PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

Abstract The preparations of chicken erythrocyte linear double-stranded DNA and superhelical plasmid pBR322 DNA were irradiated by continuous low-intensity UV radiation (I = 25-50 W/m², λ= 254 nm) as well as by highintensity picosecond laser UV radiation (I = 10¹¹-10¹³ W/m², λ= 266 nm). The effect of DNA secondary structure alterations on the formation of liquid-crystalline dispersions from UV-irradiated DNA preparations was studied. It was shown that in the case of linear DNA, watching the disappearance of abnormal optical activity characteristic for cholesteric liquid crystal we managed to detect the presence of photochemical alterations in DNA irradiated by low-intensity UV radiation at an absorbed energy of more than 20 quanta per nucleotide. In the case of superhelical DNA using enzyme treatment of liquid-crystalline dispersions and monitoring the appearance of abnormal optical activity, we detected the presence of photochemical alterations in DNA molecules after low-intensity UV irradiation at an absorbed energy of less than 4 quanta per nucleotide. Under the latter approach using picosecond UV laser irradiation at three different light intensities we were able to distinguish the different mechanisms of fine alterations in DNA secondary structure at an absorbed energy value of about 3 quanta per nucleotide.     

Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking

Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.     

Probing the Binding Region of the Single-Stranded DNA-Binding Domain of Rat DNA Polymerase β Using Nanosecond-Pulse Laser-Induced Cross-Linking and Mass Spectrometry

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase β (pol β-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol β-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.     

LASER CROSS-LINKING OF PROTEINS TO NUCLEIC ACIDS: PHOTODEGRADATION AND ALTERNATIVE PHOTOPRODUCTS OF THE BACTERIOPHAGE T4 GENE 32 PROTEIN

Pulsed laser cross-linking provides a means of introducing a covalent bond between proteins and the nucleic acids to which they are bound. This rapid cross-linking effectively traps the equilibrium that exists at the moment of irradiation and thus allows examination of the protein-nucleic acid interactions that existed. Laser irradiation may also induce photodestruction of protein and we have used the bacteriophage T4 gene 32 protein to investigate this phenomenon. Our results show that both nonspecific and specific photoproducts can occur, specifically at wavelengths where the peptide backbone of proteins is known to absorb. These results demonstrate that nonspecific photodegradation can be correlated with the formation of a specific photodegradation product. The formation of this product was monitored to show that product yield is nonlinearly dependent on laser power and wavelength. We have also investigated an unexpected photoproduct whose formation is dependent on the length of the polynucleotide to which the gene 32 protein binds and that further demonstrates the complexities of analyzing protein-nucleic acid interactions through the use of UV laser cross-linking. These data provide essential information for the establishment of appropriate conditions for future studies that use UV cross-linking of protein-nucleic acid complexes.     

Irradiation of ferrous ammonium sulfate for its use as high absorbed dose and low-temperature dosimeter

In the present paper, we study the response of crystalline ammonium ferrous sulfate as a function of the irradiation dose and temperature. The dose studied ranged from 33.5 to 546 kGy. The temperature regimen varied from 77 K (liquid nitrogen) to 311 K. The analysis of the samples was made by UV spectroscopy and EPR.The results show that the change in absorbance of the dosimeter was linear with respect to the absorbed dose in the range studied. There is a small influence of the irradiation temperature in the response of the iron salt. The dose rate and storage time after irradiation was of no importance in this application.     

Irradiation of Skin and Contrasting Effects on Absorption of Hydrophilic and Lipophilic Compounds

Increasing legal requirements for risk assessment and efficacy testing in the dermo-cosmetic field have led to the development of alternative test methods. In this study, the porcine skin model was chosen to test the effect of irradiation on the penetration habits of UV filters and caffeine. For decades, the pig has been recognized as an experimental animal in biomedical research thanks to its morphological and physiological similarities to humans. In this study, we wanted to investigate the effect of UV irradiation on the absorption of octocrylene (OC) and benzophenone-3 (B3) sunscreens used under those circumstances and a model hydrophilic molecule, caffeine (Caf). These particular compounds were chosen due to their different lipophilic profiles. The percutaneous penetration of the two UV filters and Caf was studied after two simulated solar radiation doses of 61.4 kJ m⁻². After irradiation simulation, the total absorbed dose was increased for OC while for B3 and Caf it was lower. Thus, modifications in percutaneous absorption have been observed, and it appears that UV could play a crucial role in this process. Moreover, it has been observed that the lipophilic profile of the studied compounds affects percutaneous penetration when irradiated.     

Effect of laser and LED on enzymatic production of ceramide

An enzyme (Phospholipase C Type I from Clostridium perfringens) was exposed to 0-810Jcm(-2) of energy using laser light at wavelengths 808, 532, 1064 and 1342nm and two LED light sources at wavelengths 810 and 640nm. Enzyme responses were evaluated by measuring ceramide concentration using high performance thin-layer chromatography (HPTLC) at 0.5, 1, 2, 3, 4, 6, 17, 24h after irradiation. The duration of effect was evaluated from the experimental data. The results show that enzyme activity can be increased by using both laser and LED sources whose wavelength is located within a certain range. The effect depends on the energy and wavelength of the light. The increase in enzyme activity continued for about 4h after irradiation. This study shows that the duration of irradiation should be included as one of the main laser parameters when reporting on the effects of laser irradiation on enzymes. We also find that laser sources and LED sources have the same effect on enzyme activity if the wavelength and absorbed energy are equal.     

Dosimetric characteristics of different types of saccharides: An EPR and UV spectrometric study

The time stability and dose response of the free radicals produced in various types of “less-studied” mono- and disaccharides by γ-radiation is studied by EPR (Electron Paramagnetic Resonance) and UV spectrometry. The time evolution of the shape of the EPR spectra of irradiated saccharides is investigated from 5 min to 5 months after irradiation. The intensity of the stable EPR signal is studied as a function of the absorbed γ-dose in the range 0.5–20 kGy. Aqueous solutions of irradiated solid saccharides exhibit a UV absorption maximum in the range 250–290 nm. A linear dependency is found between the magnitude of the UV absorption maximum and the absorbed γ-dose. The time stability of the UV absorption maximum is also studied for every saccharide. The results are compared with those obtained for irradiated sucrose.     

Effects of ultraviolet laser radiation on Venezuelan equine encephalomyelitis virus.

The effects of usual low-intensity continuous (lambda = 254 nm, I = 10 W/m2) UV radiation and high-intensity laser nanosecond (lambda = 266 nm, tau p = 10 ns, I = 10(9) W/m2) or picosecond (lambda = 266 nm, tau p = 23 ps, I = 10(12) W/m2) UV radiation on Venezuelan equine encephalomyelitis virus (a member of the Togaviridae family) were compared. The quantum yields of infectivity inactivation, pyrimidine dimer formation and RNA-protein crosslinking were determined.     

电子辐照及再结晶P(VDF-TrFE)共聚薄膜红外光谱研究

利用傅立叶变换红外光谱(FT-IR)研究聚偏氟乙烯与聚三氟乙烯共聚薄膜[P(VDF0.80-TrFE0.20)]的电子辐照和再结晶处理过程中分子链构型变化和化学变化, 为深入探讨辐照的改性机理提供依据. 研究发现, 辐照后薄膜分子链全反构型百分含量随吸收剂量增大而迅速减少, TG构型和T3G构型百分含量显著增多. 而当吸收达一定剂量时, 三种构型相对含量基本不再变化, 表明高剂量辐照时样品极性回升不依赖分子链构型中全反型的含量的增多, 而是和高交联度的边界效应有关. 再结晶过程中分子链构型变化恰好和辐照效应产生的变化相反, 并且形成了更加稳定的C=C共轭结构.     

Use of maximum temperature for correction of alanine dosimetry in electron beams

Alanine dosimetry is well characterized for irradiation temperature response. In use, alanine absorbed dose response is corrected for the irradiation temperature. The temperature used to correct alanine dosimetry absorbed dose response in electron beams has historically been the mean temperature occurring during irradiation (Sharpe and Miller, 2009). At lower absorbed doses, the change in temperature is relatively low; thus the absorbed dose response correction due to temperature is small. However, industrial electron beam processing often requires higher absorbed dose measurements where the change in temperature can be very large and the corresponding dose response correction for alanine becomes significant. This paper compares the impact of the temperature correction based on the use of a mean irradiation temperature (Sharpe and Miller, 2009) versus the use of a maximum irradiation temperature on the absorbed dose measurement. The results of this comparison indicate that the use of a mean temperature correction for higher absorbed doses measured with temperature corrected alanine dosimetry introduces a bias in the absorbed dose estimate.     

EFFECT OF CHRONIC UV EXPOSURE ON EPIDERMAL FORWARD SCATTERING-ABSORPTION INSK–1 HAIRLESS MOUSE SKIN

Abstract— Clinical and histological precancerous responses to UV irradiation are complicated dynamic functions of total dose, dose fractionation, fluence rate, and spectral distribution. This may be due, in large part, to the ability of UV to decrease epidermal-stratum corneum transmission by stimulation of hyperplasia. This work provides quantitative measurement of dose- and wavelength-dependent optical changes inSK–1 hairless mouse epidermis-stratum corneum occurring under irradiation with “monochromatic” UV wavebands, at 280, 290, 300, 307, and 313 nm. Mice were irradiated 5 days per week with a filtered Xenon-Hg high-intensity grating monochromator, starting with 0.9 minimal erythemal dose (MED), followed by incremental increases in the radiation dose by 20% of the original dose every tenth irradiation day, for2–8 consecutive weeks. Subsequent irradiations (for longer experiments) were followed by 30% incremental increases after the 8th week every 10th irradiation day until cessation of radiation at the end of 14 weeks. Irradiated and control full-thickness epidermis/ stratum corneum were examined histologically and by forward-scattering absorption spectroscopy. Chronic irradiation of hairless mice resulted in significant hyperplasia which was optically manifested by a general increase in forward-scattering absorbance. At moderate local doses (7.2 MED), the absorbance increase per MED was approximately the same for all excitation wavelengths, whereas at large total doses (? 100 MED) the optical increase per delivered MED progressively decreased in the order 313> 307> 300? 290> 280 nm. The increase in skin thickening, expressed as observed increase in absorption at 320 nm, correlated well with histological and clinical data. We propose that optical changes induced by UV-induced thickening can account in large part, if not entirely, for dynamic changes in action spectra for (pre) cancerous processes under chronic irradiation conditions.     

Photo-crosslinking and negative-type micropattern formation of a polymeric photobase generator containing phthalimido carbamate groups

The photo-crosslinking reaction of a polymeric photobase generator containing phthalimido carbamate (PC) groups was studied and applied to the formation of a negative type micropattern. The copolymer containing PC groups was prepared through the polymerization of methyl methacrylate and phthalimido methacryloxyethylcarbamate (PMC). The photochemical and cross-linking reaction of the copolymer film were studied using the UV and IR absorption spectral changes along with the normalized thickness measurements upon irradiation. The copolymer film was effectively cross-linked upon irradiation with 254 nm UV light, and the cross-linking reaction progressed further with increasing irradiation dose and the amount of PMC units in the copolymer. The photochemical formation of the isocyanato groups was evidenced by comparing the IR absorption spectral changes of the exposed and masked copolymer film. The photo-crosslinking reaction of the copolymer was further studied by using a model compound. The results indicated that the cross-linking reaction occurred because of the formation of urea-type chemical bonds, which were produced through the reaction of the photochemically produced isocyanato and amino groups in the copolymer. A negative micropattern was obtained by using the photo-crosslinking reaction.     

ULTRAVIOLET-B DOSE-RESPONSE CURVES FOR LOCAL AND SYSTEMIC IMMUNOSUPPRESSION ARE IDENTICAL

Irradiation of mice with UVB suppresses contact hypersensitivity either "locally", i.e. when sensitizer is applied to the UV irradiated site, or "systemically", i.e. when sensitizer is applied to a site distal to the site of irradiation. It has been suggested that local suppression requires lower doses of UV than does systemic suppression, and that different mechanisms are therefore responsible. We undertook a detailed analysis of the dose-response and kinetics of UV-induced local and systemic suppression of contact hypersensitivity to trinitrochlorobenzene in two strains of mice, C57BL/6 and BALB/c. We found that the UV dose-responses for systemic and local suppression were identical within the same strain. Comparison, however, of UV dose-responses between strains indicated that C57BL/6 mice required 6.4 times less UV than did BALB/c mice to generate an equivalent amount of suppression. In both strains, local suppression was initiated if sensitizer was applied immediately, or 1 or 3 days after completion of a single dose of UV. In contrast, systemic suppression was initiated only if sensitizer was applied 3 days after UV irradiation. Thus local suppression was generated in the absence of significant systemic suppression (but not vice versa), and this was dependent on time of application of sensitizer after UV irradiation, not on the dose of UV administered. Filtration of the UV source with Mylar indicated that UVB was responsible for initiating both local and systemic suppression. In summary, these results indicate that (1) genetically determined differences in susceptibility to UV suppression exist, (2) the time courses of generation of local and systemic suppression are identical, and therefore use of the terms "low dose" and "high dose" to refer respectively to local and systemic suppression by UV irradiation are incorrect. We conclude that a common mechanism initiates UV-induced local and systemic suppression of contact hypersensitivity by the immediate formation, at the site of UV irradiation, of an immunosuppressive signal which takes between 1 and 3 days to act systemically.     

Influence of spectral and angular sensitivity on the readout of biological dosimeters

Biological systems used as biological dosimeters can possess different angular sensitivities from the detectors usually used in physical devices. A simple experimental setup has been developed and used to measure the angular sensitivity of uracil thin-layer biological dosimeters. Results of angular sensitivity measurements for uracil thin-layer dosimeters are presented using a Xe arc lamp as the UV source. According to the experiments described here, uracil thin-layer dosimeters show a cosine-type angular dependence. In several indoor experiments broadband UV meters are used to control the applied dose rate from a given artificial UV source. The experimental setup has been designed and used to verify experimentally the importance of spectral and angular sensitivity differences of biological and physical UV meters applied in biological experiments. Model calculations for two different irradiation systems, using different geometrical arrangements of artificial UV sources, are also presented. For these arrangements relative dose rates that could be measured with dosimeters of arbitrary spectral, but different angular sensitivity have been calculated.     

DETERMINATION OF PARAMETERS OF EXCITED STATES OF DNA and RNA BASES BY LASER UV PHOTOLYSIS

Abstract— The mechanism of photodecomposition of nucleic acid bases in a neutral aqueous solution upon two-step excitation of high-lying electronic states by a powerful laser UV radiation is discussed. Experimental dependences of photodecomposition efficiency versus UV radiation intensity are measured both under picosecond and nanosecond laser UV irradiations. By comparison of experimental dependences with a theoretical model, we obtain some characteristics of excited states, such as lifetime t₁ of the first electronic excited state S₁ intersystem crossing yield φ, photosensitivity from an intermediate excited state and others for all five nucleic acid bases.     

Study of a new route by which to make fully cured thermoplastic elastomers with plastics and ultrafine powdered rubber

A series of fully cured thermoplastic elastomers (TPEs) have been developed by mixing plastics and fully cured, ultra-fine powdered rubbers (UFPRs). The fully cured UFPRs were prepared by the irradiation of rubber latexes and drying of the irradiated rubber latexes. The average particle size of the UFPRs ranged from 50 to 500nm. The influences of the dose rate, absorbed dose and content of cross-linking agent on the cross-linking of rubber latexes were studied. It was found that a new type of TPEs with excellent properties can be easily made by blending UFPRs with plastics.     

Facile method for the fabrication of robust polyelectrolyte multilayers by post-photo-cross-linking of azido groups

In this letter, we have developed a facile method to enhance the stability of polyelectrolyte multilayers. We fabricate conventional polyelectrolyte multilayers of PAH/PAA through electrostatic layer-by-layer (LbL) assembly and then postinfiltrate photosensitive cross-linking agent 4,4'-diazostilbene-2,2'-disulfonic acid disodium salt into the LbL films. After cross-linking by UV irradiation, the stability of the photo-cross-linked multilayer is highly improved as evidenced by the lack of dissolution under ultrasonication in saturated SDS aqueous solutions for 10 min. Moreover, by taking advantage of the different stability of the LbL film before and after UV irradiation, a patterned surface can be achieved.     

Selfcalibrated alanine/EPR dosimeters: A new generation of solid state/EPR dosimeters

Alanine/EPR dosimeters are well established as secondary, reference dosimeters for high-energy radiation. However, there are various sources of uncertainty in the evaluation of absorbed dose. This arises primarily from the necessity to calibrate each EPR spectrometer and each batch of dosimeters before their use. In order to overcome this disadvantage, a new generation alanine/EPR dosimeter has been developed, and its possibilities as a radiation detector are reported. Principally, it is a mixture of alanine, some quantity of EPR active substance, and a binding material. The EPR active substance, acting as an internal EPR standard, is chosen to have EPR parameters which are independent of the irradiation dose. The simultaneous recording of the spectra of both the sample and the standard under the same experimental conditions and the estimation of the ratioI _alanine/I _Mn as a function of the absorbed dose strongly reduces the uncertainties. The response of these dosimeters for⁶⁰Co γ-radiation exhibits excellent linearity and reproducibility in the range of absorbed dose, 10²−5·10⁴ Gy.