The synergistic insecticidal action of characteristic defensive substances produced by the plant family Asteraceae was investigated under controlled laboratory conditions. Sesquiterpene lactones isolated from Asteraceae that may form, through a Michael addition process, conjugates with glutathione were administered in a meridic diet to a herbivorous insect, Manduca sexta. By administering sesquiterpenes, variable in vivo reduced glutathione levels were observed in the insect larvae. When the Asteraceae-derived photooxidant α-terthienyl was co-administered, lipid peroxidation and larval mortality were significantly enhanced in the treated groups of insects with lowered in vivo glutathione levels.

We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet-C (<280 nm) and UVB/UVA (280–390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen-activated protein kinase family. Ultraviolet-C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826–829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA-irradiated plants that were subsequently wounded accumulated 2.5–4-fold higher levels of proteinase inhibitor I when compared to wounded non-irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12-oxo-phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.

The exposure of populations of Ceratitis capitata (fruit fly), Bactrocera oleae (olive fly) and Stomoxis calcitrans (house fly) to a bait containing μmolar concentrations of porphyrin-type photosensitizers resulted in a significant accumulation of the porphyrin by the insects and a consequent development of photosensitivity upon exposure to visible light. The photoinsecticidal activity appeared to increase with increasing hydrophobicity of the porphyrin molecule: thus, the amphiphilic dicationic meso-di(cis-4N-methyl-pyridyl)-cis-diphenyl-porphine (n-octanol/water partition coefficient = 20) was markedly more efficient than its tricationic analogue or the dianionic hematoporphyrin (n-octanol/water partition coefficient = 12). The observed large decrease in the acetylcholinesterase activity of the photosensitized flies suggests that the damage of the nervous system gives an important contribution to the phototoxic action of porphyrins. Studies with C. capitata indicate that the photoinsecticidal action of porphyrins can be utilized to control the population of noxious insects also in open field conditions.

Vitamin B₆ (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (¹O₂), cercosporin. We measured the rate constants (k_q) for the quenching of ¹O₂ phosphorescence by 1–4 in D₂O. The respective total (physical and chemical quenching) k_q values are: 5.5 × 10⁷ M⁻¹ s⁻¹ for 1; 7.5 × 10⁷ M⁻¹ s⁻¹ for 2, 6.2 ×10⁷ M⁻¹ s⁻¹ for 3 and 7.5 × 10⁷ M⁻¹ s⁻¹ for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by ¹O₂, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize ¹O₂. This photodegradation was completely stopped by azide, a known physical quencher of ¹O₂. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B₆ vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and ¹O₂ in the living fungus.

Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (¹O₂)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants. Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity. The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state. As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for ¹O_{2 production in a range of solvents. We found that as a ¹O2 photosensitizer, HAC was only moderately effective in organic solvents (ϕSO = 0.14–0.18) and very poor in water (ϕSO = 0.02–0.04). By contrast, the ¹O2 quantum yield of cercosporin itself was unaffected by solvent (ϕSO = 0.84–0.97). To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy. This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin. We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular ¹O2 production to levels that can be tolerated by the fungus. In addition, we have, for the first time, directly detected ¹O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating ¹O2 production in vivo and from cercosporin in culture.}

To address the deficiencies of benzophenone UV screens for preventing brightness reversion in high yield mechanical papers, we synthesized a new series of such materials with enhanced water solubility and compatibility with the lignocellulosic substrate. A series of 2,4-dihydroxybenzophenones (DHB) were synthesized containing various Mannich bases at the C3 position of one of its rings. They possess the UV-screening ability of o-hydroxylbenzophenones, and they also contain tertiary nitrogen atoms that may function as radical scavengers. Aqueous solutions of the hydrochloride salt of 3-(dimethylaminomethylene)-2,4-dihydroxylbenzophenone (1), when applied on bleached chemithermomechanical pulp (CTMP) sheets, were significantly more efficient in preventing photoyellowing than the original DHB applied on the sheets from ethanol–water solutions. This confirmed our original hypothesis that increasing the compatibility of the UV screen with the lignocellulosic matrix would increase its efficiency in preventing photoyellowing. Compound 1, however, was found to be somewhat more effective than its hydrochloride salt toward preventing photoyellowing. This was attributed to the synergistic action of the free tertiary aminic center attached on the molecule with its UV-screening ability. To comprehend further the various parameters that influence the photoyellowing inhibition performance of these compounds and DHB with bleached CTMP pulp fibers, a series of handsheets were prepared at different pH. The interactions of the protonated compound 1 with pulp fibers were then evaluated by studying their kinetics of absorption and desorption to and from the fiber matrix. This part of our study found that the adsorption of protonated Mannich derivatives of DHB onto pulp is most likely governed by a cation-exchange mechanism involving the cationic amine group with the sulfonic and carboxylic acid groups located on the surface of the fibers. The pH the paper sheet was made from was also found to affect profoundly the adsorption and retention characteristics of these compounds onto the lignocellulosic matrix.

In this report, we describe our attempt to understand the photochemical interactions that occur between dihydroxybenzophenone (DHB)-based UV screens and lignin when high-yield pulps are treated with such materials. Milled wood lignin (MWL) and filter paper were used as models, and various irradiation protocols were carried out in the presence and absence of UV screens. After irradiation, the lignin and the UV screen were extracted and the products analyzed. These experiments showed that upon irradiation, fragments of MWL-containing chromophores were linked to cellulose via an acid-labile linkage. In the presence of UV screens, these reactions were minimized. Molecular weight measurements of the extracted lignin showed that the MWL is degraded upon solid-state irradiation. The samples that contained UV screens showed a reduced tendency to degrade. Using quantitative ³¹P NMR, it was possible to probe further the detailed structural changes that occurred in MWL during irradiation. In general, DHB-based UV screens and derivatives were found to interact actively with MWL when irradiated.

We measured the end-to-end diffusion coefficient of an alkyl chain-linked donor–acceptor pair using the time-resolved frequency-domain decay of the donor. The donor was a rhenium metal–ligand complex with a mean decay time ranging from 2.1 to 7.9 μs in the absence of the Texas red acceptor. The decay time was used to measure the donor-to-acceptor distance distribution and the mutual diffusion coefficient. Using this long lifetime donor, it was easily possible to determine a diffusion coefficient near 2 × 10⁻⁸ cm²/s and diffusion coefficients as low as 1.3 × 10⁻⁹ cm²/s were measurable. Such long lifetime donors should be valuable for measuring the flexing of peptides on the microsecond timescale, domain motions of proteins and lateral diffusion in membranes. The availability of microsecond decay time luminophores now allows luminescence spectroscopy to be useful generally for studies of microsecond dynamics of biological macromolecules.

The relative induction of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4)pyrimidone photoproducts ([6–4]PD) was quantified in the duplex homopolymers polydeoxyadenosine : polydeoxythymidine, polydeoxyguanosine : polydeoxycytidine and polydeoxyguanosine :polydeoxy-5-methylcytidine irradiated with UVC or UVB radiation. Cytosine methylation significantly increased the yield of cytosine (6–4)PD after irradiation with UVC light and of cytosine CPD and (6–4)PD after irradiation with UVB light. The data suggest that CPD and (6–4)PD are preferentially induced at 5-methylcytosine bases in DNA of cells exposed to sunlight and comprise a major component of the mutation spectrum leading to the initiation of sunlight-induced skin cancer.

The excited-state intramolecular H-atom transfer reactions of hypocrellins B and A are compared by using time-resolved absorption and fluorescence upconversion techniques. The hypocrellin B photophysics are well described by a simple model involving one ground-state species and excited-state forward and reverse H-atom transfer with a nonfluorescent excited state. We suggest that excited-state conformational changes are coupled to the H-atom transfer in hypocrellin B just as gauche/anti changes are coupled to the H-atom transfer in hypocrellin A.

Nabumetone is a phototoxic nonsteroidal antiinflammatory drug used for the treatment of osteoarthritis. However, nabumetone is considered a prodrug with its metabolite 6-methoxy-2-naphthylacetic acid the active form. Photophysical and photochemical studies on this metabolite have been undertaken. It undergoes photodecarboxylation in aerated aqueous and organic solvents. In addition to the accepted photodegradation pathway for related molecules, a new mechanism that implies generation of the naphthalene radical cation from the excited singlet and addition of O₂ prior to the decarboxylation process has been demonstrated. Evidence for the involvement of the excited singlet state in this mechanism have been obtained by steady-state and time-resolved fluorescence experiments. The fluorescence quenching by O₂ and the shorter singlet lifetime in aerated solvents support this assignment. Laser flash photolysis also supports this mechanism by showing the noninvolvement of the triplet in the formation of the naphthalene radical cation. Finally, the well-known electron acceptor CCl₄ acts as an efficient singlet quencher, enhancing the route leading to the radical cation, preventing intersystem crossing to the triplet and thus resulting in a dramatic increase in the yield of 6-methoxy-2-naphthaldehyde, the major oxidative decarboxylation product; this constitutes unambiguous proof in favor of the new mechanistic proposals.

The time-resolved fluorescence spectra of the main arterial fluorescent compounds were retrieved using a new algorithm based on the Laguerre expansion of kernels technique. Samples of elastin, collagen and cholesterol were excited with a pulsed nitrogen laser and the emission was measured at 29 discrete wavelengths between 370 and 510 nm. The expansion of the fluorescence impulse response function on the Laguerre basis of functions was optimized to reproduce the observed fluorescence emission. Collagen lifetime (5.3 ns at 390 nm) was substantially larger than that of elastin (2.3 ns) and cholesterol (1.3 ns). Two decay components were identified in the emission decay of the compounds. For collagen, the decay components were markedly wavelength dependent and hydration dependent such that the emission decay became shorter at higher emission wavelengths and with hydration. The decay characteristics of elastin and cholesterol were relatively unchanged with wavelength and with hydration. The observed variations in the time-resolved spectra of elastin, collagen and cholesterol were consistent with the existence of several fluorophores with different emission characteristics. Because the compounds are present in different proportions in healthy and atherosclerotic arterial walls, characteristic differences in their time-resolved emission spectra could be exploited to assess optically the severity of atherosclerotic lesions.

Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively ≥10⁶ TCID₅₀ of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin–hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (ΔΨ_m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li–Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D₃₇ (LFS cells)/D₃₇ (normal human fibroblasts) was 2.8 ± 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 ± 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G₂ phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.

It has been proposed that the construction of a photosensitizer–polymer conjugate would lead to an increased selective retention of the drug in tumor tissue resulting in an enhancement of selective tumor destruction by light in photodynamic therapy. In this study the kinetics of a tetra-pegylated derivative of meta-tetra(hydroxyphenyl)chlorin (mTHPC–PEG) were compared with those of native meta-tetra(hydroxyphenyl)chlorin (mTHPC) in a rat liver tumor model. In addition, the time course of bioactivity of both drugs was studied in normal liver tissue. Pegylation of mTHPC resulted in a two-fold increase in the plasma half-life time, a five-fold decrease in liver uptake and an increase in the tumor selectivity at early time intervals after drug administration. However, although mTHPC concentrations in liver decrease rapidly with time, mTHPC–PEG liver concentrations increased as a function of time. This led to a loss of tumor selectivity at all but the earliest time points, whereas with mTHPC tumor selectivity increased with time. For both drugs the time course of bioactivity in the liver parallels drug concentration levels with extensive necrosis after irradiation of mTHPC–PEG-sensitized liver tissue up to drug–light intervals of 120 h. It is concluded that on balance mTHPC–PEG does not appear to show any benefits over native mTHPC for the treatment of liver tumors, as normal liver tissue accumulates the compound. However, pegylation is a potentially promising strategy with an increase in tumor selectivity and reduced liver uptake if accumulation in the liver can be prevented.

Mechanically deformed morphologic cartilage grafts undergo temperature-dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, porcine nasal septal cartilage specimens were evaluated for viability by measuring the incorporation of Na₂³⁵SO₄ into proteoglycan (PTG) macromolecules in whole tissue culture following laser-mediated reshaping. Synthesis rates of PTG were determined by scintillation counting lyophilized specimens and normalizing these values by total protein content. Positive controls were established by inducing chondrocyte apoptosis using prolonged exposure to nitric oxide (NO). In chondrocytes, apoptosis induced using NO resulted in significantly lower PTG synthesis rates compared to untreated native specimens. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm², λ = 1.32 μm) while recording simultaneously radiometric surface temperature, internal stress and back-scattered light intensity from a probe laser. Each specimen received one, two or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in back-scattered light intensity that correlate with accelerated stress relaxation. A 5 min time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Average PTG synthesis rates decreased with successive laser exposures, though these were always higher than baseline rates established for NO-treated tissues, suggesting that laser-mediated cartilage reshaping acutely does not eliminate the entire population of viable chondrocytes. The reduction in PTG synthesis is correlated with the time–temperature-dependent heating profile created during laser irradiation, supporting our hypothesis that careful monitoring of laser dosimetry is required to ensure chondrocyte viability.

A simple method has been developed to determine the optical properties of the anterior segment of the intact eye. This consists of a probe that is inserted into the posterior sclera and detects light passing through the anterior segment. The probe is connected to a charge-coupled device spectrophotometer via a fiber optic bundle. It was determined that the young rat anterior segment transmits light down to 300 nm, whereas calf and rabbit eyes transmit no UVB and only part of the UVA to the posterior segment. The absorbing species in these animals is most likely NAD(P)H, which has an absorption maximum at ∼345 nm and is associated with ζ-crystallin. A young primate anterior segment transmits almost no UV with a steep increase in transmission at CA 400 nm. Because some experiments employed a light tube that is used to illuminate the retina during vitrectomies, this method can be used to determine the transmission spectra of the anterior segment of humans in vivo.

A fluorescent protein isolated from the deep-sea luminous bacterium Photobacterium phosphoreum strain bmFP has been purified, cloned and sequenced. The protein is 96.5% identical in amino acid sequence to FP₃₉₀, the weakly fluorescent flavoprotein encoded by the luxF gene characteristic of Photobacterium species. Similar to FP₃₉₀, bmFP is a dimer of two homologous subunits binding four FMN-myristate chromophores but has the distinctive feature of emitting a bimodal fluorescence with maxima at about 488 and 517 nm, hence the name bmFP. For both bands of this fluorescence, the excitation spectrum exhibits a peak at 336 nm, not corresponding to its flavin-like absorption spectrum. Heating of bmFP in urea resulted in a decrease in the intensity of the 488 nm band along with the appearance of a new fluorescence peaking at 423 nm, partially reversible upon the removal of the urea. Upon complete denaturation, either by heat or guanidium chloride at 65°C, fluorescence characteristic of both free flavin and this 423 nm species appears. It is speculated that chromophores in different states of protonation, associated with a single protein, are responsible for the unusual spectral properties of bmFP.

The activity of the bimodal fluorescent protein (bmFP) (λ_max, 488 and 517 nm) in the in vitro luciferase reaction has been studied. The bmFP that is produced by Photobacterium phosphoreum strain bmFP is a dimer of two homologous subunits binding four riboflavin 5′-phosphate (FMN)-myristate chromophores. The addition of bmFP to the luciferase reaction in the presence of the lumazine protein prevented the lumazine protein-induced blue shift in the emission band. The bmFP reduced electrochemically serves as a substrate in the luciferase reaction in the absence of added FMN, resulting in light emission with a single maximum at about 487 nm. The bmFP was also active in lieu of FMN in the NADH/FMN oxidoreductase (flavin reductase)–luciferase coupled bioluminescence reaction in the absence of added FMN. In the coupled reaction, bioluminescence with the isolated bmFP chromophore was weaker than that with the holo-bmFP. After bmFP was used in luciferase reactions initiated either chemically or electrochemically, it was still capable of emitting bimodal fluorescence.