ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10^-4 W m^-2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad-blue light and 450 nm light as references, typical flavin-like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10^-8 W m^-2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m^-2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10^-9 W m^-2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

FLUORESCENCE OF 5-METHYLCYTOSINE

Abstract— 5-Methylcytosine and 5-methyldeoxycytidylic acid are fluorescent in aqueous solution at room temperature and neutral pH. 5-Methylcytosine, 10^-3M, pH 8.5, 25°C, has a quantum yield of 5 ×10^-4, 5-Methyldeoxycitydylic acid, 10^-4M, pH 7.5, 20°C, has a quantum yield of 8 × 10^-4. Emission maxima are 2.91 and 2.80μ^-1. At pH 14, the quantum yield of 5-methylcytosine is 1.6 × 10^-2; the emission maximum is 2.82μ^-1. At pH I, the quantum yield of both compounds is less than or equal to 10^-4. Both compounds were chromatographically homogeneous, had absorption spectra which agreed with published data, and excitation spectra which agreed closely with absorption spectra.     

FLUORESCENCE OF THYMINE IN AQUEOUS SOLUTION AT 300° K

Abstract— –Fluorescence of thymine in neutral aqueous solution at room temperature has been detected using the multiscaling operation of a multichannel analyzer. The emission maximum (2.96 μm^-1) and 0-0 transition energy (3.37-3.45 μm^-1) are close to those determined at liquid nitrogen temperature in mixed solvents. The quantum efficiency of fluorescence excited at 3.77 μm^-1 is calculated to be 1.04 × 10^-4.
The corrected relative excitation spectrum shows significant differences from the absorption spectrum when both are determined under identical conditions of concentration and spectral bandwidth on the same instrument. The quantum yield of fluorescence decreases about 2-fold as the energy of excitation is increased beyond the 0-0' transition and follows the relation 1/φ°α E _excit..
This behavior is discussed in terms of (a) n π* and ππ* states, (b) emission from a minor tautomer and (c) kinetics of competing deactivation processes.     

BIOLOGICAL CHEMILUMINESCENCE

Abstract
The nucleobase 5-methylcytosine ( I ) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2'-deoxyribonucleoside, 5-methyl-2'-deoxycytidine ( II ), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine ( Ib ) and 3-(2- erythro - d -pentopyranos-1-yl) amino-2-methylacrylamidine ( IIb ) as the main photoproducts. The structure of the photoproducts was established by spectroscopic methods (¹H and ¹³C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib , confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2–¹³C]cytosine, hydrolysis of N -carbomethoxy-3-amino-2-methylacryl-amidine and reaction of Ib with 1,1'-carbonyldiimidazole to give I ). The quantum yield for formation of Ib was determined to be 1.8 × 10^-3at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 × 10^-3 at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 × 10^-3 at pH 7.0 ( Ib ) and 0.6 × 10^-3 at pH 5.0 ( IIb ). The mechanism of formation of Ib (or IIb ) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or II ), followed by ring opening and decarboxylation of an intermediate carbamic acid.     

PHOTOELECTRON QUANTUM YIELDS AND PHOTOELECTRON MICROSCOPY OF CHLOROPHYLL AND CHLOROPHYLLIN

Abstract— The absolute PE quantum yield curves of chls a and b ,.chin, and phytol were examined over the wavelength range 500-180 nm. In the long wavelength region (500-240 nm) quantum yields are below 5 × 10^-6 electrons per incident photon. Below 240 nm the quantum yields rise sharply. The chls and chin exhibit similar yield curves; measured yields were of order 5 × 10^-6 electrons per incident photon at 240 nm, 1 × 10^-4 at 220 nm, and 1 × 10^-3 at 180 nm. These yields are at least 2 orders of magnitude greater than those of the amino acids and more than three orders of magnitude greater than that of phytol over this wavelength region. Photoemission observed in chl thin films is due to the porphyrin moiety of the molecule. High contrast is obtained in PE micrographs of chin deposited on substrates of bovine serum albumin, dipalmitoyl phosphatidylcholine, or starch. Chl is expected to be the dominant photoemissive component of thylakoid membranes and accounts for the image contrast observed previously in PE micrographs of spinach chloroplasts.     

CHEMILUMINESCENCE IN THE PROCESS OF OXIDATIVE RING-OPENING OF PURPUROGALLINQUINONES

Abstract— The oxidation of purpurogalline (PPG) by alkaline solution of H₂O₂ pH 9–11 at 298°K is accompanied by chemiluminescence (CL) in the spectral range 400–600 nm with the maximum at 500 nm and quantum yield about 10^-6. The optimal concentrations of reactants with respect to maximal intensity are: 2 × 10^-4 M PPG, 10^-2 M NaOH, 1 M H₂O₂. Activation energy calculated from the maximum intensity of CL is 8.1×0.4 kcal/mole. Light emission occurs only when OH-groups of the phenolic ring of PPG undergo oxidation and the blue anion of o -PPG-quinone is formed. The rate that determines step in the reaction associated with luminescence is the nucleophilic attack of OOH^- ion on the blue anion of o -PPG-quinone. In this exergonic step (-ΔH = 63 to 230kcal/mole) the o - and/or p -quinone ring is opened and carbonyl derivatives of α-tropolone are produced. They display fluorescence in the region 400–600 nm. The fluorescence spectrum of the reaction mixture after oxidation of PPG is very close to that of CL. It is likely that carbonyl derivatives of α-tropolone are emitters of CL.     

THE NATURAL PHOSPHORESCENCE LIFETIME OF BIACETYL AND BENZIL IN FLUID SOLUTION

Abstract— The quantum yields of phosphorescence and fluorescence and the phosphorescence lifetime have been determined for biacetyl in benzene, p-xylene, n-heptane, and water, and for benzil in n-heptane at 22C. The following values for the fluorescence quantum yield and natural phosphorescence lifetime were obtained: For biacetyl in benzene, 2.7 times 10^-3 and 7.8 msec, in p-xylene, 2.5 times 10^-3 and 10.8 msec, in n-heptane, 2.9 times 10^-3 and 10.8 msec, in water, 1.1 times 10^-3 and 10.0 msec, and for benzil in n-heptane, 1.1 times 10^-3 and 10.4 msec.     

FLUENCE-RATE DEPENDENCE OF MONOPHOTONIC REACTIONS OF NUCLEIC ACIDS IN VITRO AND IN VIVO

The Bunsen-Roscoe law, also known as the reciprocity law ( E = f(F) with F = I t ) has only limited validity for monophotonic reactions of nucleic acids. Especially at low fluence rates, the extent of in vitro and in vivo photoreactions of nucleic acids in the far-UV and near-UV range is a function of the fluence and of the fluence rate ( E = f (F;I)). In vitro experiments with poly(dA)poly(dT) clearly show that the far-UV (254 nm) response, indicated by the changes of the ellipticity at 315 nm, does not obey the Bunsen-Roscoe law at low fluence rates in the range between 1 W m^-2 and 20 W m^-2. In vivo experiments with Escherichia coli revealed very similar anomalies. Studying the growth delay after irradiation with far-UV light at 280 nm or near-UV light at 334 nm, we have confirmed the lack of reciprocity in both spectral ranges. The failure of the Bunsen-Roscoe law for the 280 nm and 334 nm UV irradiation effect at low fluence rates was in the range O < I < 40 W m^-2. In both cases reciprocity occurred at higher fluence rates (40 < I < 100 W m^-2).     

PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

Abstract The preparations of chicken erythrocyte linear double-stranded DNA and superhelical plasmid pBR322 DNA were irradiated by continuous low-intensity UV radiation (I = 25-50 W/m², λ= 254 nm) as well as by highintensity picosecond laser UV radiation (I = 10¹¹-10¹³ W/m², λ= 266 nm). The effect of DNA secondary structure alterations on the formation of liquid-crystalline dispersions from UV-irradiated DNA preparations was studied. It was shown that in the case of linear DNA, watching the disappearance of abnormal optical activity characteristic for cholesteric liquid crystal we managed to detect the presence of photochemical alterations in DNA irradiated by low-intensity UV radiation at an absorbed energy of more than 20 quanta per nucleotide. In the case of superhelical DNA using enzyme treatment of liquid-crystalline dispersions and monitoring the appearance of abnormal optical activity, we detected the presence of photochemical alterations in DNA molecules after low-intensity UV irradiation at an absorbed energy of less than 4 quanta per nucleotide. Under the latter approach using picosecond UV laser irradiation at three different light intensities we were able to distinguish the different mechanisms of fine alterations in DNA secondary structure at an absorbed energy value of about 3 quanta per nucleotide.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

PHOTOINACTIVATION OF CATALASES FROM MAMMAL LIVER, PLANT LEAVES AND BACTERIA. COMPARISON OF INACTIVATION CROSS SECTIONS AND QUANTUM YIELDS AT 406 nm

Abstract— Photoinactivation in vitro at pH 7.0 of catalases from different sources (bovine liver, spinach leaves, and Micrococcus lysodeikticus) was studied. The wavelength of the inactivating light was close to the Soret peak of catalase. No great difference in light sensitivity between soluble catalases were found; the inactivation cross sections found ranged from 3.8.10^-4 to 5.0. 10^-4Ų/molecule. The inactivation quantum yield is 2.2. 10^-5 for bovine liver catalase and 3.110^-5 for Micrococcus catalase. The quantum yield for soluble spinach catalase is of a similar order of magnitude. There are some indications of a greater resistance to photodestruction of the spinach leaf catalase activity associated with small particles.     

LONG WAVELENGTH UV RADIATION AFFECTS CHEMILUMINESCENCE OF HUMAN POLYMORPHONUCLEAR LEUCOCYTES

Abstract— Luminol-enhanced chemiluminescence in suspensions of human polymorphonuclear leucocytes stimulated with the chemotactic oligopeptide formyl-methionyl-leucyl-phenylalanine was increased by exposure of the cells to long wavelength UV radiation (mainly320–400 nm). Leucocytes treated with 0.3 × 10⁴ J m^-2 of UV responded with doubled peak values, and 4 × 10⁴ J m^-2 lead to a five-fold increase in peak chemiluminescence, as compared with non-exposed cells. Supernatants isolated from irradiated leucocytes contained increased amounts of both myeloperoxidase and lactate dehydrogenase and showed higher chemiluminescence values, when compared with supernatants from sham-irradiated cells. The results suggest that leucocyte degranulation contributes to the inflammatory properties of long wavelength UV.     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

THE PHOTODYNAMIC EFFECT OF HEMATOPORPHYRIN ON DNA

Abstract— Breakage of DNA in vitro and inside E. colt cells has been determined after exposure to monochromatic 365 nm light in the presence of 10 µM hematoporphyrin. When measured by alkaline sucrose sedimentation, the yields of breaks were 1.4 × 10^-12 per dalton and per J/m² for Col El-DNA in vitro and 5.9 × 10^-3 per dalton and per J/m² for superinfecting phage Λ DNA inside E. coli cells made permeable by toluene. No breaks were found by neutral sucrose sedimentation, demonstrating that the lesions represent alkali-labile bonds. The majority of the alkali-labile bonds were induced by singlet oxygen, as evidenced by the several-fold higher yield obtained in D₂O-containing buffer.     

MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.