Semiconductor quantum dots (QD) are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. In comparison with organic dyes and fluorescent proteins, QD are emerging as a new class of fluorescent labels with improved brightness, resistance against photobleaching and multicolor fluorescence emission. These properties could improve the sensitivity of biological detection and imaging by at least 10- to 100-fold. Further development in high-quality near-infrared–emitting QD should allow ultrasensitive and multicolor imaging of molecular targets in deep tissue and living animals. Here, we discuss recent developments in QD synthesis and bioconjugation, applications in molecular and cellular imaging as well as promising directions for future research.

Recent progress in understanding the phase-dependent reactivity of halooxides and nitrosyl halides is outlined. Halooxide reactivity is represented by the photochemistry of chlorine dioxide (OClO) and dichlorine monoxide (ClOCl). The gas phase photochemical dynamics of OClO are contrasted with the dynamics in condensed environments. The role of excited-state symmetry in defining the reaction dynamics and the observation of photoisomerization resulting in the production of ClOO are discussed. The current understanding of the excited-state reaction dynamics of ClOCl and evidence for photoisomerization of this species resulting in the production of ClClO are outlined. Finally, the photochemical reaction dynamics of the nitrosyl halide ClNO are presented. The main difference between the gas and condensed phase reaction dynamics of this species is that whereas photodissociation to form Cl and NO dominates the gas phase reaction dynamics, photoisomerization resulting in ClON production occurs to an appreciable extent in condensed environments. The observation of photoisomerization for OClO, ClOCl and ClNO suggests that this process is a general feature of the condensed phase reaction dynamics for smaller halooxides and nitrosyl halides. Finally, future areas for study in both halooxide and nitrosyl halide photoreactivity are outlined.

The ciliate Stentor coeruleus exhibits photodispersal, that is, these cells swim away from light sources and collect in dimly lighted areas. We imaged and reconstructed the tracks of 48 Stentor to determine which swimming behaviors produced their photodispersal. We observed that their photodispersal is not due to a change in their swimming speed but rather to a change in the frequency with which they reorient their swimming direction. Therefore, their photodispersal must be due to either (1) a gradual reorientation of the organism's swimming direction determined by the direction of the light beam (phototaxis) or (2) multiple randomly directed reorientations in swimming direction that occur less frequently when the cell is swimming away from the light source (biased random walk). Sixteen (19%) of the 83 observed forward swimming tracks lasting three or more seconds exhibited a gradual bending away from the light source consistent with a phototaxis. However, most tracks were interrupted repeatedly by abrupt reorientations resulting from ciliary reversals and “smooth turns” that caused cells to reorient through 5.4 times as many degrees as were needed to direct them away from the light source. When cells were swimming away from the light source, their probability of reorienting was reduced and photodispersal resulted.

We determined the biological weighting function (BWF) of the effect of UV radiation on phototaxis of the freshwater, histophagous ciliate Ophryoglena flava. Dose-effect curves were measured by exposing the cells to 12 different irradiation regimens obtained with two different levels of UV-B radiation and by using six filters with cutoff wavelengths ranging from 280 to 335 nm. The results show that there are significant damages to phototaxis at the doses used and that the effect increases when the cutoff is shifted toward short wavelengths. The data were used to calculate the BWF of phototaxis impairment by applying a nonlinear fit procedure. The BWF thus obtained decays exponentially with increasing wavelength in agreement with similar findings reported in the literature for other systems.

The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400–500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz–tungsten–halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm² were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16–62 J/cm², a value significantly lower than that for S. mutans and S. faecalis (159–212 J/cm²). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens.

UV radiation suppresses delayed-type hypersensitivity responses to intradermally injected tuberculin purified protein derivative in Mantoux-positive individuals. The effect of the topically administered isoflavonoid NV-07α, a synthetic derivative of the isoflavonoid equol, on UV-induced suppression of Mantoux reactions was assessed in 18 healthy Mantoux-positive volunteers. A single, fixed dose of solar-simulated UV radiation was delivered to the volunteers' lower backs. Different concentrations of NV-07α or its vehicle were applied to different sites within the irradiated field immediately after UV exposure and again 24 h later. Forty-eight hours after irradiation, Mantoux testing was performed at both the irradiated sites and adjacent, unirradiated sites. The intensity of Mantoux reactions was measured 72 h later with a reflectance erythema meter and by measuring the diameter of each reaction. Although lower concentrations of NV-07α (0.5 and 2 mM) did not prevent UV immunosuppression, 4 mM NV-07α partially but significantly attenuated UV-induced suppression of Mantoux-induced erythema. Minimal erythema doses were also determined for sites treated with NV-07α or its vehicle immediately after UV exposure. NV-07α had no significant effects on UV erythema. We conclude that 4 mM NV-07α prevented the suppressive effects of UV radiation on Mantoux responses in humans but did not affect UV-induced erythema at the concentrations used.

Ultraviolet-B radiation (UV-B, 280–320 nm) has important effects in urban areas, including those on human health. Broadband UV-B radiation is monitored in Baltimore, MD, as part of the Baltimore Ecosystem Study, a long-term ecological research program. We compare broadband UV-B irradiance in Baltimore with UV-B at two nearby locations: a more rural station 64 km southeast and a suburban station 42 km southwest. The monitoring station in Baltimore is on the roof of a 33-m-tall building; there are no significant obstructions to sky view. The U.S. Department of Agriculture UV-B Monitoring and Research Program provided all sensors, which were calibrated at the National Oceanic and Atmospheric Administration Central UV Calibration Facility. UV-B irradiances at the three sites generally were similar. Over all conditions, Baltimore and the suburban site measured 3.4% less irradiance than the rural site. This difference is within the anticipated ±3% calibration uncertainty of the pyranometers. On 59 days with cloud-free conditions at all three sites, average differences in measured UV-B among the three sites were even smaller; Baltimore measured 1.2% less irradiance than the rural site. High aerosol optical thickness strongly reduced daily UV-B dose, whereas [SO₂] had no influence. Surface O₃ increased with increasing UV-B dose when [NO₂] exceeded 10 ppb.

In the filamentous, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, red light (630 nm) decreased, whereas far-red light (720 nm) increased cellular adenosine 3′,5′-cyclic monophosphate (cAMP) content. To find a red and far-red light photoreceptor that triggers the cAMP signal cascade, we disrupted 10 open reading frame having putative chromophore-binding GAF domains. The response of the cellluar cAMP concentration to red and far-red light in each open reading frame disruptant was determined. It was found that only the mutant of the gene all2699 failed to respond to far-red light. The open reading frame named as aphC encoded a protein with 920 amino acids including GAF domains similar to those involved in Cph2, a photoreceptor of Synechocystis sp. PCC6803. To determine which adenylate cyclase (AC) is responsible for far-red light signal, we disrupted all AC genes and found that CyaC was the candidate. The enzymatic activity of CyaC might be controlled by a far-red light photoreceptor through the phosphotransfer reaction. The site-specific mutant of the Asp59 residue of the receiver (R1) domain of CyaC lost its light-response capability. It was suggested that the far-red light signal was received by AphC and then transferred to the N-terminal response regulator domain of CyaC. Then its catalytic activity was stimulated, which increased the cellular cAMP concentration and drove the subsequent signal transduction cascade.

Fluorescein (2-(6-hydroxy-3-oxo-(3H)-xanthen-9-yl)benzoic acid) has been prepared inside the pores of zeolite-Y via ship-in-a-bottle synthesis. Fluorescein, whose dimensions prevent it from entering through the ∼7 Å windows of the faujasite zeolite used, was prepared by the acid-catalyzed reaction of resorcinol and phthalic anhydride. In this article we report initial spectroscopic data as well as an example of the usefulness of these fluorescence-labeled nanoparticles for imaging applications such as confocal fluorescence microscopy. Encapsulated fluorescein shows a remarkable increase in photostability.

We have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 μM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 μM. However, there was a pronounced aggregation effect at 50 μM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 μM hypericin was comparable, whereas using 5 μM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.

Determining whether α-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by α-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (¹Δ_g) oxygen produced from hypericin in the presence of α-crystallin. α-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern–Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to α-crystallin does not inhibit singlet oxygen formation by hypericin. α-Crystallin reacts with singlet oxygen with a rate constant of 1.3 × 10⁸ M⁻¹ s⁻¹. Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.

We present measurements of reflectance spectra from human skin in vivo in the spectral range from 250 to 700 nm. These measurements show that the reflectance from strongly pigmented skin is higher than that from weakly pigmented skin at wavelengths shorter than approximately 300 nm. We simulate the measured results using a new radiative transfer model developed to study light propagation in skin tissue. Our simulations mimic the measured spectra when scattering from melanosomes, and fragmented melanosomes are taken into account. Scattering from microstructures with high relative refractive indices plays a major role in tissue optics. Our results show that scattering from melanosomes and fragmented melanosomes is of particular significance.

The effect of netropsin on the oxidative reactions of duplex DNA was examined. One-electron oxidation of DNA creates a radical cation that migrates through duplex DNA and reacts primarily at GG steps. Netropsin is a dication that specifically binds primarily by hydrogen bonding in the minor groove at sites that have four or more contiguous A·T base pairs. We showed that the oxidation potential of netropsin is less than that of any of the four nucleobases. We find that netropsin quenches the oxidative reactions of DNA independent of whether it is specifically bound. Within the Perrin model of static quenching, a netropsin within a rather large fixed volume around the DNA is an effective quencher.

An important feature of tryptophan phosphorescence, crucial for probing protein structure and dynamics, is the drastic reduction of the lifetime (τ) in fluid solutions. Initial reports of indole and derivatives showed that τ decreases from 6 s in rigid glasses to about 1 ms in aqueous solutions at ambient temperature. Recently a report by Fischer et al. questioned the validity of the millisecond lifetime, claiming that in millimolar electrolyte solutions τ is about 40 μs, similar to the 12–30 μs of earlier determinations based on flash photolysis. Longer lived phosphorescence was detected in pure water but because it exhibited an initial growing phase and an anomalously large triplet yield, the emission was attributed to an artifact arising from the slow, first-order, geminate recombination of the radical cation and electron generated by photochemistry. In this study, we reexamine both the phosphorescence lifetime and the triplet quantum yield of indole, N-acetyl tryptophanamide (NATA), N-methyl tryptophan and the tryptophan–glycine–glycine tripeptide under the same conditions adopted by Fischer et al. as well as over a wider range of electrolyte and buffering salts concentrations, pH, solvent and temperature. Throughout, the results show that the phosphorescence decay is slow and uniform down to the 12 μs resolution of the instrument, with no evidence of short-lived, 40 μs–like components. Most compelling was the similarity between the fluorescence-normalized triplet yield of indole derivatives in water and that of W59 in the protein ribonuclease T₁ or of NATA in rigid glasses. Its invariance over experimental conditions that varied the production of photoproducts several fold and the characteristic susceptibility of the triplet lifetime to O₂, proton and ground state quenching demonstrated that the triplet state was formed predominantly through normal intersystem crossing and that its unquenched lifetime was at least 9 ms.

Survanta is a replacement lung surfactant (LS) used in the treatment of respiratory distress syndrome (RDS), the fourth leading cause of infant mortality in the United States. It consists of purified LS from bovine sources and retains the surfactant proteins (SP) SP-B and SP-C, both thought to be important in proper respiratory function. As such, it provides a useful and biologically relevant model system to probe the structure and function of natural LS. Here, we report results from high-resolution studies on model monolayers formed from Survanta to probe the mechanism of collapse at high surface pressure. Our results show the formation of two different collapse structures. At 62 mN/m, slightly below the collapse pressure, monolayer collapse occurs through buckling. Confocal fluorescence measurements on supported films reveal regions of overlapping phase structure in the films that mark the transition from monolayer to multilayer. Simultaneous near-field scanning optical microscopy fluorescence and force measurements show that the transition seen in the fluorescence measurements accompanies corresponding ∼4–5 nm changes in membrane topography. This change in height is consistent with bilayer formation on monolayer collapse. Analysis of the phase structure near the transitions also suggests that the buckling occurs from a continuous film. However, when the film is compressed to its collapse pressure of 65 mN/m, buckling is no longer evident in the collapsed region. In addition, multilayers and lipid–protein aggregates that are up to 40 nm higher than the monolayer are observed in the collapsed film at this pressure.

The binding of Mg(II), Ca(II), Zn(II), Cu(II) and Fe(III) to ETDA-washed Sepia melanin is quantified by inductively coupled plasma mass spectrometry. By monitoring the solution pH change associated with metal uptake, it is concluded that Mg(II), Ca(II) and Zn(II) bind to carboxylic acid groups in melanin, Cu(II) binds to hydroxyl (OH) groups and Fe(III) binds to OH or amine groups. The aerobic reactivity of melanins with different metal contents is analyzed by examining their ability to cause strand breaks in supercoiled pUC18 DNA. Cu(II)- and Fe(III)-enriched melanins induce the most damage. Hydroxyl radical, ^·OH, is proposed to be one of the reactive oxygen species responsible.

We report an observation of ultraviolet (UV) surface plasmon–coupled emission (SPCE) of N-acetyl-l-tryptophanamide (NATA). The sample was spin coated from poly(vinyl alcohol) (PVA) solution on 20 nm aluminum film deposited on a quartz substrate. The directional UV SPCE occurs within a well-defined narrow angle at 52° from the normal to the coupling hemicylinder quartz prism. The NATA directional emission is highly p polarized as expected for surface plasmon–coupled radiation. The 10 nm protective SiO₂ layer deposited on top of the aluminum film significantly neutralized the fluorophore quenching by the metal surface. SPCE of NATA demonstrates a remarkable intrinsic dispersive property—the maximum of the emission spectrum depends on the observation angle. The efficient spectral resolution of SPCE can be used in the construction of miniaturized spectrofluorometers. The observation of SPCE of tryptophan opens a new possibility for the study of many unlabeled proteins with the technique complementary to surface plasmon resonance analysis.

The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)₂) and N,N-diphenylalanyl protoporphyrin (PP(Phe)₂), and the synergistic effect of 5-methoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet-A (UV-A) irradiation (λ = 365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratinocytes decreases in the order: PPIX > PP(Arg)₂ > PP(Phe)₂. A synergistic effect of 5-MOP on the phototoxicity of PPIX, PP(Arg)₂ and PP(Phe)₂ has been observed. The combination of PPIX, PP(Arg)₂ and PP(Phe)₂ with 0.1–0.5 μM 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)₂ and 5-MOP concentrations lower than 0.75 μM. Above this 5-MOP concentration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)₂ is independent of the presence of 5-MOP. On the other hand, the intracellular content of PP(Arg)₂ is decreased in a concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg)₂ phototoxic effect in the presence of 5-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structures of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)₂ whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)₂ could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)₂ cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)₂ phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg)₂ in combination with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.

To investigate the light-harvesting properties of the Photosystem II chlorophyll (chl) a–b complexes (major light-harvesting complex of Photosystem II [LHCII], CP24, CP26, CP29) in a mature leaf under natural “daylight” illumination, the absorption spectra of the isolated complexes were converted into the photon absorption spectrum (1 − T) within a leaf, using the approach of Rivadossi et al. ([1999] Photosynth. Res. 60, 209–215). In the Q_y region, significant enhancement of light harvesting by the chl b electronic transitions, with respect to the absorption spectra (optical density [OD]), as well as a large and generalized increase (between two- and four-fold) associated with the vibrational bands of both chl a and b, was observed, which acquires an important light-harvesting role (approximately 30–40% of total). In the Soret region, a small increase in light harvesting by chl b was indicated. To gain more detailed information on these aspects the light harvesting of LHCII in a leaf was investigated. This required describing the pigment absorption (chl a and b, carotenoids) in the LHCII OD spectrum in terms of spectral subbands, which were subsequently used to estimate the relative light harvesting of each pigment type in LHCII of a leaf. When the entire visible spectral interval between 400 and 730 nm is considered, the chl a light harvesting is essentially unchanged with respect to the absorption spectrum (OD) of isolated LHCII, whereas the chl b contribution is 20% higher and the carotenoids are 33% lower. The relative enhancement of the chl b absorption is principally associated with the Q_y electronic transition region, the light-harvesting contribution of which becomes prominent in the leaf.

The sensitivity of the photosynthetic apparatus to ultraviolet-B (UV-B) irradiation was studied in cultures of unicellular green alga Scenedesmus obliquus incubated in low light (low photosynthetically active radiation intensity [LL]) and high light (high photosynthetically active radiation intensity [HL]) conditions, treated or not with exogenous polyamines. Biochemical and physicochemical measurements showed that UV-B radiation induces a decrease in the thylakoid-associated putrescine (Put) and an increase in spermine (Spm), so that the reduction of Put/Spm ratio leads to the increase of light-harvesting complex II (LHCII) size per active reaction center and, consequently, the amplification of UV-B effects on the photosynthetic apparatus. The separation of oligomeric and monomeric forms of LHCII from isolated thylakoids showed that UV-B induces an increase in the oligomeric forms of LHCII, which was more intense in LL than in HL. By manipulating the LHCII size with exogenous polyamines, the sensitivity degree of the photosynthetic apparatus to UV-B changed significantly. Specifically, the addition of Put decreased highly the sensitivity of LL culture to UV-B because of the inhibitory effect of Put on the LHCII size increasing, whereas the addition of Spm enhanced the UV-B injury induced in HL culture because of the increasing of LHCII size. The ability of the photosynthetic apparatus to recover the UV-B induced changes was also investigated.

We have synthesized five carotenoid derivatives: (1) Girard's reagent P (GRP)-retinal from GRP and retinal; (2) GRP-carotenal from GRP and β-apo-8′-carotenal; (3) Girard's reagent T (GRT)-carotenal from GRT and β-apo-8′-carotenal; (4) (GRP)₂-canthaxanthin from 2 mol of GRP and 1 mol of canthaxanthin; and (5) dansyl hydrazine (DH)-carotenal from DH and β-apo-8′-carotenal. The first three derivatives are cations, whereas the fourth is a dication and the fifth is a weak base. Using K562 cells, we compared the subcellular distribution of the synthetic carotenoid derivatives with two uncharged natural carotenoids, β-carotene and β-apo-8′-carotenal. The two natural carotenoids were present mainly within the cell membranes. The synthetic carotenoid derivatives were more broadly distributed among the cell organelles. The positively charged derivatives had relatively high concentrations in mitochondria, whereas DH-carotenal had a relatively high concentration in lysosomes. We also measured the amount of photoprotection provided by the synthetic and natural carotenoids for K562 cells labeled with a photosensitizer (hypericin, protoporphyrin IX or cis-di[4-sulfonatophenyl]diphenylporphine). In this model system, only carotenoid derivatives with a permanent positive charge provided significant photoprotection. Neither the two natural carotenoids nor DH-carotenal were effective photoprotectors.

Steady-state UV irradiation of aqueous solutions containing cytochrome c (cyt c) and N,N′-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (BPNDI), a water-soluble aromatic imide, resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) oxidation state. The reaction kinetics were followed by the increase of the ferrocytochrome c absorbance band at 549 nm. The rate of the photochemical reaction was pH dependent, reaching its maximum values over the pH range 4–7. Addition of electrolyte (NaCl) at pH 5 resulted in a decrease in the reaction rate, as expected for reactions between oppositely charged species. Flash photolysis studies revealed that the actual reductant in the reaction was a photogenerated BPNDI radical anion, which transferred an electron to the cyt c heme iron. The participation of imide radicals in the process was confirmed by the ready reduction of cyt c by BPNDI radicals chemically generated with sodium dithionite.

The aim of this study was to monitor the effect of sudden frost on the photosynthetic electron transport chain in the leaves of various plant species using the thermoluminescence (TL) technique. A short period of freezing caused a decrease in the afterglow (AG) band in young maize leaves, with a slight upshift in the maximum temperature. The B band induced by far-red (FR) illumination started to decrease at a significantly lower temperature. The flash-induced B band also showed a substantial decrease in intensity after short preliminary freezing. In contrast to other species, for which there was always a well-detectable TL signal even after relatively drastic freezing, there was no TL signal at all in geranium below a threshold temperature. The behavior of the FR-induced TL curve in cucumber plants was a mixture of that found in wheat or pea, on the one hand, and maize, on the other: the AG band gradually decreased with decreasing temperature and finally totally disappeared, as in maize. The FR-induced B band showed an upshift after freezing. These results suggest that AG is a normal component of TL bands induced not only by FR, but also by single turnover flash.

Low-power lasers are commonly used in human medicine for treatment of various pathological conditions, but mechanisms of their healing effects are still poorly understood. The results of this study provide information related to these effects at the cellular level. Two different protozoan species, Euglena gracilis and Tetrahymena thermophila, were used to study changes in locomotion behavior in response to low-power lasers. The cells were irradiated at 830 and 650 nm generated by a semiconductor laser (99 J/cm², 360 mW) and a laser pointer (0.75 J/cm², 5 mW), respectively, and their locomotion was recorded by a TV camera and analyzed using computer software. Exposure to laser light, regardless of the wavelength, resulted in increased cell velocity in both species (P < 0.001). Exposure to 650 nm produced an equal increase in median cell velocity in both E. gracilis (19.0%) and T. thermophila (18.2%), and some increase persisted in the postirradiation 30 s period. Irradiation by the 830 nm laser resulted in a markedly higher response in Tetrahymena (29.4%) than in Euglena (15.2%), and the two median values remained increased after irradiation was discontinued. Different reactions found in the species studied and some mechanisms underlying the response of cells to radiation are discussed.

The photophysical and photochemical properties of the UV-A–absorbing phototoxic drug cyamemazine (CMZ) (2-cyano-10-(3-[dimethylamino]-2-methyl-propyl)-phenothiazine) have been investigated in neutral buffered aqueous solutions. The transient absorbances of the hydrated electrons, of the first excited triplet state (³CMZ*) with a characteristic absorption band peaking at 420 nm and of the radical cation (^·CMZ ) (maximum absorbance at 500 nm) have been observed by 355 nm laser flash spectroscopy of deaerated solutions. All these transient species are formed by monophotonic processes and react with oxygen. Bimolecular reaction rate constants of ^·CMZ and ³CMZ* with O₂ are 2 × 10⁷ M⁻¹ s⁻¹ and 4 × 10⁹ M⁻¹ s⁻¹, respectively. The ³CMZ* reacts only sluggishly (reaction rate constant, 9 × 10⁶ M⁻¹ s⁻¹) with tryptophan chosen as a Type-I photodynamic substrate. Steady-state irradiations with 365 nm light demonstrate that CMZ is rapidly photolyzed (quantum yield, 0.04) in O₂-saturated solutions leading to oxidation of the sulfur atom and of the side-chain nitrogen of CMZ. This photoproduct (2-cyano-10-(3-[dimethylamino, N-oxide]-2-methyl-propyl)-5-oxide–phenothiazine), is a good Type-I and Type-II photodynamic photosensitizer producing singlet oxygen in high yield (∼0.45) and could play a major role in the phototoxicity of CMZ.

The Escherichia coli protein YcgF contains a photosensory flavin adenine dinucleotide (FAD)–binding BLUF domain covalently linked to an EAL domain, which is predicted to have cyclic-di-guanosine monophosphate (GMP) phosphodiesterase activity. We have cloned, overexpressed and purified this protein, which we refer to as blue light–regulated phosphodiesterase (Blrp) for its putative activity. Blrp undergoes a reversible photocycle after exposure to light in which the spectrum of its photostationary state and kinetics of recovery of the dark state are similar to those of the isolated BLUF domain of the AppA protein. Unlike the AppA BLUF domain, the chromophore environment in the context of full-length Blrp is asymmetric, and the protein does not undergo any detectable global changes on exposure to blue light. When overexpressed in E. coli, Blrp copurifies with certain proteins which suggests that it plays a protective role in response to oxidative stress. Predicted proteins from Klebsiella pneumoniae and from a bacterium in the Sargasso Sea are similar to E. coli Blrp in both their BLUF and EAL domains, which suggests that blue light sensing in these bacteria may follow similar pathways.

In research laboratories ultraviolet radiation is widely used, particularly in photochemistry and photobiology, as a sterilizing agent and for the characterization of samples. The results of a survey conducted near several university laboratories are presented with the aim of quantifying exposure levels to UV-incoherent radiation and to assess individual risk for researchers and students. It has been shown that exposure is not negligible, especially if safety procedures are neglected and personal protective equipments, described in this study, are not used.

The spectroscopic properties of enoxacin (ENO), oxolinic acid (OXO) and nalidixic acid (NAL) were studied in various H₂O–CH₃OH and H₂O–CH₃CN mixed solvents because these solvents were thought to behave as a biological mimetic system. ENO has piperazinyl group, but OXO and NAL do not have this substituent. The fluorescence emission spectra of ENO were very sensitive to the composition of the solvents. In the Lippert–Mataga analysis of the steady-state fluorescence data, clear reverse solvatochromism was exhibited for ENO in both mixed solvents. This observation can be explained using the excited state twisted intramolecular charge transfer (TICT) from the nitrogen of the piperazinyl group to the keto oxygen. Theoretical calculations further support this observation. The nonradiative and radiative rate constants of these molecules were analyzed as a function of dipolarity–polarizability (π*) and hydrogen bond donor acidity (α) of the mixed solvents. These results for ENO were consistent with the suggested mechanism of the TICT very well. The influence of bulk dielectric effect was more significant relative to the specific hydrogen bonding interactions. The emission spectra of OXO and NAL do not exhibit any characteristic responses to the properties of the solvent.

This article proposes a new approach to the modeling of the molecular-level mechanism of ligand–receptor interaction for Ca² receptor binding site. Chemically induced dynamic nuclear polarization (CIDNP) technique has been used to unravel fine details of the reaction in the model system composed of one of the known Ca² antagonist drugs, nifedipine (NF), and isolated amino acid residuals (e.g. tyrosine [Tyr]) of Ca² receptor binding site. It has been conclusively demonstrated that the reaction between NF and Tyr resulting in the oxidation product—nitroso form of NF—obeys the radical mechanism. CIDNP data in combination with the results of mathematical modeling of the structures of ligand–receptor complexes have allowed to propose the mechanism of the interaction of NF with Ca² receptor binding site.

Aggregation of bacteriochlorophyll (BChl) c from chlorosomes, the main light-harvesting complex of green bacteria, has been studied in aqueous buffer. Unlike other chlorophyll-like molecules, BChl c is rather soluble in aqueous buffer, forming dimers. When BChl c is mixed with carotenoids (Car), the BChl c Q_y transition is further redshifted, in respect to that of monomers and dimers. The results suggest that Car are incorporated in the aggregates and induce further aggregation of BChl c. The redshift of the BChl c Q_y band is proportional to the Car concentration. In contrast, the mixture of bacteriochlorophyllide (BChlide) c, which lacks the nonpolar esterifying alcohol, does not form aggregates with Car in aqueous buffer or nonpolar solvents. Instead, the position of the BChlide c Q_y transition remains unshifted in respect to that of the monomeric molecule, and Car precipitates with the course of time in aqueous buffer. Similar effects on both BChl c and BChlide c are also observed when monogalactosyl diglyceride (MGDG), which forms the monolayer envelope of chlorosomes, is used instead of (or together with) Car. The results show that the hydrophobic interactions of the BChl c esterifying alcohols with themselves and the nonpolar carbon skeleton of Car, or the fatty acid tails of MGDG, are essential driving forces for BChl aggregation in chlorosomes.

Oxygen free radicals play a role in the aging process, and the protective effect of various antioxidants has been intensively studied, in particular for cutaneous aging. Besides hereditary factors, free radical–mediated damage to melanocytes of the hair follicle has been considered as a mechanism for aging of the hair. It was the aim of this study to evaluate the role of photosensitization reactions for hair graying and to demonstrate potential protective effects of superoxide dismutase (SOD). Mice with black hair were depilated with the fingertips on a surface of 6 × 2.5 cm on both sides of the dorsum. The right side received five applications of a SOD-containing gel before exposure to psoralen (concentration 0.5 mg/mL) plus UV-A (365 nm, 4 J/cm²). The left side was pretreated in the same way with a gel free of SOD. When the hair started growing again, the SOD-protected side was covered with black hair, whereas the hair on the vehicle-treated side was gray or white in 27 of the 30 animals studied. The 0.01% SOD concentration was as protective as the 0.1% concentration. Heat-inactivated SOD, applied in another five animals, was not protective. Using fluorescent labeling of the SOD with fluorescein isothiocyanate, epifluorescence microscopy and digital imaging processing, we show that SOD applied to the skin surface penetrates through the follicular appendages, as well as through the unbroken stratum corneum. Our findings suggest that superoxide radicals, generated by interaction of UV-A light with the sensitizer, initiated the formation of secondary products with well-known DNA-damaging effects, such as lipid peroxidation products and tumor necrosis factor alpha. SOD prevented the damage to melanocyte DNA by dismutating superoxide. Photosensitization may be another mechanism for hair graying, which can be influenced by antioxidants. Given the large number of exogenous and endogenous sensitizers, this mechanism deserves further study for human hair graying.

St. John's Wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges and is phototoxic to skin. To determine if it also could be phototoxic to the eye, we exposed human lens epithelial cells to 0.1–10 μM hypericin and irradiated them with 4 J/cm² UV-A or 0.9 J/cm² visible light. Neither hypericin exposure alone nor light exposure alone reduced cell viability. In contrast, cells exposed to hypericin in combination with UV-A or visible light underwent necrosis and apoptosis. The ocular antioxidants lutein and N-acetyl cysteine did not prevent damage. Thus, ingested SJW is potentially phototoxic to the eye and could contribute to early cataractogenesis. Precautions should be taken to protect the eye from intense sunlight while taking SJW.

Proliferating cell nuclear antigen (PCNA) is an active nuclear protein involved in DNA replication, recombination and repair. PCNA is found throughout the basal layer in normal skin and in all layers of the epidermis in malignancy. This study evaluates PCNA's expression after acute and chronic UV-B irradiation. Skh-1 hairless mice exposed to 1.5 and 4.5 kJ/m² of UV-B were sacrificed at 6, 12, 24, 48, 72 and 168 h. Immunohistochemical analysis revealed PCNA expression throughout the basal layer of untreated skin, with diminished expression at 6 h, indicative of immediate UV damage, and evidenced by the observable upregulation in pyrimidine dimer formation early on. Subsequently, PCNA immunoreactivity progressively increased, demonstrating an aberrant upward epidermal migratory pattern in association with chronic exposure. The 4.5 kJ/m² group exhibited prolonged recovery in staining and also demonstrated this altered migratory pattern with chronic exposure. Progressive reactivation of PCNA expression occurs with repair. PCNA migration to upper layers of the epidermis indicates proliferation and possibly a subsequent increased malignant potential. We conclude that PCNA can serve as a marker of DNA repair and indirectly as an indicator of UV-B–induced damage, expression being time dependent and dose related. Specific immunoreactivity patterns and the observable atypia in keratinocytes are relevant in elucidating malignant potentiation.

5-Aminolevulinic acid and its esterified analogues have been under much investigation to enhance the endogenous production of protoporphyrin IX (PpIX) in tumor cells. However, in this work, we studied the in vitro and in vivo efficacy of exogenously administered PpIX and its esterified analogue, PpIX dimethyl ester (PME), in poorly differentiated human nasopharyngeal carcinoma (NPC/CNE-2) as a photodynamic diagnostic (PDD) agent. NPC/CNE-2 at its earliest time, 1 h after incubation with PME in in vitro studies, has exhibited 64% (P < 0.01) higher tumor to normal cell (T/N) fluorescence ratio than with PpIX. In an in vivo mouse xenograft model, comparable photosensitizer concentration in tumor after intravenous administration was observed at 1–3 h time points, but at 9 h, PME had 31% (P = 0.05) greater concentration in tumor compared with PpIX. In addition, by constituting PME and PpIX in different topical gel composites, of which, PME gel composition of 8:2 Plasdone® and Gantrez® resulted in the highest T/N ratio at 6 h after application (34%; P < 0.05) in comparison with other gel composites. Evaluation of PME and PpIX constituted in the delivery vehicles investigated showed comparable selectivity for tumor at 1–3 h, thus neither photosensitizer is more efficient than the other for PDD at the early time points; however, beyond 6 h, PME had higher selectivity for tumor compared with PpIX. Thus, further investigation is warranted to improve the drug delivery vehicle for greater tumor selectivity at a shorter incubation time.

Understanding of the mechanism of ultraviolet (UV)–mediated cutaneous damages is far from complete. The cancer-specific expression of Survivin, a member of the inhibitor of apoptosis family of proteins, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a target for cancer treatment. This study was designed to investigate the modulation of Survivin during UV response, both in vitro and in vivo. We used UV-B–mediated damages in normal human epidermal keratinocytes (NHEK) cells as an in vitro model and SKH-1 hairless mouse model for the in vivo studies. For in vitro studies, NHEK were treated with UV-B and samples were processed at 5, 15, 30 min, 1, 3, 6, 12 and 24 h after treatment. Our data demonstrated that UV-B exposure (50 mJ/cm²) to NHEK resulted in a significant upregulation in Survivin messenger RNA (mRNA) and protein levels. We also observed that UV-B exposure to NHEK resulted in significant (1) decrease in Smac/DIABLO and (2) increase in p53. For in vivo studies, the SKH-1 hairless mice were subjected to a single exposure of UV-B (180 mJ/cm²), and samples were processed at 3, 6, 12 and 24 h after UV-B exposure. UV-B treatment resulted in a significant increase in protein or mRNA levels (or both) of Survivin, phospho-Survivin and p53 and a concomitant decrease in Smac/DIABLO in mouse skin. This study demonstrated, for the first time, the involvement of Survivin (and the associated events) in UV-B response in vitro and in vivo in experimental models regarded to have relevance to human situations.

A medical device using riboflavin (RB) and light is being developed for the reduction of pathogens in platelet concentrates (MIRASOL™ pathogen reduction technology [PRT]). A high-performance liquid chromatography (HPLC) method for the quantification of RB and its main photoproduct, lumichrome (LC) in blood components has been developed and validated. In addition, the same method has been used to identify and quantify the presence of additional photoproducts–catabolites of RB. Levels of these agents before and after treatment as well as endogenous levels present in normal donor blood are reported using this analytical technique. The method allows for quantitative and qualitative analysis of RB and LC in blood components using HPLC-fluorescence detection, a Zorbax® SB-CN (stable bond cyano) column and a methanol–water mobile phase. Quantitation and qualitative analysis of additional photoproducts of RB was also performed, but the method has not been validated for these other components. The method described has passed an 8 day validation and has been found to be adequate for its intended use. The range of the method for RB is 0.016–1.500 μM and for LC is 0.060–1.500 μM. The method detection limit for RB is 0.0006 μM and for LC is 0.012 μM. The acceptance criteria for repeatability were met; the relative standard deviation for RB was 0.64% and for LC was 0.76%. The acceptance criteria for bias were met with a 97% average recovery for RB and a 102% recovery for LC. Samples were centrifuged and diluted 1:50 with 0.9% saline before analysis. No protein precipitation or extraction was required. A mass balance of approximately 93.4–94.4% was achieved after exposure of products to UV light in the intended pathogen reduction treatment method. The method permitted the identification of photoproducts in blood that were both naturally occurring and produced after photolysis of blood samples treated with the PRT process. The identity of these photoproducts has been established using HPLC Tandem Mass Spectrometry (MS/MS) and UV spectroscopic methods and has been correlated with known metabolites and catabolites of RB. HPLC with fluorescence detection using a reverse phase cyano-column allows for accurate separation, identification and quantification of both RB and LC in blood products without the need for solvent extraction or protein precipitation. Additional photoproducts could also be identified and quantified using this method. The presence of these agents in normal, untreated blood suggests that their presence in blood is ubiquitous.