Small-angle X-ray scattering was used to measure the effects of chemical bleaching on the size and morphology of tyrosine-derived synthetic melanin dispersed in aqueous media. The average size as measured by the radius of gyration of the melanin particles in solution, at neutral to mildly basic pH, decreases from 16.5 to 12.5 Å with increased bleaching. The melanin particles exhibit scattering characteristic of sheet-like structures with a thickness of approximately 11 Å at all but the highest levels of bleaching. The scattering data are well described by the form factor for scattering from a pancake-like circular cylinder. These data are consistent with the hypothesis that unbleached melanin, at neutral to mildly basic pH, is a planar aggregate of 6- to 10-nm-sized melanin protomolecules, hydrogen bonded through their quinone and phenolic perimeters. The observed decrease in melanin particle size with increased bleaching is interpreted as evidence for deaggregation, most probably the result of oxidative disruption of hydrogen bonds and an increase in the number of charged, carboxylic acid groups, whereby the melanin aggregates disassociate into units composed of decreasing numbers of protomolecules.

Two methylated thienocarbazoles and two of their synthetic nitro-precursors have been examined by absorption, luminescence, laser flash photolysis and photoacoustic techniques. Their spectroscopic and photophysical characterization involves fluorescence spectra, fluorescence quantum yields and lifetimes, and phosphorescence spectra and phosphorescence lifetimes for all the compounds. Triplet–singlet difference absorption spectra, triplet molar absorption coefficients, triplet lifetimes, intersystem crossing S₁ ∼∼→ T₁ and singlet molecular oxygen yields were obtained for the thienocarbazoles. In the case of the thienocarbazoles it was found that the lowest-lying singlet and triplet excited states, S₁ and T₁, are of π,π* origin, whereas for their precursors S₁ is n,π*, and T₁ is π,π*. In both thienocarbazoles it appears that the thianaphthene ring dictates the S₁ ∼∼→ T₁ yield, albeit there is less predominance of that ring in the triplet state of the linear thienocarbazole, which leads to a decrease in the observed ϕ_T value.

The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-l-methionine (AdoMet), ¹⁴C-isotopically labeled at the S^δ methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme–AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.

Monomethine cyanine dye 4-((1-methylbenzothiazolyliliden-2)methyl)-1,2,6-trimethylpyridinium perchlorate (Cyan 40) was investigated as a two-photon–excited fluorescence probe for nucleic acids (NA). Cyan 40 has been shown to demonstrate efficient two-photon–excited fluorescence in the presence of NA in vitro in contrast to solutions without NA. Two-photon confocal laser scanning microscopy (TPCLSM) and two-photon laser scanning microspectrofluorometry were used to check the possibility of using Cyan 40 as two-photon–excited fluorescence label for NA in living cells. Study of dye effect on viability of cells was also carried out. We ascertained that Cyan 40 is a cell-permeant dye, manifesting efficient two-photon–excited fluorescence when bound to NA in living cells, without any significant influence on viability of cells. TPCLSM images obtained from stained cells indicate preferential RNA staining by Cyan 40 compared with DNA.

The in vitro photoinduced reactions of the mycosporine-like amino acids (MAA) usujirene and palythene were studied by monochromatic stationary irradiation at 366 nm. High-performance liquid chromatography analysis of the irradiated aqueous solution of usujirene indicated a low photoreactivity on the basis of the observed photodecomposition quantum yield of ϕ_−U = (2.86 ± 0.80) × 10⁻⁵, which can be partially accounted for by the cis–trans photoisomerization of usujirene to palythene (ϕ_U→P = [1.71 ± 0.13] × 10⁻⁵). However, palythene in aqueous solution showed a higher photostability than did usujirene under equivalent conditions, establishing a photostationary mixture of cis–trans isomers with a relative composition of palythene–usujirene (11:1). These results may explain the preferential in vivo accumulation of palythene relative to that of usujirene observed in several dinoflagellate species.

A tryptophan analog, dehydro-N-acetyl-l-tryptophanamide (Δ-NATA), which is produced enzymatically via l-tryptophan 2′,3′-oxidase from Chromobacterium violaceum, is newly used for time-resolved fluorescence. The absorption and emission maxima of Δ-NATA at 332 and 417 nm, respectively, in 20% dimethylformamide–water are significantly shifted to the red with respect to those of tryptophan in water, permitting us to measure its fluorescence in the presence of tryptophan residues. We demonstrate that the steady-state spectra and the fluorescence decay of Δ-NATA are very sensitive to environment, changing dramatically with solvent as the chromophore is localized within a protein and when this tagged protein binds to a peptide. The tryptophan oxidase was also used to modify the single Trp of a neurotoxin from snake (Naja nigricollis) venom. Modification of the toxin α (dehydrotryptophan-toxin α) permitted its investigation in complex with a synthetic 15–amino acid peptide corresponding to a loop of the agonist-binding site of acetylcholine receptor (AchR) from Torpedo marmorata species. The peptide α-185 possesses a single Trp at the third position (Trp187 of AchR) and a disulfide bridge between Cys192 and Cys193. A single-exponential rotational diffusion time with a constant of 1.65 ns is measured for the isolated 15–amino acid peptide. This suggests that Trp motion in the peptide in solution is strongly correlated with the residues downstream the peptide sequence, which may in part be attributed to long-range order imposed by the disulfide bond. The dynamics of the bound peptide are very different: the presence of two correlation times indicates that the Trp187 of the peptide has a fast motion (τ_r1 = 140 ps and r(0)₁ = 0.14) relative to the overall rotation of the complex (τ_r2 = 3.4 ns and r(0)₂ = 0.04). The correlation of the Trp residue with its neighboring amino acid residues and with the overall motion of the peptide is lost, giving rise to its rapid restricted motion. Thus, the internal dynamics of interacting peptides change on binding.

Lifetimes of the lowest excited singlet (S₁) electronic states of various derivatives of the pyrimidine nucleobase cytosine (Cyt) were measured by the femtosecond transient absorption technique. The bases were excited in room-temperature aqueous solution at 265 nm using approximately 200 fs pump pulses from a titanium–sapphire laser system. The decay of excited-state absorption (ESA) at visible probe wavelengths was used to determine the S₁ lifetimes of a variety of modified Cyt compounds at different pH values by global fitting. Identical lifetimes were observed for Cyt and cytidine (Cyd) within experimental uncertainty, but ESA by the ribonucleoside was considerably stronger, suggesting that the ribose group increases the oscillator strength of the S₁ → S_N transition. The S₁ lifetime of the important minor base 5-methylcytosine (m⁵Cyt) is 7.2 ± 0.4 ps at pH 6.8. The same lifetime was measured for the ribonucleoside 5-methylcytidine, but sugar substitution again increased the strength of the ESA signal. Protonation of Cyd and m⁵Cyt at low pH led to a modest decrease in their S₁ lifetimes. On the other hand, deprotonation of Cyt and m⁵Cyt significantly increased the lifetime of their respective S₁ states. These trends support the intermediacy of the n,π* state localized on the carbonyl oxygen in the nonradiative decay mechanism of Cyt. Longer S₁ lifetimes were observed for 5-fluorocytosine and N-acetylcytosine. Collectively, these results illustrate the great potential of femtosecond laser spectroscopy for investigating excited-state dynamics in DNA and DNA components.

The reaction of singlet oxygen (¹O₂) generated by ultraviolet-A (UVA)–visible light (λ > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO–hydroxyl radical spin adduct (DMPO–^·OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO–^·OH decreased in the presence of either a ^·OH scavenger or a quencher of ¹O₂ and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both ¹O₂- and DMPO-mediated formation of free ^·OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that ¹O₂, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO–^·OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of ^·OH. These observations indicate that ¹O₂ reacts first with DMPO, and the resulting DMPO–¹O₂ intermediate is immediately decomposed/reduced to give ^·OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of ¹O₂ to ^·OH. Furthermore, DEPMPO did not cause the spin-trapping agent–mediated generation of ^·OH like DMPO did.

The photoreduction of 9,10-anthraquinone (AQ), the 2-methyl, 2-ethyl, 2,3-dimethyl, 1,4-difluoro, 1-chloro and 1,8-dichloro derivatives as well as 1,4,4a,9a-tetrahydroanthraquinone, 1,2-benzanthraquinone and 6,13-pentacenequinone in nonaqueous solution at room temperature was studied by time-resolved UV–visible spectroscopy. Upon 308 nm excitation of AQ the triplet state reacts with alcohols and triethylamine (TEA). The rate constant of triplet quenching by amines is close to the diffusion-controlled limit. The semiquinone radical ^·QH/Q^·− is the main intermediate, and the half-life of the second-order decay kinetics depends significantly on the donor and the medium. Photoinduced charge separation after electron transfer from amines to the triplet state of AQ in acetonitrile and the subsequent charge recombination or neutralization also were measured by transient conductivity. The maximum quantum yield, λ_irr = 254 nm, of photoconversion into the strongly fluorescing 9,10-dihydroxyanthracenes is close to unity. The fluorescence with maximum at 460–480 nm and a lifetime of 20–30 ns disappears as a result of a complete recovery into AQ, when the dihydroxyanthracenes are exposed to oxygen. The mechanisms of photoreduction of parent AQ in acetonitrile by 2-propanol and in benzene and acetonitrile by TEA are discussed. The effects of AQ follow essentially the same pattern. The various functions of oxygen, e.g. (1) quenching of the triplet state; (2) quenching of the semiquinone radical, thereby forming HO₂^·/O₂^·− radicals; and (3) trapping of the dihydroxyanthracenes are outlined.

To provide photostabilization for entomopathogenic fungi by anionic dyes, composite matrices based on clay–biopolymer combinations were prepared. In the first step, the negative surface charge of various clays (montmorillonite, attapulgite, bentonite and kaolinite) was reversed to positive by adsorption to the polycationic biopolymer chitosan. The second step involved adsorption of the toxicologically safe anionic dyes fast green (FG) and naphthol yellow S (NYS) to the clay complexes. Compared with cytotoxic photoprotectants like berberine, palmatine and acriflavine, the anionic dyes have no adverse effects up to a concentration of 1 M. In assays using various clay–chitosan–dye matrices and UV irradiation from a lamp source, it was evident that both FG and NYS provided considerable photostabilization for conidia of the entomopathogenic fungus Aschersonia spp. that served as a model biocontrol agent. Apparently, because of the light-dispersing property, bentonite and attapulgite per se provided significant photoprotection. All clay matrices containing FG provided a substantial photostabilization effect.

Photodynamic treatment of the gram-negative bacteria Escherichia coli B and Acinetobacter baumannii and the gram-positive bacterium Staphylococcus aureus was performed using two newly devised and synthesized antioxidant carrier photosensitizers (antioxidant carrier sensitizers-2 [ACS-2] and antioxidant carrier sensitizers-3 [ACS-3]), which are butyl hydroxy toluene and propyl gallate substituted haematoporphyrins, respectively. It was found that ACS-2 is less reactive than other photosensitizers previously used for the same purpose, whereas ACS-3 is very effective against the multidrug-resistant bacterium A. baumannii, causing its complete eradication at a low fluence (∼7.5 J/cm) of blue light (407–420 nm) and a low concentration (10 μM). At a higher fluence (∼37.5 J/cm) complete eradication of E. coli B can be obtained under the same conditions. Furthermore, X-ray microanalysis and ultrastructural changes indicate that ACS-3, especially in the case of photodynamic treatment of A. baumannii, interferes with membrane functions and causes the inactivation of the bacterium. ACS-3 may be suggested as a specific photosensitization agent for photoinactivation of gram-negative bacteria.

The type-II photosensitization process is mediated by the formation of singlet oxygen (O₂[Δ_g]). The short lifetime of this species dictates that chemical reactions with biological substrates can only occur when O₂(Δ_g) is in very close proximity to the photosensitizer itself. In this study, deuteroporphyrin, a type-II, membrane-localized photosensitizer, was used to generate O₂(Δ_g) in human lymphoblast WTK-1 cells, and the range of influence was determined by a variety of biological assays. Surprisingly, the initial membrane-confined events were shown, by comet assay, to induce DNA damage in these cells. DNA damage was inhibited both by membrane-localized (α-tocopherol acetate) and by cytoplasmic (trolox) free radical scavengers. Comet formation also was inhibited by treatment at low temperature. DNA fragmentation was not influenced by treatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, showing that apoptosis was not responsible for fragmentation. Taken together, these results show that primary photosensitization reactions involving O₂(Δ_g), even when tightly confined in extranuclear locations, leads to the production of secondary reactive oxygen species, probably as a result of lipid peroxidation, that can act at greater distances from the photosensitizer itself. These experiments were carried out under conditions where cell survival was significant and raise questions regarding DNA damage and mutagenesis pathways, even when extranuclear O₂(Δ_g)-generating compounds are used.

Fabrea salina is a marine ciliate that shows photomotile responses such as positive phototaxis and a step-down photophobic reaction. We found that preilluminated F. salina cells show a phototactic response significantly greater than that of dark-adapted cells when exposed to the same phototactic light stimulus. In particular, positive phototaxis is strongly enhanced by preillumination. This enhancement effect depends on the preillumination light irradiance, on the total preillumination dose, and on the duration of the dark interval between preillumination and the phototaxis measurement. Our results show that the determining factor is the total preillumination dose given to the sample. The enhancement effect shows an asymptotic behavior over a certain range of energy values (10–200 W/m). Further, the effect is transient; after 120 s in the dark, the cells lose any memory of the preillumination, independent of the preillumination energy received. These results are tentatively discussed in terms of light-driven membrane potential or membrane channel conductances.

This work was carried out to study the antioxidant circadian system of two species of crayfish of different latitude origin. We investigated (1) whether both species possess glutathione circadian rhythms and (2) whether both species' rhythms differ in their ability to synchronize to 24 h cycles. Two batches of Procambarus clarkii and P. digueti were kept in (1) light–dark (LD) 12:12 low irradiance (LI) cycles and then exposed to (2) 72 h of complete darkness, (3) LD 12:12 high irradiance (HI), (4) LD 20:4 LI and (5) LD 20:4 HI for 2 weeks. The midgut and hemolymph were sampled and reduced and oxidized glutathione as well as glutathione reductase and glutathione peroxidase were assayed. Cosinor and analysis of variance revealed differences between both species. Procambarus clarkii robust antioxidant circadian rhythms are able to entrain to all conditions resetting to lights on or off. However, the P. digueti weak circadian glutathione system did not entrain to the LD cycles, showing a random distribution of phases. In this species, LD 12:12 and 20:4 HI evidenced significant daily rhythms indicating a damped circadian antioxidative system that is enhanced by the effect of light. This suggests that each species' photoperiodic history determines the adaptive abilities of the circadian antioxidative mechanisms.

In this report, a number of physiological aspects was examined during developmental growth of maize seedling's mesocotyl. It was found that ultraviolet B (UVB) radiation was able to significantly induce nitric oxide synthase (NOS) activities and speedup the release of apparent nitric oxide (NO) of mesocotyl and that exogenous NO donor's rhizospheric treatments may mimic the responses of the mesocotyl to UVB radiation, such as the inhibition of mesocotyl elongation, the decrease in exo- and endoglucanase activities and the increase in protein content of cell wall of mesocotyl. When the seedlings were treated with N-nitro-l-arginine, an inhibitor of NOS, the mesocotyl elongation was promoted, the exo- and endoglucanase activities were raised and the protein content was reduced. However, under UVB radiation, the effects of exogenous NO on several physiological aspects of mesocotyl were similar to those of exogenous reactive oxygen species (ROS) eliminator, N-acetyl-cysteine. All the physiological changes were associated with either the exogenous NO supply or the activities of NOS in plant. Accordingly, it is assumed that reduction in mesocotyl length caused by UVB radiation was possibly achieved through modification of the chemical properties of the cell wall polysaccharides, which was induced by NO and ROS synergically mediated changes in exo- and endo-β-d-glucanases activities in cell walls, and NO was one of the main signaling molecule of UVB radiation in inhibiting mesocotyl elongations. So NO might function as both a second messenger and an antioxidant of UVB radiation during developmental growth of the mesocotyl.

In plants, excess light has the potential to damage the photosynthetic apparatus. The damage is caused in part by reactive oxygen species (ROS) generated by electrons leaking from the photosynthetic electron transport system. To investigate the mechanisms equipped in higher plants to reduce high light (HL) stress, we surveyed the response of 7000 Arabidopsis genes to HL, taking advantage of the recently developed microarray technology. Our analysis revealed that 110 genes had a positive response to a 3 h treatment at a light intensity of 150 W m⁻². In addition to the scavenging enzymes of ROS, the genes involved in biosynthesis of lignins and flavonoids are activated by HL and actually resulted in increased accumulation of lignins and anthocyanins. Comparing the HL-responsive genes with drought-inducible genes identified with the same microarray system revealed a dense overlap between HL- and drought-inducible genes. In addition, we have identified 10 genes that showed upregulation by HL, drought, cold and also salt stress. These genes include RD29A, ERD7, ERD10, KIN1, LEA14 and COR15a, most of which are thought to be involved in the protection of cellular components.