Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts

The search for DNA biomarkers of oxidative stress has been hampered for several decades by the lack of relevant information on base oxidation products and the challenging issue of measuring low amounts of lesions, typically a few modifications within the range 10⁶?C10⁸ normal bases. In addition and this was ignored for a long time, there is a risk of artifactual oxidation of overwhelming nucleobases during DNA extraction and subsequent workup that has led to overestimation of some base damage up to 2?C3 orders of magnitude. The main aim of the survey is to critically review the available methods that have been developed for measuring oxidatively generated base damage in nuclear and mitochondrial DNA. Among the chromatographic methods, high-performance liquid chromatography associated with tandem mass spectrometry (HPLC?CMS/MS) is the most accurate and versatile approach whereas HPLC?Celectrochemical detection (ECD) is restricted to electrochemically active modifications. These methods allow measuring several single oxidized pyrimidine and purine bases, tandem base lesions and interstrand DNA cross-links in nuclear DNA. As complementary analytical tools, enzymatic methods that associate DNA repair enzymes with either the alkaline comet assay or the alkaline elution technique are suitable for assessing low variations in the level of different classes of oxidatively generated DNA lesions. Most of the immunoassays suffer from a lack of specificity due to the occurrence of cross-reactivity with overwhelming normal bases. One major exception concerns the immunodetection of 5-hydroxymethylcytosine, produced in a relatively high yield as an epigenetic DNA modification. HPLC?CMS/MS is now recognized as the gold standard for measuring oxidized bases and nucleosides in human fluids such as urine, saliva, and plasma.     

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.     

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.     

Measurement of gonadal hormones in dried blood spots versus serum: verification of menstrual cycle phase

In the present study, we show that blood spot assays for estradiol, progesterone, and testosterone are a reliable, accurate, and sensitive means for measuring circulating gonadal hormones. The lower limit of sensitivity of each blood spot assay is sufficient to determine gonadal hormone levels in adult females. Correspondence of serum to blood spot measures is high, with blood spot hormone levels explaining an average of 88.60% of the variance in serum gonadal hormones in females, but only 46.20% in males. We provide formulas for converting hormone levels in blood to hormone levels in serum (which traditional endocrinology studies report). Finally, we show that careful attempts to estimate hormone status by day-count methods are unreliable when compared to hormone assay in blood spots.     

A MIQE-compliant real-time PCR assay for Aspergillus detection

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.     

Microbioassay of Antimicrobial Agents

A previously described agar-diffusion technique for microbioassay of antimicrobial agents has been modified to increase sensitivity of the technique and to extend the range of antimicrobial agents to which it is applicable. This microtechnique requires only 0.02 ml of an unknown test sample for assay, and is capable of measuring minute concentrations of antibiotics in buffer, serum, and urine. In some cases, up to a 20-fold increase in sensitivity is gained relative to other published standardized methods and the error of this method is less than +/-5%. Buffer standard curves have been established for this technique, concurrently with serum standard curves, yielding information on antimicrobial serum-binding and demonstrating linearity of the data points compared to the estimated regression line for the microconcentration ranges covered by this technique. This microassay technique is particularly well suited for pediatric research and for other investigations where sample volumes are small and quantitative accuracy is desired. Dilution of clinical samples to attain concentrations falling with the range of this assay makes the technique readily adaptable and suitable for general clinical pharmacological studies. The microassay technique has been standardized in buffer solutions and in normal human serum pools for the following antimicrobials: ampicillin, methicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, cephaloglycin, cephalexin, cephaloridine, cephalothin, erythromycin, rifamycin amino methyl piperazine, kanamycin, neomycin, streptomycin, colistin, polymyxin B, doxycycline, minocycline, oxytetracycline, tetracycline, and chloramphenicol.     

Dynamic state of S-nitrosothiols in human plasma and whole blood

In the vasculature, nitrosothiols derived from the nitric oxide (NO)-mediated S-nitrosation of thiols play an important role in the transport, storage, and metabolism of NO. The present study was designed to examine the reactions that promote the decomposition, formation, and distribution of extracellular nitrosothiols in the circulation. The disappearance of these species in plasma and whole blood was examined using a high-performance liquid chromatography method to separate low- and high-molecular weight nitrosothiols. We found that incubation of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO) with human plasma resulted in a rapid decomposition of these nitrosothiols such that <10% of the initial concentration was recovered after 10-15 min. Neither metal chelators (DTPA, neocuproine), nor zinc chloride (glutathione peroxidase inhibitor), acivicin (gamma-glutamyl transpeptidase inhibitor), or allopurinol (xanthine oxidase inhibitor) inhibited the decomposition of GSNO. With both CySNO and GSNO virtually all NO was recovered as S-nitrosoalbumin (AlbSNO), suggesting the involvement of a direct transnitrosation reaction. Electrophilic attack of the albumin-associated thiols by reactive nitrogen oxides formed from the interaction of NO with O(2) was ruled out because one would have expected 50% yield of AlbSNO. Similar results were obtained in whole blood. The amount of S-nitrosohemoglobin recovered in the presence of 10 microM GSNO or CySNO was less than 100 nM taking into consideration the detection limit of the assay used. Our results suggest that serum albumin may act as a sink for low-molecular-weight nitrosothiols and as a modulator of NO(+) transfer between the vascular wall and intraerythrocytic hemoglobin.     

Miniaturization and validation of a high-throughput serine kinase assay using the AlphaScreen platform

Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include low sensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility of miniaturization of a serine kinase assay using generic reagents in the AlphaScreen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-microL final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z' values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the AlphaScreen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.     

The preparation of an IgG diagnostic agent based on stained polyacrolein latexes for use in the latex agglutination reaction

IgG diagnosticum for measuring the concentration of 131I-labeled IgG antibodies to enteric antigen beta 1MA by the latex agglutination inhibition (LAI) test has been prepared on the basis of polyacrolein latexes. A method for the titration of anti-IgG antibodies with the use of the above diagnosticum has been developed, based on the late, agglutination (LA) test. The optimum conditions for the microtitration variant of the LA and LAI tests have been defined. High sensitivity, specificity and simplicity of analysis with the use of latex IgG diagnosticum have been demonstrated. The newly developed methods have been successfully used in laboratory trials of a new diagnostic radiopharmaceuticals for the assay of 131I-labeled antibodies in this preparation and for the detection of side effects of immunization on the recipients.     

Bayesian inference for prevalence and diagnostic test accuracy based on dual-pooled screening

We propose a useful protocol for the problem of screening populations for low-prevalence characteristics such as HIV or drugs. Current HIV screening of blood that has been donated for transfusion involves the testing of individual blood units with an inexpensive enzyme-linked immunosorbent assay test and follow-up with a more accurate and more expensive western blot test for only those units that tested positive. Our cost-effective pooling strategy would enhance current methods by making it possible to accurately estimate the sensitivity and specificity of the initial screening test, and the proportion of defective units that have passed through the system. We also provide a method of estimating the distribution of prevalences for the characteristic throughout the population or subpopulations of interest.     

A fluorimetric semi-microplate format assay of protein carbonyls in blood plasma

Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4-dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive enzyme-linked immunosorbent assay (ELISA) methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan (HANDL) study, oxidative stress in humans was quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls, both of which were different from the value of the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R = 0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.     

A novel high throughput chemiluminescent assay for the measurement of cellular cyclic adenosine monophosphate levels

The second messenger 3', 5'-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.     

A spectrophotometric assay for measuring and detecting an epoxide hydrolase activity

In this paper we report the development of a novel and simple spectrophotometric assay which allows one to achieve the continuous, rapid, sensitive, and accurate determination of an epoxide hydrolase activity. This assay is based on the elaboration of a coupled enzymatic/chemical methodology which allows quantification of the enzymatic activity within 3min, and offers good sensitivity of about 10 micro Mmin(-1). Applicability of this test to some other aromatic epoxides has been shown and some limitations have also been explored. This assay should be particularly useful for different applications, for example (a) activity localization during purification of such enzymes, (b) very rapid determination of kinetic constants, and (c) high-throughput screening experiments.     

Imaging of chemiluminescent signals with cooled CCD camera systems

We investigated imaging of chemiluminescent signals from 1,2-dioxetanes with cooled CCD cameras. Non-radioactive detection methods for biomolecules utilizing these chemiluminescent substrates for alkaline phosphatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, immunoblotting, ELISA methods and DNA sequencing. Dephosphorylation of the dioxetane CSPD by alkaline phosphatase generates an unstable anion that decomposes resulting in light production. The wavelength of the emitted light is approximately 460nm. We have utilized Photometrics Star and MXC 200L cooled CCD cameras for direct imaging of chemiluminescent signals. Benefits of utilizing a CCD detector include rapid data digitization and more accurate quantitation of chemiluminescent signals compred to film-based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our results indicate that the digitization of chemiluminescent signal data with cooled CCD cameras is an excellent alternative to ³²P detection methods utilizing storage phosphor screen imaging systems.     

DNA链断裂检测技术的进展

DNA链损伤特别是DNA双链段裂(dsb)的检测方法是研究DNA辐射损伤的一个关键因素.已发展的检测DNA dsb的方法很多,但各种检测法均有其一定的优越性和适用范围,近年来应用较多并日益受到重视的新方法有原位杂交法,彗星试验(单细胞电泳法)以及高效毛细管电泳法等等.     

Porous silicon biosensor for detection of viruses

There is a growing need for virus sensors with improved sensitivity and dynamic range, for applications including disease diagnosis, pharmaceutical research, agriculture and homeland security. We report here a new method for improving the sensitivity for detection of the bacteriophage virus MS2 using thin films of nanoporous silicon. Porous silicon is an easily fabricated material that has extremely high surface area to volume ratio, making it an ideal platform for surface based sensors. We have developed and evaluated two different methods for covalent bioconjugation of antibodies inside of porous silicon films, and we show that the pore penetration and binding efficiency depend on the wettability of the porous surface. The resulting films were used to selectively capture dye-labeled MS2 viruses from solution, and a viral concentration as low as 2 x 10(7) plaque-forming units per mL (pfu/mL) was detectable by measuring the fluorescence from the exposed porous silicon film. The system exhibits sensitivity and dynamic range similar to the Luminex liquid array-based assay while outperforming protein micro-array methods.     

Enzymatic assay of allantoin in serum using allantoinase and allantoate amidohydrolase

A new enzymatic assay for specifically measuring allantoin concentration in serum has been developed. The currently used methods for allantoin analysis are time consuming and nonspecific or depend on the use of expensive equipment. In our method, allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5). The allantoate produced is hydrolyzed to ureidoglycine and ammonia by the action of allantoate amidohydrolase (EC 3.5.3.9). Nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (EC 1.4.1.4) subsequently acts on the ammonia produced, resulting in a change in absorbance at 340nm due to the consumption of reduced nicotinamide adenine dinucleotide phosphate. The amount of allantoin present is related to the change in the absorbance. The standard curve is linear up to at least 1mM allantoin. The procedure is simple, rapid, and accurate. The method has been used to measure serum allantoin levels after oral administration of purine nucleotides to experimental animals, including rats that have uricase catalyzing the conversion of urate to allantoin.     

Practical applications of the microphotometric quantification of histoenzyme reactions

Summary There are many histochemical reactions which can be used for the accurate topographic detection of enzymes in tissues. There are also many reports of applications of these techniques in which the results have been evaluated subjectively. However, only a few of these methods have been the subject of quantification, using different methodologies, most of them based on microphotometry. A critical analysis of these procedures has generally shown that they are valid either relatively or at an absolute level. Nevertheless, the routine application of these methods is not as widespread as one might expect.In this paper, the methodological developments that have taken place in the histochemical assay of some enzymes by microspectrophotometry are reviewed briefly. The enzymes include acid phosphatase, non-specific esterase, succinate dehydrogenase and cytochrome oxidase. These methods have given consistent and useful results when applied to our experimental models of irradiated skin, and thus seem useful for various other applications.The reasons why there have been so few applications of quantitative histochemistry are also discussed together with possible ways of making its use more general.