In order to determine whether or not tiaprofenic acid (TPA) could cause cellular DNA damage, human fibroblasts were irradiated in the presence of the drug and subsequently examined by means of the comet assay. This led to the observation that TPA actually sensitizes cellular DNA to the subsequent irradiation. When TPA was irradiated in the presence of supercoiled plasmid DNA, it produced large amounts of single-strand breaks (SSB); this is consistent with the effects observed on cellular genomic DNA by the comet assay. More importantly, low concentrations of TPA, unable to produce direct SSB, caused photo-oxidative damage to DNA as revealed by the use of excision-repair enzymes. The fact that TPA-irradiated DNA was a substrate of formamidopyrimidine glycosylase as well as endonuclease III revealed that both purine and pyrimidine bases were oxidized. This was further supported by the TPA-photosensitized oxidation of 2′-deoxyguanosine which led to a product mixture characteristic of mixed type-I/II mechanisms. Thymidine was less reactive under similar conditions, but it also decomposed to give a typical type-I product pattern. Accordingly, the TPA triplet was quenched by the two nucleosides with clearly different rate constants (10⁸ vs 10⁷ M⁻¹ s⁻¹ respectively). As cellular RNA also contains oxidizable bases, it could be the target of similar processes, thus interfering with the biosynthesis of proteins by the cells. Extraction of total RNA from TPA-irradiated human fibroblasts, followed by gel electrophoresis and PCR analysis, confirmed this hypothesis. Finally, photosensitization experiments with Saccharomyces cerevisiae showed that, in spite of an efficient drug–yeast interaction leading to cytotoxicity, neither intergenic recombination nor gene conversion took place. Thus, while TPA-photosensitized damage to nucleic acids can result in genotoxicity, the risk of mutagenicity does not appear to be significant.

The decay processes of the lowest excited singlet and triplet states of five heteropsoralens (HPS) were investigated by steady-state and shift-phase fluorometry and by laser-flash photolysis in different solvents. The emission spectra of HPS are detectable only in trifluoroethanol (TFE), where fluorescence lifetimes (τ_F) and quantum yields (ϕ_F) were measured. The triplet lifetimes (τ_T), triplet (ϕ_T) and singlet-oxygen production (ϕ_Δ) quantum yields were determined in benzene, ethanol and TFE by laser-flash photolysis. Semiempirical (INDO/1-CI) calculations allowed the nature of the lowest excited singlet and triplet states and transition probabilities to be obtained. Theoretical and experimental results indicate that the two lowest excited singlet states S₁ and S₂ of HPS are close-lying and different in nature (π,π* and n,π*). The “proximity effect” between these two states controls the photophysical properties of HPS as it does for the other furocoumarins. However, HPS have a peculiar behavior with respect to the related compounds because they are fluorescent and have, in three cases, detectable intersystem crossing only in TFE. This behavior can be tentatively explained by a different energy gap and/or order between the S₁ and S₂ states.

As part of a project to study different methods for the disinfection of effluent water, the inactivation of different microorganisms (Escherichia coli, Deinococcus radiodurans and spores of Bacillus subtilis) using a combination of a photosensitizer (Rose Bengal) with simulated sunlight and oxygen was determined under various environmental conditions (temperature, pH index). In parallel, the singlet-oxygen (¹O₂) production was also measured under the same conditions. Whereas the vegetative cells could be inactivated much more efficiently at increased temperature and altered index of pH, the production of ¹O₂ remained essentially the same under these alterations. Additionally, the relations among the sensitivities of different cell types to be killed by our photodynamic treatments (PDT) were opposite to those found after exposure to ionizing radiation. The results of photodynamic experiments do not reflect the cells' capacity to repair DNA strand breaks. Spores of B. subtilis, as a nonvegetative system, could not be inactivated by illuminations up to 100 J cm⁻². Together, these findings indicate that DNA is not the primary target, the inactivation of which leads to the killing of our test organisms. Instead, the cellular envelope appears to be the component being assaulted by our PDT.

The triplet-excited state of benzophenone and the singlet-excited state of 2,3-diazabicyclo[2.2.2]oct-2-ene (Fluorazophore-P) have been employed as kinetic probes to obtain information on the antioxidant activity of the skin and eye pigment melanin and its biogenetic precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The excited states were generated by the laser-flash photolysis technique and their reaction kinetics was examined by time-resolved transient absorption or fluorescence spectroscopy, respectively. The reaction between triplet benzophenone and DHI produced with unit efficiency the corresponding 6O-centered semiquinone radical, which was characterized by its characteristic transient absorption. The quenching rate constants for DHI (3.1 – 8.4 × 10⁹ M⁻¹ s⁻¹) and DHICA (3.3–5.5 × 10⁹ M⁻¹ s⁻¹) were near the diffusion-controlled limit, indicating excellent antioxidant properties. Kinetic solvent effects were observed. The reactivity of synthetic melanin, assessed through the quenching rate constant of Fluorazophore-P and normalized to the number of monomer units, was more than one order of magnitude lower (2.7 × 10⁸ M⁻¹ s⁻¹) than that of its precursors. The trend of the quenching rate constants, i.e. DHI > DHICA ≈ α-tocopherol > melanin, along with the preferential solubility of DHICA in aqueous environments, serves to account for several experimental results from biochemical studies on the inhibition of lipid peroxidation by these natural antioxidants.

The low-intensity steady-state (254 nm), microsecond flash and nanosecond (266 nm) laser photolysis of some guanine (Gua) derivatives in aqueous solution were studied. A photodestruction yield between 10⁻³ and 10⁻² at a base concentration of 75 μM was determined for 254 nm irradiation at room temperature using high-performance liquid chromatography. This yield decreases with increasing purine concentration. For a similar concentration of the purine bases (2 ± 1) × 10⁻⁵ M, the yield increases as follows: Gua ≈ 9-ethylguanine < deoxyguanosine ≈ guanosine (Guo) < guanosine 5′-monophosphate. At concentrations higher than 2 × 10⁻⁴ M the Gua derivatives' photodestruction yield seems to converge to a limiting value of the order of 10⁻⁴. This behavior is explained in terms of self-quenching and aggregation effects which deactivate the excited states of the bases. The yields of electron photoejection have been determined in the nanosecond laser photolysis (0.083) and in the low-intensity steady-state (5.8 × 10⁻³) for Guo. Competition experiments using electron scavengers suggest that the electron adducts of the bases are one of the principal species participating in the photodestruction mechanism of these monomeric Gua. Close to 75% of the total destruction yield has contributions from initial reactions of the photojected electron at neutral pH. The quantum yield of photodestruction of Guo increases when the pH is increased as follows: 4.7 × 10⁻³ (pH 1.1), 6.5 × 10⁻³ (pH 2.9), 7.7 × 10⁻³ (pH 7.5) and 8.1 × 10⁻³ (pH 11.9). This dependence on pH and the electron scavenger experiments provide further evidence for the radical anion or its protonated form as one of the principal species involved in the photodestruction of the bases at the different pH. Under oxygen saturated conditions a 22% increase in the destruction yield is observed for Guo. However, for the dinucleotides adenylyl (3′ → 5′)-guanosine and thymidylyl (3′ → 5′)2′-deoxyguanosine, the participation of the electron is 41 and 36%, respectively, suggesting that going into a more DNA or RNA-like structure, the participation of the electron adducts species in the photodamage of DNA and RNA decreases. A mechanism of photodestruction for the Gua derivatives is proposed which takes into account these findings.

The quantum yield of formation of guanine (Gua), one of the major products formed in the 254 nm steady-state photolysis of the following Gua derivatives (9-ethylguanine [9Et-Gua], deoxyguanosine [dGuo], guanosine [Guo], guanosine 5′-monophosphate [GMP]) was determined under different conditions. The formation yield increases in the following order: 9Et-Gua < dGuo ≈ Guo < GMP. Electron scavengers or triplet quenchers were incorporated into the irradiated solutions with the purpose of reacting specifically with postulated or previously identified intermediates in the photolysis of Gua derivatives. A decrease in the yield of formation of Gua is observed with increasing electron scavenger concentration or with pH. These results suggest a major contribution from Gua-derivative electron adducts on the process of N(9)–R bond breakage. At an acidic pH a tautomer of the radical cation (E) of Guo is proposed as the precursor for Gua formation. The relative efficiency of the radical cation for initiating the release of free Gua depends on the pH of the solution, being less than 39% in neutral pH. Reactions from OH radicals may also result in base release as shown using N₂O as additive. Finally, the formation of aggregates by the bases at concentrations used, plays an important role in the deactivation of the excited states and also in the probability of formation of the free base.

We have previously demonstrated that the oxidizing component of ultraviolet-A (UVA) plays a central role in the activation of the nuclear oncogene and transcription factor, c-fos, in cultured human skin fibroblasts. We have now shown that expression of both c-jun and c-fos (AP-1) family of transcription factors is modulated by short and long wavelength solar ultraviolet (UV) radiation in human fibroblasts and human KB cells. UVA radiation activated c-jun and c-fos in both fibroblasts and KB cells, whereas ultraviolet-B (UVB) radiation activates such oncogenes only in KB cells. Moreover, decreasing the intracellular levels of reducing equivalents in human fibroblasts by glutathione (GSH) depletion lowered the UVA dose threshold for c-jun and c-fos activation several-fold and greatly amplified the UVA-mediated activation of such genes. A more modest effect was observed in GSH-depleted KB cells. In both GSH-depleted fibroblasts and KB cells, UVB radiation failed to amplify c-jun and c-fos activation indicating that the oxidative component of UVB plays a minor role in the modulation of such oncogene expression. These findings clearly indicate that both c-jun and c-fos are activated by the oxidizing component of UVA radiation in human fibroblasts and KB cells, while UVB-mediated modulation seems to be restricted to human epithelial cells and does not involve oxidizing intermediates.

The damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.

R- and S-epimerization at the 3¹ position of bacteriochlorophyll (BChl) c and the formation of rod-like aggregates in chlorosomes of green sulfur bacteria were markedly affected in Chlorobium (Cb.) tepidum and Cb. limicola by cultivation under various light intensities (photon fluence rate). The stronger the light, the higher the ratio of the S-epimer to the R-epimer for each homolog of BChl c in the bacteria. S[P,E] BChl c_F and S[I,E] BChl c_F were found to be the major S-epimers in Cb. tepidum and Cb. limicola, respectively. R[P,E] BChl c_F decreased markedly compared to R[E,E] BChl c_F in Cb. tepidum, whereas no observable change in the ratio of R[P,E]/R[E,E] was detected for Cb. limicola. With increase in light intensity the Q_y absorption maximum of the bacteria shifted to shorter wavelengths. In vitro spectroscopic studies of the aggregates showed a marked difference in the formation of aggregates from R- and S-epimers of BChl c; the S-epimers formed aggregates much more slowly than did the R-epimers. These results suggest that the ratio of the epimers of BChl c might significantly affect the aggregation of BChl in the chlorosome. We propose different roles for the R- and S-epimers in chlorosomes of Cb. limicola and Cb. tepidum.

Low-temperature absorption, fluorescence and persistent nonphotochemical hole-burned spectra are reported for the CP29 chlorophyll (Chl) a/b antenna complex of photosystem II of green plants. The absorption-origin band of the lowest Q_y-state lies at 678.2 nm and carries a width of ∼130 cm⁻¹ that is dominated by inhomogeneous broadening at low temperatures. Its absorption intensity is equivalent to that of one of the six Chl a molecules of CP29. The absence of a significant satellite hole structure produced by hole burning, within the absorption band of the lowest state, indicates that the associated Chl a molecule is weakly coupled to the other Chl and, therefore, that the lowest-energy state is highly localized on a single Chl a molecule. The electron–phonon coupling of the 678.2 nm state is weak with a Huang–Rhys factor S of 0.5 and a peak phonon frequency (ω_m) of ∼20 cm⁻¹. These values give a Stokes shift (2Sω_m) in good agreement with the measured positions of the absorption band at 678.2 nm and a fluorescence-origin band at 679.1 nm. Zero-phonon holes associated with the lowest state have a width of ∼0.05 cm⁻¹ at 4.2 K, corresponding to a total effective dephasing time of ∼400 ps. The temperature dependence of the zero-phonon holewidth indicates that this time constant is dominated at temperatures below 8 K by pure dephasing/spectral diffusion due to coupling of the optical transition to the glass-like two-level systems of the protein. Zero-phonon holewidths obtained for the Chl b bands at 638.5 and 650.0 nm, at 4.2 K, lead to lower limits of 900 ± 150 fs and 4.2 ± 0.3 ps, respectively, for the Chl b → Chl a energy-transfer times. Downward energy transfer from the Chl a state(s) at 665.0 nm occurs in 5.3 ± 0.6 ps at 4.2 K.

Using light-induced Fourier-transform infrared (FTIR) difference spectroscopy of the photo-oxidation of the primary donor (P) in chromatophores from Rhodobacter sphaeroides, we examined a series of site-directed mutants with His M202 changed to Gly, Ser, Cys, Asn or Glu in order to assess the ability of these side chains to ligate the Mg atom of one of the two bacteriochlorophylls (BChl) constituting P. In the P Q_A⁻/PQ_A FTIR difference spectra of the mutants HG(M202), HS(M202), HC(M202) and HN(M202), the presence of a specific electronic transition at ∼2650–2750 cm⁻¹ as well as of associated vibrational (phase-phonon) bands at ∼1560, 1480 and 1290 cm⁻¹ demonstrate that these mutants contain a BChl/BChl homodimer like that in native reaction centers with the charge on P shared between the two coupled BChl. In contrast, the absence of all of these bands in HE(M202) shows that this mutant contains a BChl/bacteriopheophytin heterodimer with the charge localized on the single BChl, as previously determined for the mutant HL(M202). Furthermore, the spectra of the heterodimers HE(M202) and HL(M202) are very similar in the 4000–1200 cm⁻¹ IR range. Perturbations of the 10a-ester and 9-keto carbonyl modes for both the P and P states are observed in the homodimer mutants reflecting slight variations in the conformation and/or in position of P. These perturbations are likely to be due to a repositioning of the dimer in the new protein cavity generated by the mutation.

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Q_x and the Q_y transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Q_y region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment–pigment and pigment–protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment–protein complexes from different sources or in complexes altered chemically or genetically.

Transformation among the aggregate forms of bacteriochlorophyll (BChl) c characterized by the wavelength of the Q_y absorption, i.e. the dimer (B675), B705, B720 and B745, was traced by electronic-absorption spectroscopy for each of the isomers including R[E,E], R[P,E], R[I,E], S[P,E] and S[I,E] suspended in the mixtures of methylene chloride and n-hexane. A combination of NMR spectroscopy determining the structural motifs and calculation of the shift of the Q_y absorption reflecting the long-range transition dipole–transition dipole interactions among the macrocycles in the entire aggregate structures proposed the following models: B705d (B705d′), a linear array of straight (inclined) columns consisting of a pair of the piggyback dimers; B720d and B745d, an assembly of two and five shifted-inclined columns consisting of more than six piggyback dimers; and B720m and B745m, an assembly of one and two parallel stepwise stacking of ∼30 monomers. Calculations of the steric energies rationalized two different pathways of transformations: the dimer → B705d (B705d′) → B720d → B745d for the R isomers; and the monomer → (B720m) → B745m for the S isomers. Addition of S[I,E] seems to trigger the B745d → B745m transformation of the R isomers.

Multiwavelength ultraviolet–visible (UV–Vis) transmission spectroscopy is a relatively simple technique that can provide considerable quantitative information on the properties of micron and submicron particle suspensions. Two important particle properties are particle size distribution (PSD) and chemical composition. These properties provide characteristics for the identification and classification of biological systems ranging in size and composition from proteins and nucleic acids to cells. By measuring the complete UV–Vis spectrum, the combined scattering and absorption properties are obtained as a function of wavelength. The quantitative evaluation of the size distribution and chemical composition is accomplished through the application of light-scattering theory. This paper reports on the estimation of the optical properties of human blood platelets and their use in the interpretation of platelet UV–Vis spectra within the context of Mie theory. The model developed herein provides reliable and accurate estimates for the PSD and particle number of platelet suspensions. One potential application of this characterization method is in the analysis of platelet activation by thrombin. Quantification of spectral data with respect to average particle size and particle number provides a real-time description of the dramatic changes that accompany the platelet activation process.

Fibroblasts of the line 3T3 from swiss albino mice were exposed to ultraviolet A (UVA) irradiation. The cells were synchronized by treatment with nocodazole and mitotic shake-off, and then exposed to UVA irradiation in different stages of the cell cycle. Their photosensitivity varied through the cell cycle, being greatest in the G₂ phase. UVA irradiation was found to induce the formation of multinucleated cells. Cells in the G₁ phase were found to be most prone to multinucleation 15 min after UVA irradiation, while cells exposed to UVA irradiation in S and G₂ phases contained the largest fractions of multinucleated cells 24 h after treatment. The present results indicate that multinucleated cells are formed by fusion of two or more cells shortly after UVA irradiation of early G₁ cells, while impairment of cytokinesis is a possible explanation for the delayed formation of multinucleated cells after irradiation in S and G₂.

Mechanism for the photoinduced increase in the lysosomal K permeability is still unknown. In this study, we investigated the effect of photodamage-induced membrane rigidification on the lysosomal K permeability by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and by monitoring proton leakage with p-nitrophenol. Membrane fluidity was measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Methylene blue–mediated photodamage to lysosomes decreased their membrane fluidity and increased their K permeability. The photoinduced increase in the K permeability can be reversed by fluidizing the rigidified lysosomal membranes with benzyl alcohol. The results suggest that the membrane rigidification induced by photodamage may increase lysosomal K permeability. This conclusion is supported by the observation that rigidifying lysosomal membranes by the treatment with membrane rigidifier cholesteryl hemisuccinate also enhanced the lysosomal K permeability.

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH₃)₂-(CH₂)₃N(CH₃)₂ (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0–1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C₅ ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.

5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [¹⁴C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis–Menten kinetics (K_m = 8–10 mM and V_max = 18–20 nmol·(mg protein × h)⁻¹), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32°C. The activation energy was 112 kJ·mol⁻¹ in the temperature range 15°–32°C and 26 kJ·mol⁻¹ above 32°C. Transport of 5-ALA was Na and partly Cl⁻-dependent. Stoichiometric analysis revealed a Na :5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65–75%, by the β-amino acids, β-alanine and taurine and by γ-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [³H]-GABA and [¹⁴C]-β-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM β-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 μM) was not significantly influenced by β-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by β-amino acid and GABA carriers in this cell line.

Insects show unique adaptations to cope with oxidative challenges during larval development, metamorphosis and adulthood. Our previous findings suggested that bioluminescence may act as an auxiliary oxygen-detoxifying mechanism in larvae of Pyrearinus termitilluminans (Elateridae: Coleoptera). We now study the antioxidant status in larval P. termitilluminans, evaluated in terms of levels of chemical and enzymatic antioxidant defenses, as compared to luciferase activity in the prothorax (intensely bright) and abdomen (dim) of the larvae, during natural- and 20-hydroxyecdysone (20-HE)-induced development. In the prothorax, relative total SOD activities in small (<1 cm), medium (1–2 cm) and large (>2 cm) larvae were 1.00:0.53:0.32. Catalase activity also decreased with development (1.00:0.69:0.55). In contrast, prothorax luciferase activities and urate content increased with ratios of 1.0:2.2:2.5 and 1:15:97, respectively. No increases were found in the level of prothorax lipid and protein oxidation. In the abdomen, luciferase activity decreased markedly with development (1.00:0.33:0.17), as did other antioxidant enzymes, including dehydroascorbate reductase (1.00:0.59:0.17) and levels of lipid peroxidation products and protein carbonyls. Similar variations were observed in antioxidant enzyme activities when the larvae were treated with 20-HE, except for prothorax catalase. As observed in natural larval growth, luciferase activity was augmented (two-fold in prothorax) upon steroid treatment, and the levels of thiobarbituric acid-reactive substances were magnified in both segments. The increase of luciferase activity and a higher urate content in the prothorax during larval development may reflect metabolic adaptations to keep levels of oxyradicals low in order to compensate for decreased antioxidant enzyme activities.

The interconvertible photoreactions of recombinant phytochrome from Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below −100°C for the tranformation of red-absorbing form of phytochrome (Pr) → far-red–absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at −140°C (lumi-R) was observed. Two intermediates could be stabilized below −40°C and between −40 and −20°C, and were denoted as meta-R_a and meta-R_c, respectively. Above −20°C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below −60°C (lumi-F) and between −60 and −40°C (meta-F). The FT-IR difference spectra of the late Pr → Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-R_a were observed. A large band around 1700 cm⁻¹ in the difference spectra between the intermediates and Pr which is tentatively assigned to the C₁₉=O group of the prosthetic group indicates the Z,E isomerization around the C₁₅=C₁₆-methine bridge of the chromophore during the formation of meta-R_a. In the difference spectra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of ²H₂O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm⁻¹ in all ²H₂O spectra suggests an assignment to an N–²H bending vibration of the chromophore.

Blue light and development regulate the expression of the phr1 gene of the filamentous fungus Trichoderma harzianum. The predicted product of phr1, the DNA repair enzyme photolyase, is likely to help protect Trichoderma, which grows in the soil as a mycoparasite or saprophyte, from damage upon emergence and exposure to ultraviolet-c. phr1 is transiently expressed in mycelium and conidiophores after illumination. phr1 mRNA also accumulates in conidiophores during development and spore maturation. As no other genes displaying rapid, direct light regulation have been described previously in this organism, we have characterized the fluence and time dependence of phr1 induction, and its relation to sporulation and photoreactivation. Induction is transient following a pulse, and, with slower decay, in continuous light. This implies that the photoreceptor, transducers or response are capable of adaptation. About two-fold more light is required to induce phr1 than conidiation, but this difference is modest, so both responses could use the same or similar chromophore. Adenosine 3′:5′-cyclic monophosphate bypasses the requirement for light for sporulation, while atropine prevents sporulation even after photoinduction. Light regulation of phr1, however, is indifferent to both these effectors. Induction of photolyase expression behaves as a direct, rapid response to light, independent of the induction of sporulation. Indeed, illumination of mature spores increases their capacity for photoreactivation. Blue light seems to warn the organism against the harmful effects of short wavelengths, inducing phr1 expression and sporulation by pathways that are, at least in part, distinct.

A polyclonal, multispecific antiserum was raised against a whole 3[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate-extract of nonirradiated Blepharisma japonicum cells. It was used to reveal the composition of solutions that were hypothesized to contain the photoreceptor of the ciliate. A Bio-Gel A 1.5 m fine column chromatography of the extract allowed recovery of a single elution peak isolated by recording the 580 nm light absorbance. Fused-rocket immunoelectrophoresis of this material revealed a large number of >300 kDa coeluted proteins. Blepharismin-rich material with a molecular mass of approximately 50 kDa, consisting of at least nine proteins was obtained when the same extract underwent preparative isoelectric focusing before column chromatography separation. Purification of the pigment obtained from light-exposed cells gave blepharismin-rich material with a molecular weight of approximately 200 kDa. Comparison of the materials obtained under the same conditions, either from the dark-kept or light-irradiated cells, by means of pore-gradient electrophoresis confirmed that proteins present in the two preparations were different. It revealed only a very small amount, if any, of proteins in the chromatography fractions with the highest absorbance at 600 nm. Results are discussed on the basis of the hypothesis that a specific blepharismin-binding protein does not exist in the protozoan.