Ultraviolet light induces reactivation in a murine model of cutaneous herpes simplex virus-1 infection.

We have developed a model of cutaneous herpes simplex virus-1 (HSV-1) reactivation in SKH-1 hairless mice which closely mimics the condition in humans. Sixty plaque-forming units of HSV-1 strain 17 syn+ were applied to a superficially abraded area on the lateral body wall. More than 85% of mice developed primary HSV-1 infection characterized by a zosteriform pattern of cutaneous vesiculation and ulceration. Approximately one-third of mice with primary skin lesions succumbed to neurologic disease and in the remaining mice cutaneous lesions healed completely. Subsequent exposure of healed areas to two minimal inflammatory doses of UV resulted in recrudescence of skin lesions in the irradiated areas in almost 60% of mice. Lesions appeared approximately 4 days after irradiation, persisted for 3-5 days and then resolved completely. Reactivation rarely resulted in death due to neurologic disease. Primary lesions had a histologic appearance typical of cutaneous HSV-1 infection with vesicles and focal epithelial necrosis accompanied by the formation of epithelial syncytial cells and the presence of herpetic intranuclear inclusion bodies. In primary lesions HSV-1 was demonstrated by immunohistochemistry, polymerase chain reaction and culture. In reactivated lesions epithelial syncytia and inclusion bodies were not seen; however, virus was demonstrable by polymerase chain reaction and culture. Exposure of the uninfected side to UV did not stimulate disease recurrence suggesting that local effects of UV rather than systemic immunosuppression were responsible for reactivation. Reactivation could also be obtained with two minimal inflammatory doses of UV from a UV-340 light source which emits light approximating the solar spectrum.     

Independent generation and characterization of a C2'-oxidized abasic site in chemically synthesized oligonucleotides

Abasic lesions, which are formed endogenously and as a consequence of exogenous agents, are lethal and mutagenic. Hydrogen atom abstraction from C2' in DNA under aerobic conditions produces an oxidized abasic lesion (C2-AP), along with other forms of DNA damage. The effects of C2-AP on DNA structure and function are not well understood. A method for the solid-phase synthesis of oligonucleotides containing C2-AP lesions is reported. The lesion is released via periodate oxidation of a triol containing a vicinal diol. The triol is introduced via a phosphoramidite that is compatible with standard oligonucleotide synthesis and deprotection conditions. UV-melting studies indicate that the C2-AP lesion has a comparable effect on the thermal stability of duplex DNA as other abasic lesions. The C2-AP lesion is rapidly cleaved by piperidine at 90 degrees C. However, cleavage by NaOH (0.1 M, 37 degrees C) shows that C2-AP is considerably less labile (t(1/2) = 3.3 +/- 0.2 h) than other abasic lesions.     

Modeling the chemical step utilized by human alkyladenine DNA glycosylase: a concerted mechanism AIDS in selectively excising damaged purines

Human alkyladenine DNA glycosylase (AAG) initiates the repair of a wide variety of (neutral or cationic) alkylated and deaminated purines by flipping damaged nucleotides out of the DNA helix and catalyzing the hydrolytic N-glycosidic bond cleavage. Unfortunately, the limited number of studies on the catalytic pathway has left many unanswered questions about the hydrolysis mechanism. Therefore, detailed ONIOM(M06-2X/6-31G(d):AMBER) reaction potential energy surface scans are used to gain the first atomistic perspective of the repair pathway used by AAG. The lowest barrier for neutral 1,N(6)-ethenoadenine (εA) and cationic N(3)-methyladenine (3MeA) excision corresponds to a concerted (A(N)D(N)) mechanism, where our calculated ΔG(?) = 87.3 kJ mol(-1) for εA cleavage is consistent with recent kinetic data. The use of a concerted mechanism supports previous speculations that AAG uses a nonspecific strategy to excise both neutral (εA) and cationic (3MeA) lesions. We find that AAG uses nonspecific active site DNA-protein π-π interactions to catalyze the removal of inherently more difficult to excise neutral lesions, and strongly bind to cationic lesions, which comes at the expense of raising the excision barrier for cationic substrates. Although proton transfer from the recently proposed general acid (protein-bound water) to neutral substrates does not occur, hydrogen-bond donation lowers the catalytic barrier, which clarifies the role of a general acid in the excision of neutral lesions. Finally, our work shows that the natural base adenine (A) is further inserted into the AAG active site than the damaged substrates, which results in the loss of a hydrogen bond with Y127 and misaligns the general base (E125) and water nucleophile to lead to poor nucleophile activation. Therefore, our work proposes how AAG discriminates against the natural purines in the chemical step and may also explain why some damaged pyrimidines are bound but are not excised by this enzyme.     

Discovery and mutagenicity of a guanidinoformimine lesion as a new intermediate of the oxidative deoxyguanosine degradation pathway

Oxidative degradation of DNA is a major mutagenic process. Reactive oxygen species (ROS) produced in the course of oxidative phosphorylation or by exogenous factors are known to attack preferentially deoxyguanosine. The latter decomposes to give mutagenic lesions, which under physiological conditions are efficiently repaired by specialized maintenance systems in the cell. Although many intermediates of the degradation pathway are today well-known, we report in this study the discovery of a new intermediate with an interesting guanidinoformimine structure. The structure elucidation of the new lesion was possible by using HPLC-MS techniques and organic synthesis. Finally we report the mutagenic potential of the new lesion in comparison to the known lesions imidazolone and oxazolone using primer extension and pyrosequencing experiments.     

Detection of dysplastic lesions by fluorescence in a model of chronic colitis in rats after local application of 5-aminolevulinic acid and its esterified derivatives

5-Aminolevulinic acid (ALA) and ALA ester-induced protoporphyrin IX (PPIX) fluorescence are used for photodynamic diagnosis and therapy with promising results. The aim of the present study was to investigate the detection of dysplastic lesions by fluorescence after topical application of ALA and different esterified derivatives in a model of chronic colitis in rats. In female CD rats chronic colitis was induced by oral application of 5% dextrane sulfate sodium. ALA was used at different concentrations (0.072 and 0.036 mol/L). ALA-methylester (m-ALA), ALA-hexylester (h-ALA) and ALA-benzylester (b-ALA) were used at a concentration of 0.003, 0.002 and 0.002 mol/L, respectively. Fluorescence was examined under blue light, and histological findings of fluorescent and nonfluorescent biopsy specimens were recorded. Using ALA at a concentration of 0.072 mol/L, all dysplastic lesions (8/8) showed fluorescence (sensitivity 100%). Specificity was low at 57%. Reducing the concentration to 0.036 mol/L resulted in a sensitivity of only 56% (5/9) with an increase in specificity to 76%. On using h-ALA, sensitivity was 60% (3/5) with a specificity of 51%. Using m-ALA and b-ALA, sensitivity values were 25% and 33%, and values for specificity were 62% and 63%, respectively. Despite a low number of dysplastic lesions, the results of this study indicate that ALA ester-induced PPIX fluorescence has the potential for the detection of premaligant lesions but was not superior to ALA. ALA esters were used in 18- to 36-fold lower concentrations compared with ALA.     

Incorporation of a minimal nucleotide into DNA

Abasic sites are amongst the most frequent DNA lesions and result from spontaneous hydrolysis of the glycosidic bond or from the removal of damaged nucleobases. These depurination events can also occur on free deoxyribonucleoside triphosphates present in cells and lead to the formation of an abasic site triphosphate of which very little is known. Herein, we report the synthesis and biochemical characterization of the minimal triphosphate dФTP. Unexpectedly, dФTP is tolerated by various DNA polymerases and the incorporation efficiency obeys the A-rule. Single incorporation of dФMP units were also observed opposite abasic sites and the addition of prosthetic molecules mimicking base-pairs do not seem to favor the process.     

Chemical mediators: the remarkable structure and host-selectivity of depsilairdin, a sesquiterpenic depsipeptide containing a new amino acid

The chemical structure determination of depsilairdin, a highly selective phytotoxin produced by the plant pathogenic fungus Leptosphaeriamaculans/Phoma lingam, is described. The elucidation of the unusual chemical structure used a combination of NMR spectral data and X-ray crystallography. The absolute configuration was established using chemical degradation and synthesis of (3S,6R)-3,6-diisopropyl-2,5-morpholinedione and its (3R,6S) and (3R,6R) stereoisomers. Similar to the fungal pathogen, depsilairdin caused strong lesions only on brown mustard leaves but not on related species. [structure: see text]     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

Model compounds for (6-4) photolyases: a comparative flavin induced cleavage study of oxetanes and thietanes

Thietanes were used in the past as mimics for an unstable oxetane intermediate formed during the repair of mutagenic (6-4) lesions. The thietane derivatives were found to be not repaired, raising the question of how well thietanes are cleaved by single electron donation compared to oxetanes. We have prepared two flavin-containing oxetane and thietane model compounds for the (6-4) photolyase catalyzed repair process and we show that both are efficiently cleaved by a reduced and deprotonated flavin. Thietanes are therefore excellent models. The lack of their repair can be attributed to lack of binding.     

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV‐light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis‐syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6‐4)pyrimidone photoproducts ((6‐4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6‐4)C lesion containing DNA duplex in complex with the (6‐4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6‐4) lesions into strained four‐membered ring repair intermediates, it is surprising that the not rearranged T(6‐4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6‐4) DNA photolyases.     

Electrochemotherapy of Mycosis fungoides by interferon-alpha

Eight patients with 29 lesions of histologically verified 1st stage of Mycosis fungoides were successfully treated by electrochemotherapy with interferon-alpha. For this purpose 8 biphasic pulses were used, each of 50+50 micros duration with 900 micros interpulse intervals, resulting in a burst of 7.1 ms total duration. Compared to the traditional monoimmunotherapy with interferon-alpha applied three times weekly for a total of 4 weeks, the electrochemotherapy was very efficient. Complete response (CR) was observed in 25 (86%) of the 29 treated lesions by single-act electrochemotherapy with interferon-alpha. At the end of the 12-month period, all 29 lesions showed 100% complete response (CR). New lesions for a period of 12 months were not observed. The expected mechanism involved in multiple cytotoxic action of interferon-alpha could be the local increased concentration in the tumour and prolongation of the time of its action after the application of pulses.     

Distinctive Molecular Abnormalities in Benign and Malignant Skin Lesions: Studies by Raman Spectroscopy

Abstract— Near-infrared Fourier transform Raman spectroscopy is an analytical, nondestructive technique that provides information about the molecular structure of the investigated sample. The molecular structure of proteins and lipids differs between neoplastic and normal tissues and therefore Raman spectroscopy has been considered promising for the diagnosis of cancer. We aimed to compare the molecular structure of normal skin, benign and malignant skin lesions by the near-infrared Fourier transform Raman spectroscopy. Biopsies were obtained from the following skin lesions: skin tag, dermatofibroma, seborrhoeic keratosis, actinic keratosis, keratoacan-thoma, basal cell carcinoma, squamous cell carcinoma, nevus intradermal, nevus compositus, dysplastic nevus and lentigo maligna. Control skin was harvested from the vicinity of these lesions. In the Raman spectra, the secondary structure of the proteins was reflected by the amide vibrations of peptide bonds. The principal lipid vibrations were twisting and wagging (CH₂) and CH stretching vibrations. Histologically distinguishable lesions showed specific combinations of band changes indicating alterations in the protein conformation and in the molecular structure of the lipids. Histogenetically related lesions (actinic keratosis and sqamous cell carcinoma) produced similar but not identical patterns of spectral changes. Because the examined skin lesions produced reproducible and unique spectra, we suggest that Raman spectroscopy will be useful for diagnosis of skin lesions.     

Generation of DNA photolesions by two-photon absorption of a frequency-doubled Ti:sapphire laser

The formation of spatially localized regions of DNA damage by multiphoton absorption of light is an attractive tool for investigating DNA repair. Although this method has been applied in cells, little information is available about the formation of lesions by multiphoton absorption in the absence of exogenous or endogenous sensitizing agents. Therefore, we have investigated DNA damage induced in vitro by direct two-photon absorption of frequency-doubled femtosecond pulses from a Ti:sapphire laser. We first developed a quantitative polymerase chain reaction assay to measure DNA damage, and determined that the quantum yield of lesions formed by one-photon absorption of 254 nm light is 7.86×10(-4). We then measured the yield of lesions resulting from exposure to the visible femtosecond laser pulses, which exhibited a quadratic intensity dependence. The two-photon absorption cross section of DNA has a value (per nucleotide) of 2.6 GM at 425 nm, 2.4 GM at 450 nm, and 1.9 GM at 475 nm. A comparison of these in vitro results to several in vivo studies of multiphoton photodamage indicates that the onset of DNA damage occurs at lower intensities in vivo; we suggest possible explanations for this discrepancy.     

Tandem lesions are the major products resulting from a pyrimidine nucleobase radical

Nucleobase radicals (e.g., 1) are the major family of reactive intermediates formed when DNA is exposed to gamma-radiolysis. Independent generation of 1 in chemically synthesized oligonucleotides reveals that formation of this nucleobase radical under aerobic conditions results in the formation of tandem lesions approximately 65% of the time. The distribution of lesions formed with the 5'- and 3'-adjacent nucleotides is dependent upon the secondary structure of duplex DNA. Tandem lesions, which are defined as two contiguously, damaged nucleotides in a single DNA strand, are of significant biological interest. The yield of tandem lesions from 1 is much greater than was previously believed. The observations presented could have significant ramifications on how scientists interpret the effects of gamma-radiolysis on DNA.     

Molecular simulations reveal a common binding mode for glycosylase binding of oxidatively damaged DNA lesions

Cellular DNA is constantly exposed to oxidative stress from both exogenous and endogenous sources, creating lesions that lead to aging related diseases, including cancer. 8-Oxo-guanine (8OG) is one of the most common forms of oxidative DNA damage, and failure to repair this lesion results in G:C to T:A transversion. Another common lesion, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapydG), shares the same precursor as 8OG. In Escherichia coli, both lesions are recognized and excised by the DNA glycosylase Fpg. X-ray crystallographic studies have shown that FapydG and 8OG adopt different conformations in the active site of Fpg. Our simulations suggest that the different binding modes observed for 8OG and FapydG arise directly from response to the nonconserved E77 present in the thermophilic Fpg sequences used for the crystallography experiments. In simulations with consensus S77, these lesions adopt very similar binding modes.     

The (6–4) Dimeric Lesion as a DNA Photosensitizer

The front cover artwork is provided by Miguel A. Miranda and co‐workers at Instituto de Tecnología Química (UPV‐CSIC). The image shows that (6–4) photoproducts, primary UVB dimeric lesions, act as photosensitizers of cyclobutane pyrimidine dimers that are produced as secondary DNA lesions. Read the full text of the article at 10.1002/cphc.201600154 .     

Synthesis and plant growth inhibitory activity of both enantiomers of pyricuol, a phytotoxin isolated from rice blast disease fungus Magnaporthe grisea

Both enantiomers and racemate of pyricuol, a phytotoxin isolated from the rice blast disease fungus, Magnaporthe grisea, have been synthesized by using Stille coupling and [2,3]-Wittig rearrangement reactions as the key steps. Both enantiomers induced dark necrotic lesions on rice leaves almost equally, but did not affect the growth of rice second leaf sheath and the germination of lettuce. Only natural enantiomer promoted the root growth of rice and lettuce.     

ACETONE-SENSITIZED PHOTOINACTIVATION OF TRANSFORMING DNA

Abstract— Irradiation of Haemophilus influenzae transforming DNA with 313 nm radiation in the presence of acetone leads to inactivation of transforming activity. Some of the lesions produced are substrate for photoreactivating enzyme and the dark-repair mechanism. In addition, other lesions are produced which, if their production is not prevented by the presence of EDTA during irradiation, render the DNA less photoreactivable. The possibility is discussed that, among others, single-strand breaks are responsible for this loss in photoreactivability.     

DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: biological consequences

4,6,8,9-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (HFQ) and its isomer FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one) showed very strong antiproliferative activity in mammalian cells, about two times greater than 8-methoxypsoralen (8-MOP). Both compounds induced DNA-protein cross-links (DPC) but not interstrand cross-links. The FQ generated DPC in a biphotonic process, yielding a new kind of diadduct, whereas HFQ induced DPC by a monophotonic one, probably without its physical participation in the covalent bridge. These lesions gave different toxic responses. Sensitization of FQ led to extensive DNA fragmentation and to a number of chromosomal aberrations. Conversely, HFQ seemed to be completely inactive and 8-MOP gave intermediate results. A strict relationship between DPC formation and induction of chromosomal aberrations was observed. The HFQ did not induce light skin erythemas, whereas FQ was more phototoxic than 8-MOP, thus suggesting that FQ lesions, DPC in particular, may be implicated in skin phototoxicity. Ehrlich ascites cells, a transplantable mouse tumor, inactivated by furoquinolinone sensitization and injected into healthy mice, protected them from a successive challenge by viable tumor cells. This response appeared to be based on an immune mechanism. Comparable amounts of base substitution revertants were scored when testing furoquinolinones and 8-MOP in bacteria but no DPC were detected. This suggests that classic mutagenesis tests on bacteria are insufficient to give adequate information on furocoumarin genotoxicity. Given its features, HFQ can be regarded as an interesting new agent for psoralen plus UVA photochemotherapy and photopheresis.     

Effects of UV irradiation on skin and nonskin-associated herpes simplex virus infections in rats

Herpes simplex virus (HSV) normally causes vescular lesions on mucocutaneous surfaces but can also cause encephalitis. The virus can reactivate from the latent state in neurons to form recrudescent lesions. One common stimulus for reactivation is exposure to sunlight. In the present study, the effects of irradiating rats with suberythemal ultraviolet (UV) before or after infecting them epidermally with HSV was investigated. Preexposure to UV impaired HSV-specific cellular immune responses, as indicated by delayed type hypersensitivity (DTH) and in vitro lymphoproliferation assays. However, the number and severity of the skin lesions were not altered. In contrast, exposure after infection did not affect cellular immunity but resulted in a large increase in the severity and number of lesions. In a second series of experiments, the effects of preirradiating with UV on HSV infection was examined using a route of inoculation which was not skin-associated, namely intranasal, allowing direct non-invasive access to the nervous system. It was found that suppressed DTH resulted, together with an increase in the incidence and severity of neurological symptoms and an increased viral load in the brain. Therefore, unlike the situation in the skin, irradiation of rats before intranasal inoculation led to a suppressed immune response to HSV which correlated with increased viral load and symptoms. These results indicate that the effects of UV may be dependent on whether the animal is exposed before or after the infection, and whether the infection is skin-associated or systemic.