IN VIVO MEASUREMENTS OF THE PHYTOCHROME PHOTOSTATIONARY STATE IN FAR RED LIGHT

Abstract— The in vivo photostationary state, φ_fr= ([ P _fr]_fr/[ P ]), of phytochrome in far red light has been determined in mustard seedling cotyledons by three different methods. The φ_fr is a function of the length of time of etiolation ( t = 36 hr, φ_fr= 0·14; t = 72 – 120 hr, φ_fr= 0·075). The calculated φ_r= 0·8. The amount of P _tot is strongly dependent on the time of onset of far red light. These data imply that it would be almost impossible to maintain a constant level of P _fr in mustard cotyledons over a considerable period of time.     

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF PHOTOLYASE MOLECULES IN Saccharomyces cerevisiae CELLS–I. EFFECTS OF TEMPERATURE AND LIGHT

Abstract— The effects of temperature and light on the cellular concentration of photoreactivating enzyme (PRE) molecules in haploid Saccharomyces cerevisiae cells were investigated. (1) Temperature effect: The number of active PRE molecules per cell (N_PRE) in cells grown at 37°C was about 13% of that grown at 23°C, although the amount of proteins per cell remained the same. (2) Light effect: N_PRE in cells grown in light was about 2.8 times larger than that grown in the dark. The value of N_PRE in cells grown in the light decreased more rapidly during holding in buffer in the dark than in the light. The N_PRE decrease during holding in buffer in the dark was more rapid in cells grown in the light than grown in the dark. A comparable decrease was observed after holding in buffer in the presence of cycloheximide. (3) In cells harboring a plasmid containing the gene PHR1, N_PRE was larger in cells grown at 23 than at 30°C.     

THE EFFECT OF RED AND FAR RED LIGHT ON CAROTENOID AND CHLOROPHYLL FORMATION IN PEA SEEDLINGS

Abstract— Treatment of etiolated pea seedlings with a short exposure to red light caused a stimulation of growth (size and dry wt production) and carotenoid synthesis during the following 48 hr compared with seedlings kept entirely in darkness.The effect is nullified by a following dose of far red light and thus the phenomenon is probably phytochrome-controlled.
Similar treatment with red light one hour before continuous illumination with white light tended to reduce the lag period for chlorophyll synthesis.Again a following dose of far red light reversed this response.     

RED LIGHT INTERACTIONS WITH BLUE AND ULTRAVIOLET LIGHT IN POLAROTROPISM OF GERMLINGS OF A FERN AND A LIVERWORT

Abstract— In polarotropism of the chloronema of the fern Dryopteris filix-mas (L.) Schott and of the germ tube of the liverwort Sphaerocarpos donnellii Aust. a phytochrome action in blue and u.v. was presumed[1, 2]. In the present paper this assumption was tested by simultaneously irradiating with red and blue, and red and near u.v. Red energy is given to shift the phytochrome photoequilibrium in favour of high P _fr/ P ^total concentrations. The data obtained by simultaneous irradiation are consistent with the predictions made under the assumption of a phytochrome involvement in the blue- and u.v.-mediated polarotropic response.     

REPAIR OF UV-DAMAGED INCOMING PLASMID DNA IN Saccharomyces cerevisiae

A whole-cell transformation assay was used for the repair of UV-damaged plasmid DNA in highly transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities. Six rad alleles were selected from the three epistasis groups: rad 1-1 and rad2-1 from the RAD3 group, rad6-1 and rad18-2 from the RAD6 group, and rad52-1 and rad54-1 from the RAD52 group. Cells carrying single, double and triple rad alleles were transformed to uracil prototrophy by centromeric plasmid DNA (YCp19) modified in vitro with UV (254 nm). Surviving fractions were calculated as the number of transformants at each fluence relative to the number of transformants with unirradiated plasmid DNA. The sensitivity of incoming DNA in single rad mutants shows that most repair is carried out by excision repair and a RAD18-dependent process. In the rad52-1 host, the sensitivity of incoming DNA was intermediate between those found in RAD+ and rad2-1 hosts, suggesting the involvement of a recombinational repair process. Non-epistatic interactions were observed between rad alleles belonging to different epistasis groups. This provides validation for the classification of the three epistasis groups concerning the repair of chromosomal DNA for UV-incoming DNA. In both rad1-1 rad6-1 and rad1-1 rad18-2 rad54-1 hosts, the mean fluence for one lethal event corresponds approximately to one pyrimidine dimer per plasmid molecule, indicating that they are absolute repairless hosts for incoming DNA. A comparison between cell and plasmid survival reveals that there are differences in the repairability of both chromosomal and incoming DNA. The large effect of rad6-1 mutation on cell survival and the small effect on incoming DNA suggest that, in the RAD+ strain, the RAD6 product may be essential for the repair processes which act on chromosomal DNA, but not for those which act on incoming DNA. It is proposed that in yeasts postreplication repair of incoming DNA is limited to supercoiled molecules with 1-2 pyrimidine dimers that can initiate replication.     

RAPID STOMATAL RESPONSE TO RED LIGHT IN Zea mays

Gas exchange techniques were employed to study responses of stomatal conductance to pulses of red and blue light in the grass, Zea mays. Zea mays exhibited conductance increases following brief (< 1 min) pulses of either red or blue light, in contrast to other species (e.g. Commelina communis; Assmann, 1988, Plant Physiol. 87 , 226–231) that exhibit consistent conductance responses only to pulses of blue light. Red light pulses of 450 μmol m^?2s^?1 for x min or 225 μmol m^?2s^?1 for 2x min were used to probe the fluence dependence of the red light response. The red light-stimulated conductance increase was constant for a given fluence, and increased with increasing total fluence. The conductance response to red light was larger in field grown plants (maximum growth irradiance ? 1600 μmol m-²s^?l) than in plants raised in growth chambers (maximum growth irradiance ? 150 μmol m^?2s^?1).     

PHOTOXIDATION OF ASCORBATE TO OXALATE AND THREONATE MEDIATED BY CHLOROPLASTS IN FAR RED LIGHT

Abstract— Chloroplasts illuminated with light absorbed predominantly by Photosystem I can mediate the photoxidation of ascorbic acid to threonic and oxalic acids. Two micromoles of oxygen are consumed for each micromole of ascorbic acid added. The kinetics of this endogenous chloroplast system closely resemble the kinetics of an artificial flavin-manganese-catalase system in which flavin is the photosensitizing dye.     

REGULATION OF CHLOROPLAST DEVELOPMENT BY RED AND BLUE LIGHT

There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill-activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.     

COMPARISON OF THE EFFICACY OF PULSED AND CONTINUOUS-WAVE RED LASER LIGHT IN INDUCTION OF PHOTOCYTOTOXICITY BY HAEMATOPORPHYRIN DERIVATIVE

Abstract— The efficiency of pulsed and continuous wave laser light to induce photodynamic activity in haematoporphyrin derivative (Hpd) was compared in two systems, a tissue culture assay and a transplantable mouse tumour. No difference was found.     

SYNTHESIS AND SECRETION OF HUMAN ATRIAL NATRIURETIC PEPTIDE IN Saccharomyces cerevisiae

A chemically synthesized α-hANP gene was inserted into plasmid YFD18, which was an expression-secretion vector of yeast. The recombinant then transformed in the yeast Y33. The expression level of yeast transformants was about 700 μg ANP/L detected by RIA. More than 99% of expression products were secreted in the culture medium. N-terminal analysis of purified product showed that the first 4 amino acid residues of α-hANP were deleted.     

STIMULATION OF Escherichia coli DIVISION BY LOW-INTENSITY MONOCHROMATIC VISIBLE LIGHT

Abstract— Bacterial suspensions in a phosphate buffer were irradiated at 364, 404, 434, 454, 484, 520, 560. 570, 599, 619, 630, 633, 640, 693, 730 and 750 nm, incubated in Hottinguer broth, and assayed for viability by the standard surface plating technique. When irradiated at 404, 454, 560, 619, and 750 nm, the cells started dividing more rapidly, the growth stimulation effect reaching170–180% 60 min after irradiation. The shape of the action spectrum and the reduced-minus-oxidized difference spectrum of the bacterial suspensions suggest that respiratory chain components of the bacterium such as dehydrogenases and cyt d might be the photoacceptors involved. The growth stimulation effect is believed to be due to the ability of the irradiation-activated respiratory chain to produce the necessary pH gradient more rapidly, thus enabling the cells to start dividing earlier.     

THE EFFECT OF RED AND FAR-RED LIGHT ON NUCLEIC ACID METABOLISM IN RYE SEEDLING SHOOTS

Abstract— The quantitative changes in the levels of RNA and DNA in shoots treated with red and/or far-red light are presented.
The results indicate that red light, but not far-red light, stimulated cell division. The changes in RNA are discussed in relation to the nature of the light treatment.     

可见光照射下染料茜素红的光催化降解机理

研究了 可见光（ ≥４２ ０ ｎ ｍ ） 照射下 染料茜素 红在空气 饱和 的 Ｔｉ Ｏ２ 水溶 液中 发生 的光 催化降解过 程． 借 助紫外 、核磁、顺 磁、红外 、色质 联用等手 段,发现 染料茜素 红的光 催化 降解 过程 符合 Ｌａｎｇ ｍ ｕｉｒ Ｈｉｎｓｈｅｌｗ ｏｏｄ 模型,在 降解过程 中产 生了 过氧 化 物． 核磁 、红 外和 色质联 用等 结 果证 实,主要降解 中间产 物为邻苯 二甲酸 ． Ｃ Ｏ Ｄ 测试 结 果表 明, 其矿 化产 率 达３ ０ ％ , Ｅ Ｓ Ｒ 结 果证 实, 在降解过程 中有 ⒒ Ｏ Ｈ 自由基 生成,说 明其与 染料的降 解机理有 密切联 系．     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

METABOLISM OF 5-METHOXYPSORALEN BY Saccharomyces cerevisiae

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.     

POSSIBLE INVOLVEMENT OF MEMBRANE DAMAGE IN THE IN ACTIVATION BY BROAD-BAND NEAR-UV RADIATION IN Saccharomyces cerevisiae CELLS

Multiple cellular effects of near-UV radiation (300-380 nm) on inactivation, disruption of the permeability barrier and induction of gene conversion at the trp 5 locus were simultaneously measured in the same culture of a diploid strain of the yeast Saccharomyces cerevisiae in order to assess the critical lethal damage. Inactivation of exponential phase cells in water appeared to be closely related to the disruption of the permeability barrier. Inactivation and membrane damage were remarkably oxygen dependent, whereas the induction of genetic changes was very low and dependent much less on oxygen. The dependence on the temperature for inactivation and membrane damage was both low, conforming with the expectation that the processes are mainly photochemical and not enzymatic. These features are very contrasted with the characteristics of far-UV radiation effects. Possible involvement of membrane damage in near-UV inactivation of exponential phase yeast cells is discussed.     

CHARACTERIZATION OF THE RED LIGHT INDUCED REDUCTION OF A PARTICLE ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Methylene blue transfers electrons to a membrane-associated b-type cytochrome in particulate fractions from corn coleoptiles. The K_m for methylene blue is less than 1 µM under optimal conditions. This reaction is destroyed by boiling, but not by 7 M urea. Kinetic analyses of the influence of light intensity on cytochrome reduction suggest that a first order photochemical reaction is limiting. Free EDTA may serve as an electron donor in this system at least at high methylene blue and protein concentrations. The photoactivity does not coincide either with mitochondrial or endoplasmic reticulum markers, and may be localized in plasma membrane. There is an estimated 5 times 10^-10 mol photoreducible cytochrome per g coleoptile tissue. Studies on the effect of pH on the reaction in the presence of methylene blue or thionine indicate that dye photoreduction itself is not rate-limiting. Wavelength dependence studies suggest that it is methylene blue monomer and not dimer which mediates the reaction. Although oxygen is apparently required for the reaction, neither superoxide nor excited singlet oxygen appear to be involved. The reaction mechanism is still unknown.     

TRANSPORT OF LIGHT IN TISSUE IN PHOTODYNAMIC THERAPY

Abstract The dose rate in photodynamic therapy is proportional to the energy fluence rate and the concentration of the photosensitizer. Calculations of the energy fluence rate have been performed in slab, cylindrical and spherical geometries with the discrete ordinates transport method and diffusion theory. The attentuation of the energy fluence rate is least in slab geometry and greatest in spherical geometry. Violet (405 nm) light is attenuated much more rapidly than red (630 nm) light. Small tissue dimensions or narrow beam irradiation further decrease the energy fluence rate with radius and depth. Anisotropic scattering increases the energy fluence rate at large depths, but decreases it near the source. Measurements of the absolute energy fluence rate vs depth in a mouse tumor model exhibit an order of magnitude attenuation through the skin and a 3 mm thick tumor. Calculations of the energy fluence rate of the DHE fluorescence have been carried out to guide measurement of the concentration. Violet light excitation is much more efficient than red light excitation.     

IMPAIRMENT OF RED CELL MEMBRANE CYTOSKELETON BY PROTOPORPHYRIN-IX: LIGHT AND DARK EFFECTS

Abstract— Membrane cytoskeletons were separated by use of TritonX–100 from freshly isolated red cells, fixed with glutaraldehyde and their morphology was followed by scanning electron microscopy. At 37°C and pH 6.5 cytoskeletons retained cell-like shapes for at least 2 h, but at higher pH values, they lost stability after 30 min, appearing as amorphous protein material. Irradiation in the presence of1–20 μ M protoporphyrin-IX at pH 6.5 caused crosslinking of the proteins when organized as cytoskeletons, but not when separated. Scanning electron microscopy also revealed that the cytoskeletal proteins conserved their cell-like shape even at pH values higher than 7.0. It was concluded that illumination in the presence of porphyrin causes membrane rigidity by crosslinking of the cytoskeletal proteins, and their sensitivity to crosslinking is the result of their mutual arrangement in the membrane. At concentrations higher than 100 μ M protoporphyrin-IX induced, even in the absence of light, the opposite effect, namely dissociation of the cytoskeletal proteins. The data suggest that the changes observed in this study provide an explanation for both dark and light induced injuries of red cells in porphyria disorders.     

THE PHOTOBIOLOGY OF Paphiopedilum STOMATA: OPENING UNDER BLUE BUT NOT RED LIGHT

Abstract— The responses of stomata from Paphiopedilum harrisianum , Orchidaceae, to light and CO₂ were studied in epidermal peels. Stomatal opening under red light was indistinguishable from that in darkness, whereas blue light promoted opening above dark levels. The ineffectiveness of red light in causing stomatal opening was confirmed in the presence of 100 μ M KCN; average apertures in both darkness and red light were 53% of those measured in the absence of the inhibitor, whereas under blue irradiation, the KCN inhibition was only 30%, with average apertures two-fold of those measured under red light or darkness. Fluence rate response curves under blue light were typical of a single photoreceptor; removal of CO₂ increased aperture values without a significant light-CO₂ interaction. The lack of a stomatal red light response contrasts with results obtained in species with chlorophyllous stomata in which red light consistently causes stomatal opening, and suggests that the previously reported red light responses in stomata from intact Paphiopedilum leaves resulted from indirect effects, such as depletion of intercellular CO₂ by mesophyll photosynthesis. In isolation, Paphiopedilum stomata appear to rely on a blue light photosystem for their responses to light and fail to open under red light because of their lack of guard cell chloroplasts.