Changes of phage T7 nucleoprotein structure at low ionic strength. A Raman spectroscopic study.

To detect changes in DNA and/or protein structures of phage T7 under different ionic strength, Raman spectra of phage T7 have been recorded in solutions of three different NaCl + Tris concentrations. Iterative Jansson-Van Cittert deconvolution, as well as decomposition methods have been used to quantify changes in DNA structure. Significant modifications in ratios of contributions from 675 and 685 per cm vibrations, as well as in the DNA backbone vibrations, characteristic for B-DNA, near 835 per cm frequency have been found. Changes of the base electronic structure were identified in the interval between 1280 and 1400 cm(-1). Estimation of the overall protein structure suggests predominant beta-sheet content.

Properties of gramicidin A channels in erythrocyte membranes.

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.

The effect of UV irradiation on uracil thin layer measured by optical waveguide lightmode spectroscopy.

The polycrystalline uracil thin-layer dosimeter is a well-established method to monitor the biological effects of the environmental ultraviolet (UV) radiation. It is based on the optical density (OD) decrease of the uracil layer in the UV absorption band due to photodimerization of the crystal caused by UV irradiation. In the present study, we report measurements made with optical waveguide lightmode spectroscopy (OWLS) to characterize the changes in the optogeometrical parameters of the uracil layer caused by an artificial UV source. It is shown that UV irradiation causes a decrease in the refractive index and an increase of the optical anisotropy. The determined kinetic parameters of the UV dose-sensor response curves correlate well with results of OD measurements, but the sensitivity of OWLS is about ten times higher. The results show that OWLS is capable of analyzing the UV response of the uracil layer and opens the way for dosimetrical applications.

Use of uracil thin layer for measuring biologically effective UV dose.

Dimerization of uracil monomers in a polycrystalline state by UV radiation changes the absorption characteristics of a thin layer of the material. The change in optical density, measured by spectrophotometry in the 250-400 nm range, as a function of the exposure time is evaluated in terms of the biologically effective UV dose. A statistical evaluation of a great number of uracil dosimeters irradiated with a TL01 lamp from Philips establishes the possibility of evaluating the biologically effective UV dose using a uracil dosimeter. Nonlinear regression procedures were introduced to correct the absorption spectra for contributions due to light scattering and to determine the optical density values required to calculate the UV dose expressed in HU units. Comparison of cumulative daily doses and long-term monitoring measured by the uracil thin-layer dosimeter and a phage T7 dosimeter are given, which allow the determination of conversion factors between various biological dosimeters under different irradiation conditions.

Comparisons of spore dosimetry and spectral photometry of solar-UV radiation at four sites in Japan and Europe.

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (+/- 0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.

Influence of phage proteins on formation of specific UV DNA photoproducts in phage T7.

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.

Assessment of the effects of various UV sources on inactivation and photoproduct induction in phage T7 dosimeter.

The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in HT7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200-280 nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.

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.

Dark and photoreactivity of 4'-aminomethyl-4,5',8-trimethylpsoralen with T7 phage.

The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

Phage T7 in biological UV dose measurement.

An experimental method complete with theoretical considerations is presented for the measurement of different biological UV doses. The method is based on the high sensitivity of phage T7 activity to UV light. A precisely determined T7 inactivation action spectrum is presented over a wide optical range (240-514 nm). Using the T7 spectral sensitivity in relation to the minimal erythema dose (MED) and the effective spectral irradiance from solar radiation for the MED, an example is given to determine the MED value based on the measurement of T7 inactivation for a given case. The advantages and applicability of the method are discussed.

Action spectra for photoinduced inactivation of bacteriophage T7 sensitized by 8-methoxypsoralen and angelicin.

The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method. For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum. For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different. The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation. The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA. The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7. Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated. Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens.

Phage nucleoprotein-psoralen interaction: quantitative characterization of dark and photoreactions.

The irradiation of the phage T7 system containing psoralen as photosensitizer causes many processes, each of them leading to phage inactivation. These processes include the UV-induced photoreactions in the phage nucleic acid, and photoreactions in the nucleic acid sensitized by either psoralen or psoralen photobreakdown products. In addition the intercalation of the psoralen molecule itself in the phage nucleic acid as well as the psoralen photobreakdown products cause phage inactivation. Under appropriate experimental conditions these reactions can be studied and characterized separately. The quantitative characteristics (e.g. inactivation cross-section, action spectra and index for dark genotoxicity) are demonstrated for different linear and angular psoralens. Some theoretical and practical consequences of the results obtained are discussed.

Construction of spectral sensitivity function using polychromatic UV sources.

A procedure is presented for constructing the spectral sensitivity functions of biological dosimeters, using five polychromatic UV sources possessing different emission spectra. Phage T7 and uracil biological dosimeters have been used for measuring the dose rates of the lamps. Their spectral sensitivity functions consisting of two exponential terms have been constructed. The parameters of the spectral sensitivity functions have been determined by comparing the directly measured and calculated dose-rate values. The parameters of the sensitivity function are accepted as correct values when the deviation of the measured and calculated values is a minimum. Based on the deviations between the constructed and the experimentally determined spectral sensitivities with monochromatic sources, the differences between the measured and calculated results are interpreted. The importance of the correct spectral sensitivity data is demonstrated through the effectiveness spectra of a TL 01 lamp for phage T7 killing, uracil dimerization and erythema induction.

Biophysical and biological properties of newly synthesized dioxinocoumarin derivatives. II. Dark and photoinduced effects on T7 phage, yeast and HeLa cells.

The dioxinocoumarin derivatives 5H-[2]benzopyrano-[3,4-g][1,4]benzodioxin-5-one (I), 5H-[2]benzopyrano-[3,4-g][2,3]-dihydro-[1,4]benzodioxin-5-on e II, 6H-[2]benzopyrano[3,4-f]-1,4-benzodioxin-6-one (III) and 6H-[2]benzopyrano[3,4-f]-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized. Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells. The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity. The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen. Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins. Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP. At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

Biological UV dosimeters in the assessment of the biological hazard from environmental radiation.

To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage.

Stability of nucleic acid under the effect of UV radiation.

Nucleic acids (combined with protein molecules) are essential constituents of the living systems playing an important role in the early evolution of life as well. A specific feature of these molecules has been found and directly confirmed recently: under the influence of short-wavelength UV radiation bipyrimidine photoproducts (cyclobutane dimers and 6-4 bipyrimidines) are induced and the reversion of them can be provoked by the same photons. However, reversion is preferred by the shorter wavelengths. With increasing ratio of the longer wavelength components of the radiation (using artificial UV sources and solar light on the Earth's surface) the impact of the reversible photoproducts in the harmful biological effect decreases and other photoproducts are dominant. Assuming the photoinduced reactions (dimerisation and reversion) are statistical events, during the irradiation the chance for a number of nucleoprotein molecules to survive the radiation damage can be reality. The theoretical and experimental basis of these assumptions will be discussed in the case of bacteriophage T7 nucleoprotein.

Monitoring of environmental UV radiation by biological dosimeters.

As a consequence of the stratospheric ozone layer depletion biological systems can be damaged due to increased UV-B radiation. The aim of biological dosimetry is to establish a quantitative basis for the risk assessment of the biosphere. DNA is the most important target molecule of biological systems having special sensitivity against short wavelength components of the environmental radiation. Biological dosimeters are usually simple organisms, or components of them, modeling the cellular DNA. Phage T7 and polycrystalline uracil biological dosimeters have been developed and used in our laboratory for monitoring the environmental radiation in different radiation conditions (from the polar to equatorial regions). Comparisons with Robertson-Berger (RB) meter data, as well as with model calculation data weighted by the corresponding spectral sensitivities of the dosimeters are presented. Suggestion is given how to determine the trend of the increase in the biological risk due to ozone depletion.

Salt effects on bacteriophage T7-I.

The thermal denaturation as a measure of the structural stability of the nucleoprotein in bacteriophage T7 has been studied in dependence of the ionic environment. Optical density and circular dichroism melting curves measured at wavelengths characterizing either the DNA or protein conformational changes were compared to identify different steps of the denaturation and to follow the effect of the ions. Monovalent salts strengthen the helical structure of intraphage DNA logarithmically in the way as they do in the case of isolated double-stranded DNA. Mg2+ and Ca2+ at very low concentrations stabilize the DNA helicity. Higher divalent ion concentrations decrease the stability of the double helix because of the repulsive ionic interactions. The high structural sensitivity of DNA in the presence of Mg2+ and Ca2+ in this "in situ" environment can be related to the biological role of these ions.

Phage T7-inactivation test. A possibility of quantitative mutagenicity screening.

A new method is described for the quantitative characterization of the genotoxic effect of chemicals. The method is based on the determination of the inactivation of bacteriophage T7 and on the application of a simple mathematical model valid for the processes during, or at least in the initial stage of the interaction of chemicals and phages. A value characteristic for the chemical is defined and it is determined from the inactivation kinetics. Typical inactivation kinetic curves and some problems of the application of the model as well as the mutagenicity index values determined for about 30 substances are presented. The substances examined have mutagenicity index values covering a range of six orders of magnitude. The obtained values are compared with the results of different mutagenicity/carcinogenicity tests and discussed on the basis of data in the literature. The presented method is proposed to be applied for quantitative mutagenicity screening of chemicals.

Salt effects on the bacteriophage T7-II structure and activity changes.

Optically detected thermal stability and biological activity of phage T7 has been compared as the function of the ionic composition and strength of the buffers. The ionic strength range was studied between 20-140 mmol/1. In Tris buffer containing only monovalent ions the biological activity of the phages decreases abruptly below 50 mmol/1 ionic strength. Structural studies show a logarithmic dependence between the ionic strength and the intraphage DNA stability and no significant change in the thermal stability of the whole phage. Mg2+ and Ca2+ ions at low concentration (1 mmol/1) given into a Tris buffer of 20 mmol/1 original ionic strength highly stabilize the biological activity, which stabilization is also to be seen in the intraphage DNA and also in the whole phage thermal denaturation process.