Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK^+/− assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.     

Quantitative analysis of FRET assay in biology—new developments in protein interaction affinity and protease kinetics determinations in the SUMOylation cascade

F(o)rster resonance energy transfer (FRET) techniques have been widely used in biological studies in vitro and in vivo and are powerful tools for elucidating protein interactions in many regulatory cas...     

Identification of PrP sequences essential for the interaction between the PrP polymers and Aβ peptide in a yeast-based assay

Alzheimer disease is associated with the accumulation of oligomeric amyloid β peptide (Aβ), accompanied by synaptic dysfunction and neuronal death. Polymeric form of prion protein (PrP), PrP^Sc, is implicated in transmissible spongiform encephalopathies (TSEs). Recently, it was shown that the monomeric cellular form of PrP (PrP^C), located on the neuron surface, binds Aβ oligomers (and possibly other β-rich conformers) via the PrP_23–27 and PrP_90–110 segments, acting as Aβ receptor. On the other hand, PrP^Sc polymers efficiently bind to Aβ monomers and accelerate their oligomerization. To identify specific PrP sequences that are essential for the interaction between PrP polymers and Aβ peptide, we have co-expressed Aβ and PrP (or its shortened derivatives), fused to different fluorophores, in the yeast cell. Our data show that the 90–110 and 28–89 regions of PrP control the binding of proteinase-resistant PrP polymers to the Aβ peptide, whereas the 23–27 segment of PrP is dispensable for this interaction. This indicates that the set of PrP fragments involved in the interaction with Aβ depends on PrP conformational state.     

Allelic discrimination using fluorogenic probes and the 5′ nuclease assay

Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5′ nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5′ nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.     

The relative biological activities of α- and β-ecdysone and their 3 dehydro derivatives in the chromosome puffing assay

The relative biological activity of α- and β-ecdysone and their 3-dehydro derivatives in inducing changes in puffing activity in D. melanogaster salivary glands in vitro is approximately constant. Although the six different puffing assays differed considerably in their sensitivity to ecdysones, no stage-specific or locus-specific differential response to the four compounds was detected.     

A kinetic aggregation assay allowing selective and sensitive amyloid-β quantification in cells and tissues

The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for quantifying Aβ fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated Aβ fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ(1-40) reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half-time (t(50)) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.     

Synthesis of alkyl and cycloalkyl α-d-mannopyranosides and derivatives thereof and their evaluation in the mycobacterial mannosyltransferase assay

The synthesis of a series of alkyl (having from C6 to C20 aglycones), cyclohexyl, and cyclohexylalkyl α-d-mannopyranosides, 6-deoxygenated analogs, thioglycosides, and sulfones derived thereof, is reported. Here, under the in vitro assay conditions used, none of the 15 tested compounds acted as an inhibitor of the mannose transfer catalyzed by the enzymes present in mycobacterial membrane and cell wall fractions. Mannopyranosides comprising shorter aliphatic, up to 8 carbon atoms long linear, or cyclic aglycone served as the acceptor substrates in the mycobacterial mannosyltransferase reaction. The thioglycosides exhibited similar behavior, in contrast to the sulfones, which were essentially not recognized by the mycobacterial enzymes. 6-Deoxygenated glycosides were not processed by the enzymes, suggesting that the mannose transfer occurs at position 6 of the acceptors.     

Expression and purification of RHC–EGFP fusion protein and its application in hyaluronic acid assay

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM–enhanced green fluorescence protein (RHC–EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC–EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.     

High-performance liquid chromatographic assay for methyl-β-cyclodextrin in plasma and cell lysate

This paper describes a high-performance liquid chromatographic method with fluorescence detection for the analysis of methyl-β-cyclodextrin (MEBCD) in plasma and cell lysate, after in situ complexation with 1-naphthol. The size-exclusion HPLC column packed with TSK 3000 SW gel, was equilibrated with an eluent mixture composed of methanol and purified water (2:98, v/v) containing 10⁻⁴ M 1-naphthol as a fluorophore. The detection is based on fluorescence enhancement caused by the formation of inclusion complexes and was performed at 290 and 360 nm for excitation and emission, respectively. The method involved a simple treatment of the samples with chloroform. Daunorubicin was used as internal standard. Limits of quantitation were 0.8 μM in plasma and 0.5 μM in cell lysate. Detection limits of 0.5 μM (50 pmol) and 0.3 μM (30 pmol) were obtained for MEBCD in the two media, respectively. Linear detection response was obtained for concentrations ranging from 1 to 100 μM in plasma and cell lysate. Recovery from plasma proved to be more than 40%. Precision, expressed as C.V. was in the range of 4 to 11%. Accuracy ranged from 89 to 105%.     

Wheat-infectingFusarium species in Poland — their chemotypes and frequencies revealed by PCR assay

ThreeFusarium species:F. graminearum, F. culmorum andF. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identifyFusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates ofF. graminearum, the 15Ac-DON chemotype dominated, and among those whereF. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates ofF. graminearum tested, displayed the NIV chemotype. An increase in the frequency ofF. graminearum and a decrease in the frequency ofF. culmorum were found during 1998 to 2006.     

Novel and recurrent mutations in the tyrosinase gene and the P gene in the German albino population

Albinism is a heterogeneous group of genetic disorders resulting from deficiencies in pigmentation. Clinically, it is divided into ocular (OA) and oculocutaneous albinism (OCA). OCA involves lack of pigment in the skin, hair, and eyes and results from mutations in the tyrosinase gene or in the P gene. OA mainly affects pigmentation in the visual system and may be a mild form of OCA or may be caused by other genetic defects. Clinical diagnosis of albinism type is difficult, because of the observed range of phenotypic variation. Thus, genetic analysis may be helpful with respect to a more accurate diagnosis. Here, we report the mutational profile, determined by genetic analysis of the tyrosinase and P genes, of a large German albino population. We have revealed a total of 42 distinct mutations, 19 of which are novel. Of the 74 unrelated patients screened, 32 (43%) had mutations in the tyrosinase gene, 16 (22%) had P gene mutations, and 26 (35%) patients had no detectable genetic abnormalities. This defines a population of albino patients who are tyrosinase-gene- and P-gene-negative and who thus may represent a good study group for searching for additional genes associated with albinism.     

Extended-term cultures of human T-lymphocytes and the comet assay: a useful combination when testing for genotoxicity in vitro?

Extended-term cultures of human lymphocytes provide a source of uniform human cells that can be used for several experiments performed over a long time, avoiding the variability arising from taking blood samples for individual experiments. The use of extended-term cultures of human T-lymphocytes in the alkaline single-cell gel electrophoresis assay (comet assay) was evaluated as a test for the potential genotoxicity of chemicals. The DNA-damaging effects of five DNA-reactive mutagens and clastogens (benzo[a]pyrene, cyclophosphamide, formaldehyde, 4-nitroquinoline-N-oxide (4NQO) and N-nitrosopiperidine) was determined and compared with the effects of one non-DNA-reactive mutagen (5-hydroxyurea), and one non-mutagenic agent (ethanol). The alkylating and/or DNA-adduct forming agents N-nitrosopiperidine, cyclophosphamide, 4-nitroquinoline-N-oxide and benzo[a]pyrene increased the DNA migration in a dose-dependent manner. In contrast, the DNA/protein-crosslinking agent formaldehyde decreased the migration of DNA during the electrophoresis. The lowest observed effect levels (LOELs) under the experimental conditions used in the present study, were: 0.0001 mM (4-nitroquinoline-N-oxide without S9), 0.05 mM (benzo[a]pyrene with S9), 0.1mM (formaldehyde without S9), 0.25 mM (cyclophosphamide with S9), and 0.5mM (N-nitrosopiperidine with S9), respectively. The antimetabolite 5-hydroxyurea was also found to increase the tail moment, but only in cells that had been exposed to rather high concentrations (> or =10mM) of the compound. Ethanol did not affect the tail moment, not even in cells that had been exposed to an apparently cytotoxic concentration (500 mM). The results of the present study are in qualitative agreement with those obtained using other cells in the alkaline comet assay and it is therefore concluded that extended-term cultures of human T-lymphocytes and the alkaline version of the single-cell gel electrophoresis assay is a useful combination when testing for the potential genotoxicity of chemicals.     

Liquid chromatography–tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid

BackgroundAnalysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.MethodsThe method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (¹³C₃-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients’ samples with known differences in SA metabolites like meningitis (n = 6), brain tumour (n = 2), leukaemia (n = 5), and Salla disease (n = 1).ResultsLimit of detection (LOD) was 0.54 μM for FSA and 0.45 μM for TSA. Intra- and inter-assay variation for FSA (21.8 μM) were 4.8% (n = 10) and 10.4% (n = 40) respectively. Intra- and inter-assay variation for TSA (35.6 μM) were 9.7% (n = 10) and 12.8% (n = 40) respectively. Tested patients showed values of TSA above established reference value.ConclusionThe validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.     

Detection of the genotoxicity of air pollutants in and around the city of São Paulo (Brazil) with the Tradescantia-micronucleus (Trad-MCN) assay

Tradescantia pallida cv. purpurea, a popular garden plant in Brazil, was used for the Tradescantia micronucleus (Trad-MCN) assay. In situ monitoring of the genotoxicity of air pollutants was carried out by sentinel approach, using the plant grown in the field or using the plants in pots which were carried to the monitoring sites. Two highly polluted sites, in S?o Paulo city (Cerqueira Cesar and Congonhas) and two rural sites (the cities of Pirassununga, 200 km and Caucaia do Alto, 50 km from S?o Paulo, respectively) were chosen for this study, in order to determine the gradient difference of the air pollution levels. Sentinel plants in Congonhas site presented the highest frequency of micronuclei (4.4%), in comparison with 2.2 and 2.3% found in plants from Pirassununga and Cerqueira Cesar sites, respectively (Kruskal-Wallis; P<0.020). Significant increases (F test; P<0.0001) in the frequency of micronuclei were observed in plants exposed in the polluted urban sites (Cerqueira Cesar: 5.7%; Congonhas: 7.1% and Caucaia do Alto: 2.3%). The increase in the frequency of micronuclei observed indicates the potential risk of mutagenicity in presence of high concentrations of pollutants.     

Leukocyte 8-oxo-7,8-dihydro-2′-deoxyguanosine and comet assay in epirubicin-treated patients

Epirubicin fights cancer through topoisomerase II inhibition, hence producing DNA strand breaks that finally lead to cell apoptosis. But anthracyclines produce free radicals that may explain their adverse effects. Dexrazoxane—an iron chelator—was proven to decrease free radical production and anthracycline cardiotoxicity.

In this article, we report the concentrations of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) relative to 2′-deoxyguanosine (dGuo), and comet assay results from a study including 20 cancer patients treated with epirubicin. Plasma concentrations of vitamins A, E, C and carotenoids are also reported. All data were obtained before and immediately after epirubicin infusion. The ratios of 8-Oxo-dGuo to dGuo were measured in leukocyte DNA by HPLC-coulometry after NaI extraction of nucleic acids. Vitamins A and E and carotenoids were measured by HPLC-spectrophotometry. Vitamin C was measured by HPLC-spectrofluorimetry.

Median 8-oxo-dGuo/dGuo ratios increased significantly from 0.34 to 0.48 lesions per 100,000 bases while per cent of tail DNA increased from 3.47 to 3.94 after chemotherapy 8-Oxo-dGuo/dGuo and per cent of tail DNA medians remained in the normal range. Only vitamin C decreased significantly from 55.4 to 50.3?μM Decreases in vitamins A, E, lutein and zeaxanthin were not significant, but concentrations were below the lower limit of the normal range both before and after chemotherapy. Only the correlation between comet assay results and vitamin C concentrations was significant ( ).

This study shows that cellular DNA is damaged by epirubicin-generated free radicals which produce the mutagenic modified base 8-oxo-dGuo and are responsible for strand breaks. However, strand breaks are created not only by free radicals but also by topoisomerase II inhibition. In a previous study we did not find any significant change in urinary 8-oxo-dGuo excretion after adriamycin treatment. However, 8-oxo-dGuo may have increased at the end of urine collection as DNA repair and subsequent kidney elimination are relatively slow processes. In another study, authors used GC-MS to detect 8-oxo-dGuo in DNA and did not find any change after prolonged adriamycin infusion. Reasons for these apparent discrepancies are discussed.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan? assay

A Serratia plymuthica-specific TaqMan? assay was designed based on the consensus nucleotide sequence from the 3??- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan? assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan? assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100?cfu ml^?1) in vitro, down to 50 bacterial cells per reaction (1,000?cfu ml^?1) in spiked potato root extracts and down to 5 bacterial cells per reaction (100?cfu ml^?1) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan? assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.