COMPARISON OF PHOTODYNAMIC ACTION BY ROSE BENGAL IN GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA

Abstract— The photodynamic inactivation by illuminated Rose Bengal of a number of bacterial species was compared. The gram-positive species, Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis and Streptococcus salivarius, were inactivated about 200x more quickly (99% inactivation) than a Salmonella typhimurium wildtype strain. The Salmonella inactivation curve exhibited an initial lag time during which bacteria were not significantly inactivated. The lag time for inactivation of a derivative of the wildtype Salmonella strain that is deficient in a large portion of its cell wall lipopolysaccharide coat was approximately half of the lag time for the wildtype strain but the subsequent rate of inactivation was approximately the same for the two strains. Dark preincubation of both Salmonella strains with Rose Bengal before illumination shortened the lag time, but did not increase the final rate of inactivation. Dark preincubation prior to illumination did not measurably change the inactivation curve of the gram-positive species. The lag time observed in the inactivation curves for Salmonella bacteria may reflect the time required for penetration of the Rose Bengal anion through the outer portion of the gram-negative cell wall to a critical location within the cell for effective photosensitization.     

A PHOTON COUNTING TV CAMERA EQUIPPED WITH AN IMAGE GUIDE FOR RAPID DETECTION AND COUNTING OF SINGLE BACTERIA IN A WIDE FIELD

Abstract— We developed a photon counting TV camera system, which uses an image guide consisting of tapered optical fibers, for the rapid detection and counting of microscopic sized luminous particles in a wide field. Using a luminous bacterium as a standard, we compared this image guide-coupled TV camera to a lens-coupled TV camera by determining their light collecting powers and detection abilities for single bacteria on a membrane filter. The image guide shortened the detection time by an order of magnitude, making the photon counting TV camera a practical system for rapid counting of bacteria.     

A POSSIBLE CONTROL BY PHYTOCHROME and OTHER PHOTORECEPTORS OF PROTEIN ACCUMULATION IN THE GREEN ALGA Ulva rigida

Abstract— The effects of red, blue and green light pulses on total protein accumulation in the green alga Ulva rigida following transfer from a low to high nitrate medium in darkness were examined. Red light pulses prior to transfer to darkness increased protein accumulation by about 55%. Blue and green light pulses also stimulated protein accumulation, but to a lesser extent (40-30% respectively). Stimulation of protein accumulation by red, blue or green light was largely (red light) or partially (blue or green light) reversible by far-red. The role of phytochrome and a red/green photoreversible system in the control of protein synthesis is discussed.     

PHOTODYNAMIC EFFECTS INDUCED BY FUROCOUMARINS ON A MEMBRANE SYSTEM. COMPARISON WITH HEMATOPORPHYRIN

Abstract Isolated rat liver mitochondria have been used as a model to investigate the photodynamic action of psoralen (Ps) and 4,5',8-trimethylpsoralen (TMP) on cellular membrane systems in comparison with hematoporphyrin (Hp). Oxidation was detected by the consumption of free oxygen as measured polarographically in the respiratory chamber when irradiated with UV light (320-380 nm). In the presence of Ps, singlet oxygen was produced in the respiratory medium but neither the respiration nor the oxidative phosphorylation were affected. On the contrary the hydrophobic derivative TMP impaired the respiration with rapid uncoupling of oxidative phosphorylation as did Hp. The ineffectiveness of Ps as well as the effectiveness of TMP vs. Hp is explained on the basis of photophysical properties of the molecules and their partition coefficient. These results may indicate that, in the photochemiotherapy of skin diseases, furocoumarins can drive photodynamic reactions at various subcellular levels according to their hydrophobicity.     

石墨管原子吸收法测定原子簇化合物中钼铁含量

不久前,我们研究了火焰原子吸收测定Mo-Fe-S原子簇化合物中钼铁元素的方法,收到了较好的效果^[1]。但待测液中铁的含量小于20μg/ml时,铁对钼测定所产生的正干扰消除得不理想。并且当钼、铁浓度小于10μg/ml时,测量的准确度较差.为了克服以上不足,我们研究了石墨管原子吸收法测定低含量钼、铁元素的方法,取得了满意的结果.     

TIME-RESOLVED STUDIES OF SINGLET-OXYGEN EMISSION FROM L1210 LEUKEMIA CELLS LABELED WITH 5-(N-HEXADECANOYL)AMINO EOSIN. A COMPARISON WITH A ONE-DIMENSIONAL MODEL OF SINGLET-OXYGEN DIFFUSION AND QUENCHING

Abstract— Time-resolved measurements were made of near-infrared emission from 5-( N -hexadecanoyl)amino-eosinlabeled L1210 leukemia cells following pulsed-laser excitation. The cells were suspended in phosphate-buffered saline made with deuterium oxide solvent. A significant fraction of the emission occuring10–80 μs after the laser pulse was due to singlet oxygen. This singlet-oxygen emission is believed to result from singlet oxygen generated near the cell-membrane surface, where 5-( N -hexadecanoyl)amino eosin is known to concentrate, and then diffusing out into the buffer. The intensity and the kinetics of the experimentally observed singlet-oxygen emission were in excellent agreement with the predictions of a theoretical one-dimensional model of singlet-oxygen diffusion and quenching.
During the10–80 μs time period studied, most of the singlet oxygen was located in the buffer. Thus, the use of water-soluble singlet-oxygen quenchers, such as histidine, provide one means of separating the singlet-oxygen emission quenchers, such as histidine, provide one means of separating the singlet-oxygen emission from other sources of light during this time interval.     

ENHANCEMENT OF PHOTOREPAIR OF ULTRAVIOLET-INDUCED PYRIMIDINE DIMERS BY PREILLUMINATION WITH FLUORESCENT LIGHT IN THE GOLDFISH CELL LINE. THE RELATIONSHIP BETWEEN SURVIVAL AND YIELD OF PYRIMIDINE DIMERS

The enhancement of photorepair of UV-induced pyrimidine dimers by preillumination with fluorescent light, previously reported with RBCF-1 cells derived from caudal fin of a goldfish, was studied in terms of clonogenic ability and yields of dimers. In the logarithmic growth phase, the ability of photorepair increased with the time after preillumination, reached a maximum at 8 h, and gradually declined. At 8 h, the dose decrement with the photorepair-treatment for 20 min at 7.5 J/m2 UV increased by preillumination for 1 h from 1.6 to 3.1 J/m2 in terms of restoration of survival and from 1.2 to 4.3 J/m2 in terms of the disappearance of dimers. Incubation of the preilluminated cells in the medium containing cycloheximide (0.5 microgram/mL) after preillumination until UV-irradiation diminished their enhancement of photorepair. In the density-inhibited state, the ability of photorepair was higher than in the log phase, and it was hardly enhanced by preillumination.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-II. PHOTOSENSITIZATION BY CHLOROPHYLL,PHEOPHYTIN AND BACTERIOCHLOROPHYLL OF A ONE-ELECTRON TRANSFER REACTION BETWEEN ETHANOL AND QUINONE*

Abstract— Chlorophyll, pheophytin and bacteriochlorophyll sensitize a one-electron transfer in the presence of quinones in ethanol, to produce a ternary complex of ground state porphyrin analog, alcohol cation radical and semiquinone anion radical as the primary photo-product at low temperature. A similar photo-oxidation of alcohol to produce a binary complex is caused by direct excitation of quin-one in the absence of chlorophyll. The mechanisms of these reactions and their implications for photosynthesis are discussed.     

CORRELATION BETWEEN INACTIVATION OF HUMAN CELLS and NUMBERS OF PYRIMIDINE DIMERS INDUCED BY A SUN LAMP and 254 nm RADIATION

Abstract— Nondividing human fibroblasts are inactivated by radiation from a source (a Westinghouse sun lamp) that simulates the UV spectrum of sunlight. Survival curves determined for a DNA excision repair-proficient and a repair-deficient strain (XP12BE) are related to those determined using germicidal light (254 nm) by constant fluence modification factors. In addition, the same fraction of XP12BE cells are killed per pyrimidine dimer by 254 nm and sun lamp light. These results, when related to other survival and photoreactivation studies, suggest that the mechanism for inactivation of nondividing human cells by sun lamp light is the same as that by 254 nm and that pyrimidine dimers are the major responsible photolesion. Repair reverses some of the lethal effects of this light. We suggest that these conclusions apply to sunlight-irradiated skin cells in vivo.     

UVB-INDUCED PHOTOPEROXIDATION OF LIPIDS OF HUMAN LOW and HIGH DENSITY LIPOPROTEINS. A POSSIBLE ROLE OF TRYPTOPHAN RESIDUES

Ultraviolet radiation of the UVB region readily destroy tryptophan (Trp) residues of low (LDL) and high (HDL) density lipoproteins. The photooxidation of tryptophan residues is accompanied by the peroxidation of low and high density lipoproteins unsaturated fatty acids, as measured by the thiobarbituric acid assay. Moreover, low and high density lipoproteins are natural carriers of vitamin E and carotenoids. These two antioxidants are also rapidly bleached by UVB. The UVA radiation promotes neither tryptophan residue destruction nor lipid photoperoxidation. The redox cycling Cu2+ ions considerably increase lipid photoperoxidation. The synergistic action of photo and auto (Cu2(+)-induced) peroxidation induces marked post-irradiation modifications of apolipoproteins as illustrated by the degradation of most tryptophan residues after overnight incubation in the dark of pre-irradiated samples.     

SINGLET OXYGEN GENERATION FROM LIPOSOMES: A COMPARISON OF TIME-RESOLVED 1270 NM EMISSION WITH SINGLET-OXYGEN KINETICS CALCULATED FROM A ONE DIMENSIONAL MODEL OF SINGLET-OXYGEN DIFFUSION and QUENCHING

Abstract— Time-resolved measurements of 1270 nm singlet-oxygen emission following pulsed-laser excitation were made from unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes labeled with zinc phthalocyanine. The effect of the hydrophobic quenchers, β-carotene and ethyl β-apo-8' trans carotenoate, and the hydrophilic quenchers, histidine and methionine, upon the kinetics of the 1270 nm singlet-oxygen emission was studied. Hydrophobic quenchers principally lowered the intensity of the 1270 nm emission and caused only modest changes in the lifetime of the 1270 nm emission. The decrease in 1270 nm emission caused by hydrophobic quenchers was related to the size of the liposomes. The larger the radius of the liposome, the greater the decrease in 1270 nm emission caused by a given concentration of hydrophobic quencher. In contrast, hydrophilic quenchers principally decreased the lifetime of the 1270 nm emission. The effect of hydrophilic quenchers was independent of the size of the liposomes.
There was good agreement between the experimental results and the kinetics of the singlet-oxygen emission calculated using a one dimensional model of singlet-oxygen quenching and diffusion. The kinetics of singlet-oxygen emission from liposomes without added hydrophobic quenchers closely approximated the theoretical kinetics of singlet oxygen in a homogeneous aqueous solution.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY AMINES—I. A FLASH PHOTOLYSIS STUDY OF THE REACTION BETWEEN TRIPLET METHYLENE BLUE AND AMINES

Abstract— Flash photolysis was used to study the reduction of the triplet state of methylene blue by both alkyl- and aryl-amines. The extent of the formation of the semireduced form of the dye yielded rate constants of interaction between the triplet state and the amine ( k ₅). A correlation between log k ₅ and ionization potentials for alkylamines (slope = -1.75 eV^-1) was interpreted as evidence for the formation of a partial charge-transfer intermediate. The rate constants ( k ₅) calculated for aryl-amines approached the rate of diffusion in many cases. A Hammett plot for a series of N, N-dimethyl-anilines yielded a moderately large p value (– 3.28) consistent with the formation of a charge-transfer intermediate. It was concluded that reaction of amines with triplet methylene blue leads to the formation of a partial charge-transfer intermediate which may undergo complete electron transfer to yield radicals, or undergo spin inversion and return to the ground state.     

ANALYSIS OF FLASH PHOTOLYSIS DATA BY A GLOBAL FIT WITH MULTI-EXPONENTIALS–I. DETERMINATION OF THE MINIMAL NUMBER OF INTERMEDIATES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN BY THE 'STABILITY CRITERION'

A major problem in establishing a kinetic scheme for photo-induced reaction sequences is the determination of the unknown number of important intermediates. It is shown that in addition to the χ²-value and the distribution of the residuals obtained from a least-squares fit of the experimental curves by a sum of exponentials, the stability of the apparent rates towards an increase of the number of exponentials with arbitrary test values can be used as an additional criterion. Experimental data describing the photocycle of bacteriorhodopsin after the appearance of intermediate K ( t > 1 μs) are analysed with the described algorithm. It is found that 5 exponentials are necessary and sufficient for the fit.     

UNCOUPLING OF PHOTOPHOSPHORYLATION BY NITROGENOUS BASES. NO CORRELATION Ofb UNCOUPLING WITH pKb and PARTITION COEFFICIENT

Twenty six nitrogenous compounds were tested for uncoupling activity from water to methylviologen in spinach chloroplasts. No correlation was found between photophosphorylation and pK_b or partition coefficient. Thus basic strength is not related with uncoupling phenomena, suggesting that nitrogenous bases are not protonated during uncoupling. Steric effect is also unimportant for uncoupling. New discovered uncouplers were acetamide, hexylamide and trichloroacetamide, which are all very efficient uncouplers. Acetamide accelerates electron transport and decreases ATP synthesis. However, it increases net proton uptake at low concentration, and collapses proton gradient at high concentration. 1,12 Diaminododecane is also a very efficient uncoupler'but it does not have any relationship with other diamines tested; it causes reversible shrinkage to the chloroplast, which suggests that it is not a detergent type of uncoupler.     

[PMe3(CH2CH(OMe)Ph)]+ A PHOSPHONIUM SALT WITH AN ASYMMETRIC CARBON ATOM SYNTHESIZED BY REACTION OF PHENYLACETYLENE WITH COBr(PMe3)3 IN METHANOL. STRUCTURAL CHARACTERIZATION

Abstract

Reaction of HC[tbnd]CPh in excess with a mixture of CoBr(PMe₃)₃/NaBPh₄ in methanol gave rise in 40% yield to the cationic phosphonium salt [PMe₃(CH₂CH(OMe)Ph)]BPh₄. It has been characterized in the solid state by X-ray diffraction and in solution by ¹H and ³¹P NMR.     

ANALYSIS OF FLASH PHOTOLYSIS DATA BY A GLOBAL FIT WITH MULTI-EXPONENTIALS–II. DETERMINATION OF CONSISTENT NATURAL RATE CONSTANTS and THE ABSORPTION SPECTRA OF THE TRANSIENT SPECIES IN THE BACTERIORHODOPSIN PHOTOCYCLE FROM MEASUREMENTS AT DIFFERENT TEMPERATURES

A procedure is described which allows one to deduce from flash photolysis data (absorbance change vs time) recorded at different temperatures the natural rate constants for the elementary reaction steps and the transient absorption spectra of the intermediates within a given kinetic scheme. The selected solutions fulfil two requirements: (i) the rate constants for different temperatures follow Arrhenius'law; (ii) the absorption spectra of the intermediates are independent of temperature. The method is applied to the photocycle of bacteriorhodopsin; the selected model comprises two L-species, a branching at the M-intermediate directly back to BR and an equilibrium between M and O.     

SIMILARITIES IN THE INDUCTION OF SYNTHESIS OF A CELL-SURFACE POLYPEPTIDE IN Arthrobacter sp. BY NEAR-UV IRRADIATION and PHOTODYNAMIC CONDITIONS

Irradiation of aerobic suspensions of Arthrobacter sp. with near-UV light (310-400 nm) induced synthesis of a 21 000 dalton, cell-surface polypeptide. Synthesis of this polypeptide also was induced by visible light in the presence of photodynamic dyes, as shown previously (Hoober, 1978). Induction of the polypeptide in near-UV light and with visible light plus dyes was inhibited by histidine. Hemin inhibited induction in near-UV light and in visible light with methylene blue, neutral red and acrifiavin, which are cationic dyes, but failed to inhibit induction in visible light with rose bengal, an anionic dye. These results suggested that inhibition by hemin required electrostatically favored interaction between the anionic porphyrin and the sensitizer, and that the near-UV light effect was mediated by a cationic or neutral endogenous sensitizer. The similarities in the responses of the cells to near-UV irradiation and visible light plus dyes suggested that the mechanism of induction under the two conditions was the same.     

THE INTERACTION OF MELANIN WITH 8-METHOXYPSORALEN: EFFECT ON RADIATIVE and NONRADIATIVE TRANSITIONS. A FLUORESCENCE and PULSED PHOTOACOUSTIC STUDY

Abstract –The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluorescence and pulsed photoacoustic calorimetry. Due to the strong overlap of the absorption bands of melanin and 8-MOP, a method is presented to account for the systematic errors introduced by the optical filter effect exerted by each absorbing species in the fluorescence and the photoacoustic measurements. As a preliminary step to the understanding of the nonradiative behavior of the psoralen-melanin complexes, the photoacoustic parameters of 8-MOP in various solvents were determined. Spectroscopic data indicate the absence of interaction at the ground-state level, whereas the singlet excited state of 8-MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static quenching mechanism. The photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.     

PHOTOCHEMISTRY IN A CYCLODEXTRIN CAVITY. PRIMARY PROCESSES IN THE PHOTOREDUCTION OF 3- and 4-BENZOYLPYRIDINE STUDIED BY LASER FLASH-PHOTOLYSIS*

The photochemistry of 3- and 4-benzoylpyridine-cyclodextrin inclusion complexes (BPyCDx) was examined by nanosecond laser flash-photolysis and stationary techniques. The lifetimes of the triplet complex and of the triplet radical pair, formed by H-abstraction from a glucose unit of the CDx, have been measured in β-CDx complexes. The reactivity of the heterocyclic ketones with CDx is higher than that of benzophenone, but the lower binding ability of the macrocycle toward these more hydrophylic molecules induces faster separation of the geminate radicals. The quantum yields of the escaped radicals and their decay kinetics have been determined. The β-CDx cage favours geminate recombination reactions, while α- and -γ-CDx tend to release the guest molecule. Cage products have been spectroscopically characterized in the case of the 3-BPy-β-CDx system.