages were measured and compared to the results of [³H]thymidine and [³²P]orthophosphate solutions, respectively. In all of our measurements a scintillation mixture based upon Triton X-100/toluene (0.5 kg/1 liter) was used. We explain the different effects of the chemical quench (e.g., by CCl₄) and of the absorption of β energy inside the specimen (e.g., phages and bacteria) on the pulse height spectra by means of Bethe's theory of electron stopping power. We measured also the dependence of the relative efficiency on the content of aqueous suspensions of [³H]DNA and [³²P]DNA phages in the sample, and compared the results to the relative efficiencies of aqueous [³H]thymidine and [³²P]orthophosphate solutions, respectively.     

Method for measuring 32P-labeled cAMP levels in intact tissue.

A new method has been developed for prelabeling tissue ATP pools with ³²P inorganic phosphate (P_i) and for the subsequent isolation of [³²P]cAMP and [³²P]ATP. The new method of prelabeling eliminates the need to separate trace amounts of radioactive cAMP from radioactive breakdown products of adenine formed in tissues prelabeled with [³H]- or [¹⁴C]adenine. The effect of epinephrine to increase [³²P]cAMP levels in rat ventral prostate tissue fragments has been studied in terms of increase in the ratio of [³²P]cAMP/[³²P]ATP in the absence and presence of various phosphodiesterase inhibitors. Tissue prelabeling with ³²P_i labels GTP as well as ATP (and other nucleoside triphosphates); thus the method lends itself to the isolation of [³²P]cGMP as well as [³²P]cAMP from the same tissue sample.     

Sequence analysis of small amounts of nonradioactive oligoribonucleotides containing ribose-methylated nucleosides by a combination of 3H- and 32P-labeling techniques

The oligonucleotides A-G-A-Cm-U and Gm-A-A-Y-A-ψ were used as model compounds to demonstrate how the complete nucleotide sequence of small amounts of nonradioactive oligoribonucleotides (0.2–0.3 nmol) can be derived by a combination of ³H-labeling procedures previously published and a new method for the characterization of 2′-O-methylated nucleosides based on enzymatic ³²P labeling. The newly developed method for the identification of ribose-methylated nucleosides entails ³²P labeling by [γ-³²P]ATP/polynucleotide kinase of the 5′-terminus of a ribonuclease T₂-stable 2′-O-methylated dinucleotide derived from the polyribonucleotide, conversion of the labeled dinucleotide to the ³²P-labeled 2′-O-methylated nucleoside 5′-monophosphate, and identification of the monophosphate by its chromatographic properties on a polyethyleneimine-cellulose thin layer. The novel method is simple, fast, and sensitive and, at present, represents the only way by which ribose-methylated nucleosides can be analyzed in small amounts (0.01 nmol) of nonradioactive oligonculeotides or RNA.     

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC–UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw–eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 μL), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC–UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL⁻¹. The LOQ values ranged from 10 to 25 ng mL⁻¹. The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC–UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC–UV method was applied to analysis of plasma samples from elderly depressed patients.     

Properties of Single- and Double-stranded Ribonucleic Acid from Barley Plants Infected with Bromegrass Mosaic Virus

Incorporation of ³²P into nucleic acids in barley plants infected with bromegrass mosaic virus (BMV) was analyzed by chromatography on methylated albumin kieselguhr (MAK) columns. Treatment with actinomycin D reduced the synthesis of ribosomal ribonucleic acid (RNA) to low levels and allowed the detection of the three components of BMV-RNA in vivo. The kinetic study on ³²P incorporation into these BMV-RNA components suggested that a single cleavage occurred in some of the intact RNA shortly after completion of its synthesis, giving rise to the small and medium components. Chromatographic analyses also revealed a double-stranded, ribonuclease-resistant RNA which has been purified by differential extraction, sucrose-density gradient centrifugation, and MAK column chromatography. This RNA sediments at approximately 14S, is alkali-labile, and has a sharp thermal transition with a T_m of 96 C in 0.1 × standard saline citrate buffer, as determined by susceptibility to ribonuclease. The RNA is absent in uninfected barley plants.     

Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase.

Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]_o > K_m indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [³²P]phosphate ion. Incubation of wheat germ acid phosphatase with ³²P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of ³²P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A ³²P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme.     

Measurement of microbial biomass phosphorus in rhizosphere soil

³²P-labelled monocalcium phosphate solution was supplied by point injection to the root system of wheat plants grown in soil cores in a controlled environment. There was no detectable incorporation of³²P into organic P fractions in the soil remaining after roots were removed, confirming field observations. The techniques used to measure organic P (including biomass P) could detect an incorporation of³²P into soil microbial biomass equivalent to 0.3 μgP.g^?1 soil, compared to a total soil biomass P content estimated to be ca. 6.5 μgP.g^?1 soil. The limited incorporation of the added P into microbial biomass in the root-free soil may be due partly to a limited diffusion of³²P into the non-rhizosphere soil and partly to the removal of³²P-labelled microbial biomass adhering to or in very close association with the root surface. it is proposed that in studies of soil nutrient status, total soil biomass P (roots + soil flora + microfauna) should be measured, rather than attempting an estimate of microbial P. A sequential extraction procedure using a single soil sample, where a biocide is added to the extracting solution, is proposed as an alternative to the conventional procedure for measuring soil biomass P where two soil samples, one treated with a biocide, are extracted simultaneously.     

X-ray intensifying screens greatly enhance the detection by autoradiography of the radioactive isotopes 32P and 125I

Detection of both γ-ray (¹²⁵I) and high energy β (³²P) particle-emitting radioisotopes by autoradiography is significantly enhanced by the use of X-ray intensifying screens. We have compared five commercially available intensifying screens (containing different inorganic phosphors) in combination with a number of films and exposure conditions. One calcium tungstate (Dupont, Cronex Lighting Plus intensifying screen produces an 8- to 10-fold enhancement in the detection of ³²P and a 30- to 40-fold enhancement in the detection of ¹²⁵I when an autoradiogram is made with Kodak RP ROYAL X-OMAT film at ?70°C. With one intensifying screen, 10 dpm of ³²P and 50 dpm of ¹²⁵I in an area of 10 mm² are readily detectable in a 20-hr exposure.     

Long Distance Transport in Macrocystis integrifolia: II. Tracer Experiments with C and P

Discs from mature regions of Macrocystis blades picked up significantly more [³²P]phosphate from the ambient medium than similar discs from young meristematic regions, and this uptake was higher in light than in darkness. Double-labeling experiments with NaH¹⁴CO₃ and [³²P]phosphate, using intact fronds as well as cut frond segments, indicated that ³²P was translocated from mature blades to sink regions at velocities of 25 to 45 centimeters per hour, velocities comparable to ¹⁴C translocation velocity in the same material. There was a slight delay in transport of ³²P which may be due to a delay in loading or to a high metabolism of ³²P in the transporting channels. Histoautoradiography of stipe segments in the translocation pathway indicated that transport of label occurred in the peripheral parts of medulla. An analysis of ³²P-labeled compounds in the fed blade and in the sieve tube sap, collected from basal cut ends of stipes, indicated major differences in labeling patterns. In the blade, a high proportion of ³²P was recovered as inorganic phosphate and relatively small amounts were found in hexose mono- and diphosphates, UDPG and ATP. In the sieve tube sap, however, only a small amount of ³²P was present as inorganic phosphate, a large proportion was found in hexose mono- and diphosphates, and appreciable amounts were present in ATP and UDPG.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.