RETARDATION OF ULTRAVIOLET LIGHT ACCELERATED CHLOROSIS BY VISIBLE LIGHT OR BY BENZYLADENINE IN NICOTIAN A GLUTINOSA LEAVES: CHANGES IN AMINO ACID CONTENT AND CHLOROPLAST ULTRASTRUCTURE*

Abstract— Low doses (180–720 Jm^-2) of ultraviolet light (254 nm) are known to accelerate the chlorosis of detached leaves in darkness. The development of such chlorosis is prevented by a photoreactivation treatment. However, we found that delayed light exposure or benzyladenine treatments (which were not effective in photorepair of UV-induced thymine dimers in cell DNA) were also effective in retarding the UV-accelerated chlorosis. Small drops of benzyladenine solution placed on the UV irradiated leaf formed green islands which acted as strong sinks for the accumulation of free amino acids during dark incubation. To a lesser degree, non–irradiated green tissues surrounded by irradiated yellow leaf tissue also acted as sinks for amino acid accumulation. The accelerated chlorophyll loss in UV-irradiated leaves was correlated with degradation of chloroplast ultrastructure. Visible light or benzyladenine retarded this chloroplast degradation. The accelerated senescence of UV irradiated leaf tissue, therefore, is ultrastructurally and physiologically similar to normal senescence of detached dark-incubated leaves, but progresses at a faster rate. When the lower leaf surface was irradiated with high UV doses (3600–10,800 Jm^-2), the chloroplast ultrastructure of the spongy cells (except the envelope) was preserved for 3 days after dark incubation. However, the chloroplasts of the palisade cells were in a late stage of senescence. Since the spongy cells were dead (plasmalemma, tonoplast and chloroplast envelope disappeared), the maintenance of green color and ultrastructure of chloroplasts could have been due to inhibition of degrading enzymes normally associated with senescence.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

PHOTOSENSITIZATION BY ANTITUMORAGENTS–6. PRODUCTION OF SUPEROXIDE RADICAL AND HYDROGEN PEROXIDE DURING ILLUMINATION OF DIAMINOANTHRACENEDIONES IN THE PRESENCE OF NADH IN AQUEOUS SOLUTIONS: AN EPR STUDY

Abstract— Photosensitizing capabilities of anthracenedione anticancer agents to oxidize NADH in aqueous solutions have been studied by EPR and spin trapping techniques. It is demonstrated that 1,4-diamino substituted anthraquinones, like mitoxantrone and ametantrone, do not photosensitize NADH oxidation while 1,5- and l,8-bis[[(diethylamino)ethyl]amino]anthraquinones do, undergoing simultaneous one-electron reduction to their semiquinone radical forms upon illumination with visible light. In aerated aqueous solutions the reaction leads to the production of superoxide ion and hydrogen peroxide.     

PHOTOEXCITATION OF RHODOPSIN: CONFORMATION CHANGES IN THE CHROMOPHORE, PROTEIN AND ASSOCIATED LIPIDS AS DETERMINED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsin's function is to transduce the light induced isomerization (ll-cis to all-trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen-out-of-plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all-frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.     

In vitro PHOTODYNAMIC EFFECTS OF LYSYL CHLORIN p6: CELL SURVIVAL, LOCALIZATION AND ULTRASTRUCTURAL CHANGES

The in vitro cell survival, localization and ultrastructural changes following irradiation were examined in 9L glioma cells sensitized with a new photosensitizer, lysyl chlorin p₆ (LCP). In clonogenic assays, LCP was 10–100-fold more phototoxic than photofrin II on a μg/mL basis. Lysyl chlorin p₆ uptake was blocked when cells were incubated at 2°C. In view of the chemical properties of LCP, this finding indicates that uptake probably occurred through the endocytic pathway. Fluorescence studies showed LCP localized in a region of the endocytic compartment similar in size, shape and distribution to that labeled by lucifer yellow CH (LY), as well as localizing diffusely throughout the perinuclear cytoplasm. Cells stained with both LY and LCP, however, had distinctly separate regions of staining. Lysyl chlorin p₆ localization differed from that of fluorescent probes labeling the mitochondria, Golgi apparatus and endoplasmic reticulum. Ultrastructural changes at both 2 and 30 min after laser irradiation were similar. Mitochondria were often condensed or swollen and also had constrictions and cytoplasmic invaginations. The Golgi apparatus, perinuclear space and rough endoplasmic reticulum (RER) were dilated. These data demonstrate that LCP localizes in a portion of the endosomal compartment, but that morphologic damage initially occurs in the mitochondria, Golgi apparatus and RER.     

SOME RELATIONSHIPS BETWEEN ULTRAVIOLET LIGHT AND HEME-PROTEIN-INDUCED PEROXIDATIVE LIPID BREAKDOWN IN LIPOSOMES, AS REFLECTED BY FLUORESCENCE CHANGES: THE EFFECT OF NEGATIVE SURFACE CHARGE

Abstract— The water soluble, photolabile nitrene precursor,azidonaphthalene–2,7-disulfonic acid (ANDS) was encapsulated in small unilamellar, isoelectric (egg PC) or negatively charged (egg PC + dihexadecylphosphate) liposomes. The individual and combined effects of heme-proteins and UV irradiation on the fluorescence of these vesicles under aerobic conditions were studied. Consistent with the catalytic action of heme-proteins on lipid peroxidation and peroxide decomposition, addition of cytochrome c (positively charged) or catalase (negatively charged) to the vesicles elicited immediate formation of a fluorescence band at 470 nm, characteristic of Schiff bases that form from aldehyde byproducts of decomposing hydroperoxides. Ultraviolet irradiation of liposomes for 5 min caused no significant changes in the fluorescence spectrum, in spite of the radiolysis of ANDS inside the vesicles with consequent formation of nitrene radicals. When isoelectric vesicles were irradiated with UV light in the presence of cytochrome c or catalase, Schiff base formation was further increased by2–3 fold, which effect was not observed in the absence of internal ANDS, or in the presence of negative surface charge on the vesicles. These findings suggest that (a) UV irradiation, by itself, cannot trigger lipid decomposition even when it is assisted by photoproduced nitrene radicals, (b) there is a ternary synergism between UV light, heme-proteins and nitrene radicals in promoting peroxidative lipid breakdown, and (c) negative surface charge inhibits the above synergism, which effect is unlikely to be due to electrostatic interaction between the vesicles and the protein or the ANDS.     

MULTI-SITE PHOSPHORESCENCE OF α, β-ENONES:LEVEL INVERSION IN 6β, 19-EPOXYCHOLEST-4-EN-3-ONE BY SOLVATION AND AGGREGATION

Millisecond time-resolved emission spectroscopy was used to probe the phosphorescence kinetics of the α-β-enone 6β, 19-epoxycholest-4-en-3-one (1) as a function of concentration in several paraffinic and hydroxylic glasses at 77 K. Only in methylcyclohexane/methylcyclopentane glass at low concentration (10^?4M) does the phosphorescence decay exponentially. It is interpreted as emission from the ³n* state. Upon increasing the concentration a second emission grows which is characterized by a longer lifetime, a decreased fine structure and a hypsochromically shifted S₀→¹nπ* excitation spectrum. This phosphorescence is ascribed to ³ππ* emission of aggregates of 1. In hydroxylic glasses the phosphorescence decay is multiexponential, even at 10^?4M concentration; from emission band shapes and lifetimes it follows that both ³nπ* and ³ππ* type emissions are present, the latter increasing with the alcohol concentration in the solvent. The two types of phosphorescence have different excitation spectra: that of the structureless and long-lived ³ππ* emission is shifted to the blue in the S₀→¹nπ* region and to the red in the S⁰→¹ππ* region. This emission is ascribed to complexes of 1 with the alcoholic solvent. The results of time-resolved measurements of the circular polarization of the luminescence are consistent with the assignments given above and indicate that in the H-bonded and possibly also in the free species ³ππ* and ³nπ* states are intermixed to a considerable extent.