EXTRARETINAL PHOTORECEPTION IN CHICKENS: ENTRAINMENT OF THE CIRCADIAN LOCOMOTOR ACTIVITY RHYTHM

Abstract— The Circadian locomotor activity rhythm of blinded chickens entrained to, or was synchron ized by, light in light dark cycles.     

CIRCADIAN RHYTHM OF Robinia pseudoacacia LEAFLET MOVEMENTS: ROLE OF CALCIUM AND PHYTOCHROME

Abstract— The effect of external calcium level, calcium ionophore A23187 and red light on the circadian rhythm of Robinia pseudoacacia leaflet movements has been studied. Fifteen minute red light pulses shifted the phase of leaflet rhythmic movement with a phase-response curve type 0. Maximum advances and delays (about 10 h and 8 h, respectively) were obtained between circadian time (CT) 10 and CT 12 at the end of a subjective day. An almost null effect was obtained at the end of a subjective night. Phytochrome is the photoreceptor involved in phase shifting since this effect of red light is reversed by 5 min of far red light. Two hour pulses of external calcium, applied as CaCl₂ (10 m M ), and 2 h pulses of calcium ionophore A23187 (10–50 μM) also shifted the phase of leaflet circadian movement and caused the same type of phase-response curve, with maximum advances and delays at the same time as those produced by red light. Two hour pulses of an external calcium chelator, EGTA (5 m M ), and a calcium channel blocker, LaCl₃ (10–50 m M ), damped the circadian rhythm or did not change the phase when they were applied at lower concentration. These results indicate that phytochrome could control the circadian oscillator, which drives Robinia leaflet movements by increasing the intracellular calcium concentration.     

CIRCADIAN RHYTHM OF LOCOMOTOR BEHAVIOR IN A POPULATION OF Paramecium multimicronucleatum: ITS CHARACTERISTICS AS DERIVED FROM CIRCADIAN CHANGES IN THE SWIMMING SPEEDS AND THE FREQUENCIES OF AVOIDING RESPONSE AMONG INDIVIDUAL CELLS

Abstract The locomotor behavior of Paramecium is determined by two components: swimming speed and the frequency of avoiding responses. The circadian oscillations of these two components were examined in order to interpret characteristics of the circadian locomotor rhythm, previously found in a Paramecium population using an originally defined parameter, "traverse frequency" (Hasegawa et al ., 1978, 1982). In our present study, a modified version of the previously developed, fully computerized, close-up video/photoamplifier system was used. Results indicate that individual specimens swam fast and unidirectionally during the day, while at night, in a light–dark cycle, they swam slowly and frequently turned. This oscillatory pattern was sustained in continuous darkness, where fluctuation in the frequency of avoiding responses was a dominant characteristic. The time structure of the "random walk" of Paramecium behavior was also examined by constructing and stochastically testing histograms of the interval times between specimens consecutively traversing beneath an observation point. Statistical analyses of observation data indicated that the circadian organization of the two components by individual specimens resulted in a circadian accumulation/dispersal rhythm of the entire population. It was concluded that the circadian "traverse frequency" rhythm principally represented this circadian accumulation/dispersal rhythm.     

EXTRARETINAL PHOTORECEPTION IN ENTRAINMENT OF CRUSTACEAN ORCADIAN RHYTHMS*

Abstract— An attempt was made to determine whether entrainment of the circadian rhythms of locomotor activity and ERG amplitude of the crayfish involved extraretinal photoreception. The results of a variety of experiments involving surgical lesions and localized illumination provided evidence that both rhythms can be entrained via an extraretinal pathway. The data also demonstrate that the caudal photoreceptor is unnecessary for entrainment. Our evidence to date suggests that the extraretinal photoreceptor is located in the supraesophageal ganglion; however, the possibility of photoreceptive input from other regions of the CNS, particularly the optic lobe, has not been eliminated. It is also tentatively concluded that the circadian oscillators for both the locomotor and the ERG amplitude rhythms are located within the supraesophageal ganglion, but more data are needed to confirm this conclusion.     

EXTRARETINAL PHOTORECEFTION IN INSECTS

Abstract— Extraretinal photoreceptors are widespread among insects and function in the photoperiodic control of development and in the entrainment of circadian rhythms. The effects of light on the daily and seasonal regulation of brain neuroendocrine activity are mediated solely through extraretinal photoreceptors. In primitive insects, the eyes participate in the entrainment of nonendocrine circadian rhythms such as the locomotor rhythm. In more advanced forms, however, extraretinal pathways appear to be the only pathway for the entrainment of all rhythms thus far examined. But even in this latter case, the eyes sometimes effect a masking of the expression of the overt rhythm. An exact localization of the extraretinal receptors has not been accomplished, but in all studies to date they appear to be associated with the cerebral lobe region of the brain. Action spectra for photoperiodic responses have been determined for a number of insects. In general the responses are maximally sensitive in the blue with a marked decline in the red although exceptions do exist. Complete action spectra for circadian responses have been determined only for two insects. In both cases a plateau of sensitivity extends through the blue with a steep drop at longer wavelengths. From the action spectra data, the extraretinal receptors appear to have a threshold sensitivity less than 3 times 10^-2 J/m². The pigment nature of the receptor is unknown although it appears not to be a carotenoid derivative.     

EXTRARETINAL PHOTORECEPTION IN BIRDS

Abstract— It is clearly established that in birds, as in the other lower vertebrates, brain photoreception is a critically important perceptual mode. In this review, the variety of extraretinally-mediated responses has been described, and the physiological properties and anatomical location of extraretinal photoreceptors have been briefly discussed. In order to deepen our understanding of its place in the sensory armamentarium, several unresolved problems have been pointed out.     

PHOTOSENSITIVITY IN THE SKIN OF THE LIZARD, Anolis carolinensis

Skin of the lizard, Anolis carolinensis, is remarkably sensitive to light. Illuminated in vitro with visible light from a tungsten source (110 W (m-2)), skin changes from brilliant green to dark brown (50% reduction in reflectance) within 2-4 min as a result of dispersion of melanin from a perinuclear position within dermal melanophores into superficial dendritic processes. Reversal of the process, reaggregation of pigment, will occur within 2.0 min upon return to darkness. This photic response can be initiated with light levels as low as 5.0 W m(-2) and is maximized by light levels only 5% that of midwinter sunshine. Pigment dispersion in response to both melanocyte simulating hormone and to light is inhibited by cytochalasin-B, indicating that microfilaments may be the motor element for pigment movement in that direction. Colchicine, but not cytochalasin-B, totally blocks pigment reaggregation following melanocyte stimulating hormone exposure and partially blocks it in the dark phase of the photic response. The results of this study are consistent with a model for pigment movement in A. carolinensis that provides microfilaments for pigment dispersion and microtubule involvement in both dispersion and aggregation. Finally, because it is readily visible, easily quantified, rapid and reversible, photic response in the skin of A. carolinensis is recommended as a valuable model system for the study of saltatory movement of organelles within cells.     

ACTIVATION OF BENZOPORPHYRIN DERIVATIVE IN THE CIRCULATION OF MICE WITHOUT SKIN PHOTOSENSITIVITY

In vitro experiments with benzoporphyrin derivative monoacid ring A (BPD) confirmed earlier studies that it was taken up rapidly (within 30 min) to maximum concentrations by all cells tested. It was also confirmed that rapidly dividing tumor cell lines and mitogen-activated murine T lymphocytes took up significantly more (5-10-fold) BPD than did normal splenic lymphocytes. Further experiments were undertaken to determine whether BPD could be activated by whole-body irradiation with red light in the blood of animals, shortly after intravenous (i.v.) administration, in the absence of skin photosensitivity. It was found that shaved and depilated mice injected i.v. 60 min earlier with BPD at between 0.5 and 1.0 mg/kg could tolerate 160 J/cm² of broad-band red light (560-900 nm) delivered, at a relatively low rate, over a 90 min time interval without developing skin photosensitivity or general phototoxicity. During the treatment time, plasma levels of BPD were between 0.7 and 1.0 μg/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating BPD. When LI 210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total-body light treatment (160 J/cm² of 590-900 nm light delivered over 90 min), significant reductions in circulating clonogenic tumor cells were observed in blood samples taken immediately following treatment. This indicated that sufficient light was being delivered to BPD in the blood flowing in the peripheral vasculature to effect cytotoxicity to cells containing the photosensitizer without causing either vascular or skin photosensitivity. Thus, activation of this photosensitizer in the circulation can be achieved by transdermal light exposure without causing skin photosensitivity provided that light exposure is performed at a time when the first phase of plasma clearance is complete and when the drug has not yet accumulated in skin.     

INFLUENCE OF THE LOCATION OF TRYPTOPHANYL RESIDUES IN PROTEINS ON THEIR PHOTOSENSITIVITY

The environmental effect on Trp residues photolysis was investigated on four proteins containing a single Trp residue in environments of various polarities: glucagon (exposed residue), nuclease (partially buried residue), RNase T₁ (fully buried residue) and melittin (exposed or partially buried residue depending on the salt concentration). Direct photolysis was performed in neutral N₂-saturated phosphate solution at 20°C using 302 nm monochromatic light. Tryptophan loss was monitored by both absorption and fluorescence spectroscopy and by amino acid analysis. The results suggest that tryptophan photodegradation depends on the location of the residue in the protein, with regard to the exposure to the aqueous medium and to the neighbouring amino acids in the primary amino acid sequence and in the three dimensional structure. Photochemical products were not analysed but fluorescence spectra indicate that they vary with protein.     

AUGMENTATION OF ULTRAVIOLET ERYTHEMA BY INDOMETHACIN IN ACTINIC PRURIGO: EVIDENCE OF MECHANISM OF PHOTOSENSITIVITY

Abstract— The effect of topical indomethacin on the intensity of erythema induced by ultraviolet radiation was measured by reflectance spectrophotometry in six patients with actinic prurigo. The intensity of UV-C erythema was decreased by indomethacin in five patients. The intensity of UV-B erythema was increased by indomethacin in five patients, and UV-A erythema was increased by indomethacin in all patients. The increased inflammatory response induced by UV-B and UV-A with indomethacin application was related to erythemal sensitivity at these wavelengths. Topical indomethacin caused no change in the intensity of UV-A erythema in a group of non-photosensitive subjects.
That inhibition of cyclo-oxygenase augments the inflammatory response to ultraviolet radiation suggests that lipoxygenase metabolites of arachidonic acid may be involved in the mechanism of photosensitivity in actinic prurigo.     

THE ACTION SPECTRUM IN DRUG INDUCED PHOTOSENSITIVITY

Abstract— A new method is proposed for expressing the data obtained from in vivo studies of drug-induced photosensitivity. It is shown that consideration of the underlying normal photobiological responses that occur in the absence of the photosensitising drug can yield much closer agreement between the in vitro absorption spectrum of the drug and the action spectrum obtained in photosensitised skin than is apparent from many published reports.     

MULTIPLE ACTION OF FAR-RED LIGHT IN PHOTOPERIODIC INDUCTION and CIRCADIAN RHYTHMICITY

Abstract— It is generally accepted that phytochrome influences the photoperiodic induction of flowering through its interaction with the circadian clock mechanism. We have attempted to separate the effects of phytochrome on the clock mechanism from those that mediate flowering directly by examining a number of responses that are unrelated to flowering but are also regulated by the circadian clock. Gas exchange measurements of both CO₂ and H₂0 vapor were monitored under light conditions (200 μmol m ² s⁻¹) where the addition of far-red energy is required for the maximal promotion of flowering. In addition, photosynthetic capacity and maximal transpiration rates were measured in plants grown under continuous dim (20 μmol m⁻² S⁻') light, with or without supplemental far-red, by exposing them briefly to saturating fluxes (1000 μmol m⁻² s^-l) of light. Net CO₂ fixation was very weakly rhythmic in plants grown under both high and low light and this weak oscillation was completely suppressed by far-red light. Far-red also suppressed the rhythm in transpiration under high light, but the rhythm was immediately reinstated when the far-red light was removed. The phase of this rhythm was also reset with the next peak always occurring15–18 h after the far-red was turned off. When grown under dim light, the transpiration rhythm was not suppressed and the amplitude of the oscillation was more than doubled. Far-red light appears to interact with the rhythm in transpiration in a manner suggesting that the stomatal rhythm may be coupled to the same clock oscillator that regulates the flowering rhythm.     

PHOTOSENSITIVITY OF CERIC ION INITIATED ACRYLAMIDE POLYMERIZATION

<正> Polymerization of acrylamide initiated by ceric ammonium nitrate alone has been studied in aqueous medium. The effects of UV light irradiation on the initial rates of polymerization, the activation energy and on the polymer molecular weights have been investigated. Compared with that in the dark, the rate of polymerization under UV light was accelerated to eleven times higher, and the overall activation energy was lowered markedly.     

A DIURNAL RHYTHM IN THE RATE OF LIGHT-INDUCED ELECTRON FLOW IN MAIZE BUNDLE SHEATH STRIPS

Abstract— A technique for the rapid mechanical isolation of bundle sheath strips from Zea mays was used to study the rate of light-induced electron flow as a function of time of day. Seedlings were raised for 10 days in light/dark cycles and then exposed to various intensities of light. The rate of light-induced electron flow was measured five times during the 24-h cycle using a whole cell, H₂O to methyl viologen assay in the presence of gramicidin. The uncoupled rate of electron flow reached a maximum at midday and then decreased for the remainder of the cycle.
If at the end of the 14-h dark period plants were either maintained in darkness, or exposed to very low irradiance white light. then no rhythmicity was observed. If continuous light at higher irradiance was used, then only one additional peak of activity was observed. The minimal light irradiance necessary to allow the continued expression of the rhythm in light/dark cycles is in the range between 11 to 54 W m_-2. A 15-min bright-light (54 W m_-2) pulse administered at dawn is not sufficient to stimulate the expression of the rhythm indicating that the light during the light/dark cycle is doing more than just acting as a "light-on" synchronizing signal. An imposed dark period is also necessary if the rhythm is to be expressed for more than one cycle.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

AFLOQUALONE PHOTOSENSITIVITY. IMMUNOGENICITY OF AFLOQUALONE-PHOTOMODIFIED EPIDERMAL CELLS

Abstract Afloqualone (AQ) is an oral muscle relaxant and evokes ultraviolet A (UVA)-induced photosensitivity dermatitis as a side effect. Histologic observations of the skin eruption suggest that AQ photosensitivity is mediated not only by phototoxic but also by photoallergic mechanisms. To address the immunological mechanisms of AQ photosensitivity, we examined the immunogenicity of AQ-photomodified epidermal cells in mice. Afloqualone was covalently coupled with bovine serum albumin by irradiation with UVA but not UVB. Because of this ability of AQ to photobind to protein, murine epidermal cells were easily modified with AQ by exposure to UVA. Subcutaneous inoculation of AQ-photomodified epidermal cells successfully induced an antigen-specific delayed-type hypersensi-tivity in mice. These findings suggest that AQ-photoderivatized epidermal cells are highly immunogenic and that photomodification of epidermal cells with AQ is an initial event to evoke AQ photosensitivity dermatitis.     

PHOTOSENSITIVITY IN CAROTENOIDLESS MUTANTS OF MICROCOCCUS LUTEUS (SARCINA LUTE A): NON-PHOTODYNAMIC MENAQUINONE DESTRUCTION IN WHITE LIGHT

–The photosensitivity of a carotenoidless mutant strain has been examined in terms of its loss of viability and loss of menaquinone when subjected to high intensity visible light. Cell viability and menaquinone are protected by cyanide (an inhibitor of cytochrome oxidation), by malate (a substrate for the respiratory chain) and by nitrogen although the protection of menaquinone is less in nitrogen. Evidence is presented in support of the view that oxidised membrane-bound menaquinone is more photolabile in visible light than the reduced form. Loss of menaquinone is seen as the primary cause of death in these obligately aerobic carotenoidless cells and the role of oxygen as indirect by converting reduced menaquinone to the oxidised form. Evidence is also presented that the cause of cell death in red light in the presence of an exogenous photosensitizer, toluidine blue, is different from that in white light.     

PHOTODYNAMIC ACTIVITIES and SKIN PHOTOSENSITIVITY OF THE bis(DIMETHYLTHEXYLSILOXY)SILICON 2,3-NAPHTHALOCYANINE IN MICE

The photodynamic therapy (PDT) activity of the bis(dimethylthexylsiloxy)silicon 2,3-na-phthalocyanine (SiNc 8 ) was evaluated against the EMT-6 tumor implanted intradermally in BALB/c mice. The SiNc 8 was formulated in aqueous emulsions based on Cremophor EL or Solutol HS 15. The formulation was shown to affect plasma clearance and overall pharmacokinetics. Compared to Cremophor, Solutol promoted rapid plasma clearance and high liver retention of the dye, combined with a slight increase of dye tumor concentrations. The PDT action spectrum for tumor response of SiNc 8 in Cremophor (190 mW cm², 200 J cm², 24 h postinjection [p.i.] of 1 (jimol kg¹) showed a maximum at 780 nm, which corresponds to the absorption maximum of the monomelic dye as well as the in vivo maximum change in the “diffuse optical density” produced by the dye. The extent of tumor necrosis increased with augmented dye and light doses. Regardless of the formulation, at 1 h p.i. of 0.1 μmol kg^?! SiNc 8 , PDT efficiency (190 mW cm'², 400 J cm²) was high but accompanied by severe damage to normal tissues, at 24 h PDT resulted in complete tumor regression in 80% of the animals without adverse effects to adjacent tissues, while at 72 h p.i. PDT induced no tumor response with Cremophor and only a partial response with Solutol. At the latter time point, plasma dye clearance was nearly complete while tumor tissue levels remained high, suggesting that tumor response correlates with plasma rather than tumor dye levels. Skin sensitivity of SKhl mice to solar-simulated radiation was lower with SiNc 8 as compared to Photofrin®. Our data suggest the potential of SiNc 8 as a far-red absorbing photosensitizer in clinical PDT.     

SKIN PHOTOSENSITIVITY AND PHOTODESTRUCTION OF SEVERAL POTENTIAL PHOTODYNAMIC SENSITIZERS

The major side effect associated with porphyrins (Photofrin II) in clinical photodynamic therapy is skin photosensitivity. In order to avoid this deleterious reaction, patients must remain out of the sunlight for approximately 1 month. A possible procedure to reduce the amount of skin photosensitivity is to photodegrade (photobleach) the compound in the skin. In this study, we report a series of experiments describing the photodegradation rates of two photosensitizers currently receiving attention due to their potential for use in PDT (mono L-aspartyl chlorin e6 and chloroaluminum sulfonated phthalocyanine). These compounds are compared to Photofrin II (PfII). Experiments consisted of determining photodegradation rates and efficiencies of the sensitizers in (i) phosphate buffered saline (PBS), (ii) PBS with fetal calf serum (to enhance absorption and simulate cellular binding or deaggregation), (iii) Chinese Hamster Ovary cells, and (iv) Balb/c mice. We performed two standardized skin sensitivity assays using the Hartely albino guinea pig irradiated with a UV blue point lamp and Balb/c mice irradiated with the therapeutic wavelength of each sensitizer. In addition, we performed a cell clonogenicity assay comparing photodegraded and fresh PfII on CHO cells. The photodegraded PfII exhibited significant phototoxicity, although the fluorescence was bleached by more than 70%. The results show that PfII causes major skin photosensitization and that the other compounds produce no substantial skin sensitivity. Our studies suggest that photodegradation of PfII with 630 nm light has little influence on the phototoxicity of the compound. In addition, skin sensitivity was not alleviated with prior photobleaching with 405 nm light.