Assay for the determination of urinary 8-hydroxy-2′-deoxyguanosine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic procedure with electrochemical detection is described for the determination of urinary 8-hydroxy-2′-deoxyguanosine, a major oxidative DNA lesion induced by radical forming agents. A two-step solid-phase extraction procedure was followed for extracting 8-hydroxy-2′-deoxyguanosine from human urine and the analysis was performed on a RP-18 analytical column under isocratic conditions. The limit of detection of 8-hydroxy-2′-deoxyguanosine in urine was found to be 0.9 nM. The non-invasive assay provides an indirect measurement of oxidative DNA damage.     

Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA

The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, ¹H and ¹³C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx₂-dG) and N²-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx₁-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx₂-dG were found to be unstable and partly transformed to Gx₁-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx₁-dG was the only stable adduct. The transformation of Gx-dG to Gx₁-dG seemed to take place in single-stranded DNA and therefore, Gx₁-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.     

P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The ³¹P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5-C5′ bond (O4′-C5′-C5-C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds.The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV-visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.     

Photophysical characterization of methyl viologen ion-exchanged within a zirconium phosphate framework

The photophysical properties of 1,1′-dimethyl-4,4′dipyridinium (methyl viologen, MV²⁺) intercalated within zirconium phosphate (ZrP) were investigated. The intercalation of MV²⁺ within ZrP was achieved by ion-exchange using a hydrated form of ZrP with six water molecules per formula unit and an interlayer distance of 10.3 Å. The intercalation yields a new phase with an interlayer distance up to 10.6 Å. The MV²⁺-exchanged ZrP material was characterized using elemental analysis, XRPD and IR data. The MV²⁺-exchanged ZrP materials show a red shift in the UV-Vis spectra in contrast with solution. The photoexcitation of nitrogen purged, MV²⁺-exchanged ZrP water suspensions with UV light leads to fluorescence emission with a maximum at 337 nm. The photoexcitation of MV²⁺-exchanged ZrP suspensions without nitrogen purging yields two fluorescence emissions with maxima at 337 and 450 nm. The emission in the visible region can be attributed to a photodecomposition product. The fluorescence quantum yields indicate that the emission of MV²⁺-exchanged ZrP is of the same order of magnitude as that of MV²⁺ in water indicating a strong deactivation of the excited state by non-radiative pathways.     

A Structural Basis for the Recognition of 2′-Deoxyguanosine by the Purine Riboswitch

Riboswitches are noncoding RNA elements that are commonly found in the 5′-untranslated region of bacterial mRNA. Binding of a small-molecule metabolite to the riboswitch aptamer domain guides the folding of the downstream sequence into one of two mutually exclusive secondary structures that directs gene expression. The purine riboswitch family, which regulates aspects of purine biosynthesis and transport, contains three distinct classes that specifically recognize guanine/hypoxanthine, adenine, or 2′-deoxyguanosine (dG). Structural analysis of the guanine and adenine classes revealed a binding pocket that almost completely buries the nucleobase within the core of the folded RNA. Thus, it is somewhat surprising that this family of RNA elements also recognizes dG. We have used a combination of structural and biochemical techniques to understand how the guanine riboswitch could be converted into a dG binder and the structural basis for dG recognition. These studies reveal that a limited number of sequence changes to a guanine-sensing RNA are required to cause a specificity switch from guanine to 2′-deoxyguanosine, and to impart an altered structure for accommodating the additional deoxyribose sugar moiety.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

Detection of broad-spectrum aminoglycoside antibiotics through fluorescence-labeling aminoglycoside acetyltransferase(6')-Ii

Aminoglycosides are among the most commonly used antibiotics. The intensive use of aminoglycoside antibiotics has led to the problem of food contamination and the development of antibiotic-resistant bacteria. In the present study, we developed an effective method for easy sensitive detection of broad-spectrum aminoglycoside antibiotics. Aminoglycoside 6′-N-acetyltransferase family catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to the 6′ amino group of the aminoglycoside, which is one of the most widespread determinants of aminoglycoside resistance. Because acetyl-CoA is naturally present only in living organisms, it is expected that the enzyme can bind with aminoglycoside antibiotics without catalysis in vitro. The enzyme was mutated for the introduction of a cysteine residue to flexible loops close to the binding site, which was then labeled with thio-labeling reagent fluorescein-5-maleimide. The labeled enzymes were characterized with kinetic and binding studies of various known aminoglycoside antibiotics. The binding of the labeled enzyme with aminoglycoside antibiotics causes a conformational change of the enzyme, which subsequently changes the hydrophobicity and hydrophilicity environment of fluorescent labeling reagent resulting in emission of fluorescence. This study provides a sensitive detection method for residual aminoglycoside antibiotics and strategies to screen and discover new effective aminoglycoside antibiotics.     

Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification

DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acids. Here, an enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4 PNK. A hairpin oligonucleotide (hpDNA) was designed as a probe whose stem can be degraded from the 5′ to 3′ direction by lambda exonuclease (λ exo) when its 5′ end is phosphorylated by PNK. So, the 3′ stem and loop part of hpDNA was released as an initiator strand to open a molecular beacon (MB) that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by the nicking endonuclease (Nt.BbvCI) to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of 1.0 × 10⁻⁴ U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK as well as for studying the activity of other nucleases.     

A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with “detector” proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2′-O-(N-methylanthraniloyl) guanosine 5′-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.     

High-performance capillary electrophoretic method for the quantification of global DNA methylation: Application to methotrexate-resistant cells

Global DNA hypomethylation in tumor tissue is a common characteristic in a variety of malignancies such as breast, colon, oral, lung, and blood cancers. A rapid and sensitive method has been developed for the determination of global DNA methylation in cells. Five substances—2′-deoxycytidine (dC), 5-methyl 2′-deoxycytidine (mdC), 2′-deoxyadenosine (dA), 2′-deoxythymidine (dT), and 2′-deoxyguanosine (dG)—were completely separated by high-performance capillary electrophoresis in 10 min. Intraday coefficient of variation was less than 1%, and interday coefficient of variation was less than 2%. The minimal detection limit was 1 μM. Acquired drug resistance to methotrexate (MTX) is one of the most serious problems in cancer chemotherapy. Under optimal conditions, we analyzed global DNA methylation levels in A549 and A549/MTX cells, and only 10⁵ cells are needed to obtain reliable results. The percentage of 5-methyl-2′-deoxycytidine (5-mC) was 4.80 ± 0.52% in A549 cells, and this decreased to 4.20 ± 0.44% in A549/MTX cells. It was considered as statistically significant. This demonstrated that the mechanisms of acquired drug resistance to MTX might be concerned with DNA methylation.     

Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence

Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) following excitation at 280 nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280 nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration. Stronger quenching of the intrinsic BSA and IgG fluorescence in steady state than in time-resolved fluorescence by bis-ANS and ANS pointed toward static quenching being the dominant mechanism in addition to dynamic quenching and/or FRET. In conclusion, one should consider the role of direct excitation of ANS and bis-ANS at 280 nm to ensure a proper interpretation of fluorescence signals resulting from dye-protein interactions. When ANS or bis-ANS is to be used for protein characterization, we recommend selectively exciting the dyes at the higher absorption wavelength maximum (370 or 385 nm, respectively).     

Development of a fast ELISA for quantifying polio D-antigen in in-process samples

A fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20 h-protocol was optimized by minimizing the total incubation time to 1 h, and by replacing the signal reagent 3,3′,5,5′-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range.     

Fluorescence derivatisation of urinary corticosteroids for high-performance liquid chromatographic analysis

Corticosteroids containing a C₂₁ primary hydroxyl group were derivatised with 9-anthroyl cyanide. The reagent was prepared as a solution in acetonitrile, containing 0.1% triethylamine, at a concentration of 2 mg/ml. Approximately 1 μg of corticosteroid was reacted with 100 μl of this reagent, at 45°C for 2 h. The fluorescent derivatives were separated by HPLC on a silica column, 250×4.6 mm I.D., by stepwise elution, with a mobile phase of 2-propanol–hexane (2:98) for 20 min, followed by 2-propanol–hexane (7:93) from 20 to 40 min. The fluorescence detector was set to 370-nm excitation and 470-nm emission. The relatively low temperature for derivatisation avoided reaction with secondary hydroxyl groups and also prevented thermal degradation of the corticosteroids.     

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488 nm was recorded on excitation at 385 nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP-SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.     

Pyrene-labeled deoxyguanosine as a fluorescence sensor to discriminate single and double stranded DNA structures: Design of ends free molecular beacons

A novel fluorescent DNA probe containing pyrene-labeled C8 alkylamino-substituted 2′-deoxyguanosine was designed in order to discriminate single stranded and double stranded regions in DNA. This fluorescent sensor was used for the design of practically useful 3′- and 5′-ends free self-quenched molecular beacon (MB). Unique MB detectable by pyrene excimer fluorescence was also demonstrated.     

Structural and fluorescent study of zinc complexes of dansyl aminoquinoline

We have shown previously that 8-(5′-N,N-dimethylamino-1′-naphthalene)-sulfonamidoquinoline (DANQUIN) demonstrated a remarkable selectivity and sensitivity for the Zn(II) ion. In this work, the crystal structures of DANQUIN, Cu(DANQUIN)₂ and Cu(DANPY)₂ (DANPY, N-2-picolyl-(5′-N,N-dimethylamino-1′-naphthalene)-sulfonamide) are reported and compared with the simulated structure of Zn(DANQUIN)₂, which is important for the understanding of the factors that govern the fluorescence of DANQUIN. Free DANQUIN mainly displays the fluorescence of the dansyl group at 547 nm while the Zn(II)-DANQUIN complex mainly shows the enhanced fluorescence of aminoquinoline at 469 nm, while the emission of the dansyl group shifted to 517 nm with an almost constant intensity. This result demonstrates the advantage of this hybrid fluorescent chemosensor for Zn(II), and also makes it a potential candidate for ratiometric Zn(II) detection.     

Synthesis of 3'-azido-3'-deoxythymidine (AZT)--Cinchona alkaloid conjugates via click chemistry: Toward novel fluorescent markers and cytostatic agents

Novel nucleoside-Cinchona alkaloid conjugates were synthesized using ‘click’ chemistry approach based on the copper(I) catalyzed Huisgen azide-alkyne cycloaddition. Two series of conjugates were prepared employing 3′-azido-3′-deoxythymidine (AZT) as the azide component and the four 10,11-didehydro Cinchona alkaloids as well as their 9-O-propargyl ethers as the alkyne components. All obtained conjugates showed strong fluorescence emission and some of them exhibited marked cytotoxic activity in vitro.