S-0 and S-1 State Structure, Methyl Torsional Barrier Heights, and Fast Intersystem Crossing Dynamics of 5-Methyl-2-hydroxypyrimidine

We report the analysis of the SI So rotational band contours of jet-cooled 5-methyl-2-hydroxypyrimidine (5M2HP), the enol form of deoxythymine. Unlike thymine, which exhibits a structureless spectrum, the vibronic spectrum of 5M2HP is well structured, allowing us to determine the rotational constants and the methyl group torsional barriers in the S-0 and S-1 states. The 0(0)(0), 6a(0)(1), 6b(0)(1), and 14(0)(1) band contours were measured at 900 MHz (0.03 cm(-1)) resolution using mass-specific two-color resonant two-photon ionization (2C-R2PI) spectroscopy. All four bands are polarized perpendicular to the pyrimidine plane (>90% c type), identifying the S-1 <- S-0 excitation of 5M2HP as a 1n pi* transition. All contours exhibit two methyl rotor subbands that arise from the lowest 5-methyl torsional states 0A '' and 1E ''. The S-0 and S-1 state torsional barriers were extracted from fits to the torsional subbands. The 3-fold barriers are V-3 '' = 13 cm(-1) and V3' = SI cm(-1); the 6-fold barrier contributions V-6 '' and V-6' are in the range of 2-3 cm(-1) and are positive in both states. The changes of A, B, and C rotational constants upon S-1 <- S-0 excitation were extracted from the contours and reflect an "anti-quinoidal" distortion. The 0(0)(0) contour can only be simulated if a 3 GHz Lorentzian line shape is included, which implies that the S-1(1n pi*) lifetime is similar to 55 ps. For the 6a(0)(1) and 6b(0)(1) bands, the Lorentzian component increases to 5.5 GHz, reflecting a lifetime decrease to similar to 30 ps. The short lifetimes are consistent with the absence of fluorescence from the 1n pi* state. Combining these measurements with the previous observation of efficient intersystem crossing (ISC) from the Si state to a long-lived T-1((3)n pi*) state that lies similar to 2200 cm(-1) below [S. Lobsiger, S. et al. Phys. Chem. Chem. Phys. 2010, 12, 5032] implies that the broadening arises from fast intersystem crossing with k(ISC) approximate to 2 x 10(10) s(-1). In comparison to 5-methylpyrimidine, the ISC rate is enhanced by at least 10 000 by the additional hydroxy group in position 2.

Vibronic Spectra of Jet-Cooled 2-Aminopurine center dot H2O Clusters Studied by UV Resonant Two-Photon Ionization Spectroscopy and Quantum Chemical Calculations

For understanding the major- and minor-groove hydration patterns of DNAs and RNAs, it is important to understand the local solvation of individual nucleobases at the molecular level. We have investigated the 2-aminopurine center dot H2O. monohydrate by two-color resonant two-photon ionization and UV/UV hole-burning spectroscopies, which reveal two isomers, denoted A and B. The electronic spectral shift delta nu of the S-1 <- S-0 transition relative to bare 9H-2-aminopurine (9H-2AP) is small for isomer A (-70 cm(-1)), while that of isomer B is much larger (delta nu = 889 cm(-1)). B3LYP geometry optimizations with the TZVP basis set predict four cluster isomers, of which three are doubly H-bonded, with H2O acting as an acceptor to a N-H or -NH2 group and as a donor to either of the pyrimidine N sites. The "sugar-edge" isomer A is calculated to be the most stable form with binding energy D-e = 56.4 kJ/mol. Isomers B and C are H-bonded between the -NH2 group and pyrimidine moieties and are 2.5 and 6.9 kJ/mol less stable, respectively. Time-dependent (TD) B3LYP/TZVP calculations predict the adiabatic energies of the lowest (1)pi pi* states of A and B in excellent agreement with the observed 0(0)(0) bands; also, the relative intensities of the A and B origin bands agree well with the calculated S-0 state relative energies. This allows unequivocal identification of the isomers. The R2PI spectra of 9H-2AP and of isomer A exhibit intense low-frequency out-of-plane overtone and combination bands, which is interpreted as a coupling of the optically excited (1)pi pi* state to the lower-lying (1)n pi* dark state. In contrast, these overtone and combination bands are much weaker for isomer B, implying that the (1)pi pi* state of B is planar and decoupled from the (1)n pi* state. These observations agree with the calculations, which predict the (1)n pi* above the (1)pi pi* state for isomer B but below the (1)pi pi* for both 9H-2AP and isomer A.

Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were < 4.4 and 5.3%, respectively, the LOQ was 20 ng/mL, and the assay was linear in the range of 0.020-20 microg/mL with an accuracy of > or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma.

Hypermethylation of the DPYD promoter region is not a major predictor of severe toxicity in 5-fluorouracil based chemotherapy

BACKGROUND: The activity of dihydropyrimidine dehydrogenase (DPD), the key enzyme of pyrimidine catabolism, is thought to be an important determinant for the occurrence of severe toxic reactions to 5-fluorouracil (5-FU), which is one of the most commonly prescribed chemotherapeutic agents for the treatment of solid cancers. Genetic variation in the DPD gene (DPYD) has been proposed as a main factor for variation in DPD activity in the population. However, only a small proportion of severe toxicities in 5-FU based chemotherapy can be explained with such rare deleterious DPYD mutations resulting in severe enzyme deficiencies. Recently, hypermethylation of the DPYD promoter region has been proposed as an alternative mechanism for DPD deficiency and thus as a major cause of severe 5-FU toxicity. METHODS: Here, the prognostic significance of this epigenetic marker with respect to severe 5-FU toxicity was assessed in 27 cancer patients receiving 5-FU based chemotherapy, including 17 patients experiencing severe toxic side effects following drug administration, none of which were carriers of a known deleterious DPYD mutation, and ten control patients. The methylation status of the DPYD promoter region in peripheral blood mononuclear cells was evaluated by analysing for each patient between 19 and 30 different clones of a PCR-amplified 209 base pair fragment of the bisulfite-modified DPYD promoter region. The fragments were sequenced to detect bisulfite-induced, methylation-dependent sequence differences. RESULTS: No evidence of DPYD promoter methylation was observed in any of the investigated patient samples, whereas in a control experiment, as little as 10% methylated genomic DNA could be detected. CONCLUSION: Our results indicate that DYPD promoter hypermethylation is not of major importance as a prognostic factor for severe toxicity in 5-FU based chemotherapy.

Radiation metabolomics. 2. Dose- and time-dependent urinary excretion of deaminated purines and pyrimidines after sublethal gamma-radiation exposure in mice

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.

Sodium hydride-mediated cascade reaction towards the synthesis of 1,5-disubstituted uracil from cyanamides derived from the Baylis-Hillman ad-ducts

Sodium hydride-mediated cascade reaction towards the synthesis of 1,5-disubstituted uracil from cyanamides derived from the Baylis-Hillman ad-ducts

Intron involvement in the regulation of MuSVts110 RNA splicing

The murine sarcoma virus MuSVts110 exhibits an alternative RNA splicing pattern. Like other simple retroviruses, MuSVts110 pre-mRNA splicing is balanced to allow the production of both spliced and unspliced RNA during the replicative cycle. In addition to balance, MuSVts110 RNA splicing exhibits a unique growth-temperature restriction to splicing; temperatures below 33$\sp\circ$C are permissive for splicing while temperatures of 37$\sp\circ$C or above are non-permissive. Previous work has established that this thermosensitive splicing phenotype is mediated in cis by viral transcript features. Here we show that at least three sequence elements regulate the MuSVts110 splicing phenotype. First, the MuSVts110 branchpoint (BP) and poly-pyrimidine tract (PPT) were found to be determinants of overall splicing efficiency. Wild-type MuSVts110 possesses a weak BP and PPT adjacent to the 3$\sp\prime$ splice site. Introduction of a strong BP caused MuSVts110 splicing to proceed to virtual completion in vivo, thus losing any vestige of balance or thermosensitivity. In in vitro splicing extracts, the strong BP overcame a blockade to wt MuSVts110 splicing at both the first and second catalytic steps. Weakening the consensus nature of the strong BP allowed the recovery of thermosensitive splicing in vivo, and reinstated the blockades to splicing in vitro, arguing that a suboptimal BP is an unusual manifestation of the proportional splicing pattern of retroviruses. The PPT is essential for accurate recognition of the BP sequence by the splicing machinery. Lengthening the PPT of MuSVts110 from 9 to 19 consecutive pyrimidines increased the overall efficiency of splicing in vivo dramatically, but was less effective than the strong BP in overriding the restriction on splicing imposed by high growth temperatures. Finally, decreasing gradually the overall size of the intron unexpectedly reduced splicing efficiency at growth temperatures permissive for splicing, suggesting that non-conserved sequences within the intron of MuSVts110 participate in splicing regulation as well. Taken together, these results suggest a mechanism of control in which MuSVts110 splicing is modulated by the entire intron, but principally by suboptimal signals at the splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing. ^

Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstable

Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues--synthesis and biochemical characterization.

Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO–), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the MNCO– ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3–)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3– loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO– and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar modified ONs.

Patho-genetics of Clostridium chauvoei

The genomic sequence of Clostridium chauvoei, the etiological agent of blackleg, a severe disease of ruminants with high mortality specified by a myonecrosis reveals a chromosome of 2.8 million base-pairs and a cryptic plasmid of 5.5 kilo base-pairs. The chromosome contains the main pathways like glycolysis/gluconeogenesis, sugar metabolism, purine and pyrimidine metabolisms, but the notable absence of genes of the citric acid cycle and deficient or partially deficient amino acid metabolism for Histidine, Tyrosine, Phenylalanine, and Tryptophan. These essential amino acids might be acquired from host tissue damage caused by various toxins and by protein metabolism that includes 57 genes for peptidases, and several ABC transporters for amino acids import.

Recent improvements in antigene technology

DNA triple helix based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother like treatment in comparison to the flourishing field of oligonucleotide based control of translation (antisense technology). This was mostly due to lack of affinity of triplex forming oligonucleotides (TFOs) to their DNA target, to sequence restriciton constraints imposed by the triple helical recogniton motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine-purine inversion sites as well as sugar modifications, e.g. the 2'-aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid (LNA) sugar unit, that greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence specific genomic DNA manipulation starts to become a useful tool in biotechnology

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^

A comprehensive assessment of environmental ultraviolet radiation induced DNA damage in marine microorganisms

There is evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern is the annual pattern of ozone depletion over Antarctica and the Southern Ocean. Reduction of ozone concentration selectively limits absorption of solar UV-B (290–320 nm), resulting in higher irradiance at the Earth's surface. The effects of ozone depletion on the human population and natural ecosystems, particularly the marine environment, are a matter of considerable concern. Indeed, marine plankton may serve as sensitive indicators of ozone depletion and UV-B fluctuations. Direct biological effects of UVR result from absorption of UV-B by DNA. Once absorbed, energy is dissipated by a variety of pathways, including covalent chemical reactions leading to the formation of photoproducts. The major types of photoproduct formed are cyclobutyl pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. Marine plankton repair these photoproducts using light-dependent photoenzymatic repair or nucleotide excision repair. The studies here show that fluctuations in CPD concentrations in the marine environment at Palmer Station, Antarctica correlate well with ozone concentration and UV-B irradiance at the Earth's surface. A comparison of photoproduct levels in marine plankton and DNA dosimeters show that bacterioplankton display higher resistance to solar UVR than phytoplankton in an ozone depleted environment. DNA damage in marine microorganisms was investigated during two separate latitudinal transects which covered a total range of 140°. We observed the same pattern of change in DNA damage levels in dosimeters and marine plankton as measured using two distinct quantitative techniques. Results from the transects show that differences in photosensitivity exist in marine plankton collected under varying UVR environments. Laboratory studies of Antarctic bacterial isolates confirm that marine bacterioplankton possess differences in survival, DNA damage induction, and repair following exposure to UVR. Results from DNA damage measurements during ozone season, along a latitudinal gradient, and in marine bacterial isolates suggest that changes in environmental UVR correlate with changes in UV-B induced DNA damage in marine microorganisms. Differences in the ability to tolerate UVR stress under different environmental conditions may determine the composition of the microbial communities inhabiting those environments. ^