Mitochondrial signaling in mammalian cells activated by red and near-IR radiation

Abstract Mitochondrial signaling is an information channel between the mitochondrial respiratory chain and the nucleus for the transduction signals regarding the functional state of the mitochondria. The present review examines the question whether radiation of visible and near-IR (IR-A) radiation can activate this retrograde-type cellular signaling pathway. Experimental data about modulation of elements of mitochondrial retrograde signaling by the irradiation (mitochondrial membrane potential DeltaPsi(m), reactive oxygen species ROS, Ca(2+), NO(*), pH(i), fission-fusion homeostasis of mitochondria) are reviewed. The terminal enzyme of the mitochondrial respiratory chain cytochrome c oxidase is considered as the photoacceptor. Functions of cytochrome c oxidase as a signal generator as well as a signal transducer in irradiated cells are outlined.     

Reaction Mechanism of Enzyme Studied by Pulse Radiolysis

By virtue of its ability to generate hydrated electrons (e_aq ^?) and various radicals as reductants, the pulse radiolysis technique has been employed for investigating the mechanism of action of peroxidase, cytochrome P-450, and cytochrome oxidase. The oxy forms of hemoproteins, such as myoglobin, peroxidase, and cytochrome P-450, were reduced by hydrated electrons to form the higher oxidation states of these hemoproteins. From these results, the reactive oxygen intermediate of cytochrome chrome P-450 is discussed. The reduction of cytochrome oxidase by the 1-methylnicotinamide radical was investigated. A decrease of the 830-nm band was detected due to the reduction of “visible” copper. After the first phase of the reduction of copper, the return of the 830-nm band corresponding to oxidation of copper was observed. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme α increased. This suggests that 1-methylnicotinamide radical reacts with the “visible” copper and subsequently flows to heme α by intramolecular migration.     

A novel mitochondrial signaling pathway activated by visible-to-near infrared radiation

The number of cells attached to glass substratum increases if HeLa cell suspension is irradiated with monochromatic visible-to-near infrared radiation before plating (the action spectrum with maxima at 619, 657, 675, 700, 740, 760, 800, 820, 840 and 860 nm). Treating of cell suspension with sodium azide (2 x 10(-5) M), sodium nitroprusside (5 x 10(-5) M), ouabain (1 x 10(-6) M) or amiloride (1.7 x 10(-5) M) before irradiation significantly modifies the spectrum of cell attachment enhancement. A light-induced mitochondrial signaling pathway can be regulated by small ligands directly binding to the catalytic center of cytochrome c oxidase (N(3), NO) as well as by chemicals specifically binding to plasma membrane enzymes (ouabain, amiloride). The comparative analysis of action spectra allows the conclusions that first, Cu(A) and Cu(B) chromophores of cytochrome c oxidase could be involved as photoacceptors and second, various signaling pathways (reaction channels) between cytochrome c oxidase and cell attachment regulation are at work.     

A COMPARISON OF PHOTO-KILLING AMONG CAROTENE AND CYTOCHROME c ACCUMULATING STRAINS OF THE SMUT FUNGUS Ustilago violacea AT SPECIFIC WAVELENGTHS FROM 400 TO 650 nm

Abstract— Variously pigmented strains of Ustilago violacea were exposed to monochromatic light from 400 to 650 nm in 50-nm increments to determine which wavelengths were most effective for photo-killing. In addition, liquid nitrogen temperature absorption spectra were determined for intact cells of the fungal strains. The high cytochrome c containing pink strains 1.C429, AB278a-1, 1.C425 and 2.C427 were more sensitive to cytochrome absorption wavelengths than were their low cytochrome c counterparts 15.10, 1.C2 y , 1.C2 w and 1.C427, while the carotene accumulating strains 1.C2 y and AB278a-1 were more resistant to photo-killing than their carotene lacking counterparts 15.10, 1.C2 w , 1.C427 and 1.C429, 1.C415, 2.C428 even at wavelengths not absorbed by carotenes. The results suggest that cytochrome c and other cytochromes are photosensitizers and that carotene-related protection is through the quenching of the photosensitizers as well as by absorption.     

Photoreactions of cytochrome C oxidase

The photoreduction of oxidized bovine heart cytochrome c oxidase (CcO) by visible and UV radiation was investigated in the absence and presence of external reagents. In the former case, the quantum yields for direct photoreduction of heme A (heme a + heme a(3)) were 2.6 +/- 0.5 x 10(-3), 4 +/- 1 x 10(-4), and 4 +/- 2 x 10(-6) with pulsed laser irradiation at 266, 355 and 532 nm, respectively. Within experimental uncertainty, the quantum yields did not depend on pulse energy, implying that the mechanism is monophotonic. Irradiation with 355 nm light resulted in spectral changes similar to those produced independently by reduction with dithionite, whereby the low-spin heme a and Cu(A) are reduced first. Extended illumination at 355 and 532 nm yielded substantial amounts of reduced heme a(3). Heme decomposition was noted with 266 nm light. In the presence of formate and cyanide ions, which bind at the binuclear heme a(3)/copper center in CcO, irradiation at 355 nm caused selective reduction of only the low-spin heme a and Cu(A). The addition of ferrioxalate ion dramatically increased the efficiency of cytochrome c oxidase photoreduction. The quantum efficiency for heme A reduction was found to be near unity, significantly greater than for other known methods of photoreduction. The active reductant is most likely ferrous iron, and its reduction of the enzyme is thermodynamically driven by the reformation of ferrioxalate in the presence of excess oxalate ion. Other metalloenzymes with redox potentials similar to those of cytochrome c oxidase should be amenable to indirect photoreduction by this method.     

Comparing the Impact of NIR,Visible and UV Light on ROS Upregulation via Photoacceptors of Mitochondrial Complexes in Normal,Immune and Cancer Cells

The effect of UV/visible/NIR light (380/450/530/650/808/1064 nm) on ROS generation, mitochondrial activity and viability is experimentally compared in human neuroblastoma cancer cells. The absorption of photons by mitochondrial photoacceptors in Complexes I, III and IV is in detail investigated by sequential blocking with selective pharmaceutical blockers. Complex I absorbs UV/blue light by heme P450, resulting in a very high rate (14 times) of ROS generation leading to cell death. Complex III absorbs green light, by cytochromes b, c1 and c, and possesses less ability for ROS production (seven times), so that only irradiation lower than 10 mW cm⁻² causes an increase in cell viability. Complex IV is well-known as the primary photoacceptor for red/NIR light. Light of 650/808 nm at 10–100 mW cm⁻² generates a physiological ROS level about 20% of a basal concentration, which enhance mitochondrial activity and cell survival, while 1064 nm light does not show any distinguished effects. Further, ROS generation induced by low-intensity red/NIR light is compared in neurons, immune and cancer cells. Red light seems to more rapidly stimulate ROS production, mitochondrial activity and cell survival than 808 nm. At the same time, different cell lines demonstrate slightly various rates of ROS generation, peculiar to their cellular physiology.     

Modeling the active site of cytochrome oxidase: synthesis and characterization of a cross-linked histidine-phenol

A cross-linked histidine-phenol compound was synthesized as a chemical analogue of the active site of cytochrome c oxidase. The structure of the cross-linked compound (compound 1) was verified by IR, (1)H and (13)C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pK(a) of the phenolic proton on compound 1 (8.34) was lower than the pK(a) of tyrosine (10.1) or of p-cresol (10.2). This decrease in pK(a) is consistent with the hypothesis that a cross-linked histidine-tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time-resolved optical absorption spectra of compound 1 at room temperature, generated by excitation at 266 nm in the presence and absence of dioxygen, indicated a species with absorption maxima at approximately 330 and approximately 500 nm, which we assign to the phenoxyl radical of compound 1. The electron paramagnetic resonance (EPR) spectra of compound 1, obtained after UV photolysis, confirmed the generation of a paramagnetic species at low temperature. Because the cross-linked compound lacks beta-methylene protons, the EPR line shape was dramatically altered when compared to that of the tyrosyl radical. However, simulation of the EPR line shape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground-state Fourier transform infrared (FT-IR) spectrum of compound 1 showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. We postulate that phenol oxidation produces a small delocalization of spin density onto the imidazole nitrogen of compound 1, which may explain its unique optical spectral properties.     

傅立叶变换激光腔内吸收光谱方法研究吸收谱线的强度与线形

Based on Fourier-transform intra-cavity laser absorption spectroscopy (FT-ICLAS) setup reported, a signal differential module is introduced to reduce the noise rising from the fluctuation of the laser power and to improve the quality of the interferogram obtained in the experiment. The capability to do quantitative measurement was demonstrated by recording the atmospheric oxygen absorption near 760 nm. The v=6 local mode stretching overtone spectra of phosphine were recorded with different sample pressure. The pressure caused self-broadening and line shift parameters of this band were achieved.     

Amperometric detection of superoxide dismutase at cytochrome c-immobilized electrodes: xanthine oxidase and ascorbate oxidase incorporated biopolymer membrane for in-vivo analysis.

Amperometric measurement of superoxide dismutase (SOD) was carried out at cytochrome c-immobilized monolayers and ascorbate oxidase (AOD)/xanthine oxidase (XOD)/cytochrome c- and (AOD, XOD)/cytochrome c-multilayers. Cytochrome c was covalently immobilized on mercaptopropionic acid-containing self-assembled monolayers on gold. A biopolymer membrane of poly-L-lysine confining XOD and AOD was cast on the monolayer of cytochrome c. While both the cytochrome c-immobilized monolayer and multilayer electrodes show anodic current responses to the generation of superoxide radical, the sensitivity of the multilayer system for the detection of superoxide radical was high relative to that of the monolayer system. In the case of the cytochrome c-multilayer electrodes, the generation of superoxide radical near the sensing element, cytochrome c, resulted in high sensitivity for the detection of superoxide. The use of a XOD and AOD-incorporated poly-L-lysine membrane enabled the detection of the generation of superoxide radical in the presence of L-ascorbic acid. Though L-ascorbic acid could scavenge superoxide radical, the biopolymer membrane confined with AOD will oxidize any L-ascorbic acid that permeated into the membrane. By using the multilayer electrodes, one could measure the activity of SOD in the presence of L-ascorbic acid.     

Changes in the structure of chlorophyll-protein complexes and excitation energy transfer during photoinhibitory treatment of isolated photosystem I submembrane particles

The activity of light-induced oxygen consumption, absorption spectra, low temperature (77 K) chlorophyll fluorescence emission and excitation spectra were studied in suspensions of photosystem (PS) I submembrane particles illuminated by 2000 microE m(-2) s(-1) strong white light (WL) at 4 degrees C. A significant stimulation of oxygen uptake was observed during the first 1-4 h of photoinhibitory treatment, which rapidly decreased during further light exposure. Chlorophyll (Chl) content gradually declined during the exposure of isolated PSI particles to strong light. In addition to the Chl photobleaching, pronounced changes were found in Chl absorption and fluorescence spectra. The position of the major peak in the red part of the absorption spectrum shifted from 680 nm towards shorter wavelengths in the course of strong light exposure. A 6-nm blue shift of that peak was observed after 5-h illumination. Even more pronounced changes were found in the characteristics of Chl fluorescence. The magnitude of the dominating long-wavelength emission band at 736 nm located in untreated particles was five times reduced after 2-h exposure, whereas the loss in absolute Chl contents did not exceed 10% of its initial value. The major peak in low-temperature Chl fluorescence emission spectra shifted from 736 to 721 nm after 6-h WL treatment. Individual Chl-protein complexes differed in the response of their absorption spectra to strong WL. Unlike light-harvesting complexes (LHC), LHCI-680 and LHC-730, which did not exhibit changes in the major peak position, its maximum was shifted from 678 to 671 nm in CPIa complex after PSI submembrane particles were irradiated with strong light for 6 h. The results demonstrated that excitation energy transfer represents the stage of photosynthetic utilization of absorbed quanta which is most sensitive to strong light in isolated PSI particles.     

Absorption spectra of viral components of Sendai virus in the wavelength region from 130 to 320 nm.

Using synchrotron radiation as a light source, the absorption spectra of purified viral components of the Sendai virus, i.e. messenger RNA, lipids, spike (envelope) proteins, reconstructed envelopes, core proteins and whole virions, were obtained in the wavelength region 130-320 nm by measuring the transmission of thin films. Viral (messenger) RNA two peaks at 260 and 190 nm, and a large increase below 160 nm. The absorption spectrum of lipids exhibited a broad peak at 190 nm and a very sharp increase below 160 nm. With spike proteins, a slight peak at 280 nm and a shoulder at 230 nm were observed in addition to a sharper peak at 190 nm and a rather slow increasing absorption below 160 nm. Reconstructed envelopes showed the features of a combination of lipids and proteins. The absorption spectra of core proteins and whole virions exhibited similar characteristics to spike proteins. Conventional UV data were also obtained in the wavelength range 210-320 nm with RNA and lipids. The UV and synchrotron radiation data were in good agreement in terms of the mass absorption coefficients. The molecular splitting of spike proteins was also examined. Proteins gave more diffuse reflection than their subunits, causing a reduction in absorption. This was explained by a loss of transparency with increasing molecular weight.     

Polychromatic action spectrum for the induction of a mycosporine-like amino acid in a rice-field cyanobacterium, Anabaena sp

A polychromatic action spectrum for the induction of an ultraviolet-absorbing/screening mycosporine-like amino acid (MAA) has been determined in a filamentous and heterocystous nitrogen-fixing rice-field cyanobacterium, Anabaena sp. High-performance liquid chromatographic (HPLC) studies revealed the presence of only one type of MAA, which was identified as shinorine, a bisubstituted MAA containing both glycine and serine groups having a retention time at 2.8 min and an absorption maximum at 334 nm. Exposure of cultures to simulated solar radiation in combination with various cut-off filters (WG 280, 295, 305, 320, 335, 345, GG 400, 420, 455, 475, OG 515, 530, 570, RG 645, 665 and a broad-band filter, UG 11) clearly revealed that the induction of the MAA takes place only in the UV range. Photosynthetic active radiation (PAR) had no significant impact on MAA induction. The ratio of the absorption at 334 nm (shinorine) to 665 nm (chlorophyll a) and the action spectrum also showed the induction of MAA to be UV dependent peaking in the UV-B range at around 290 nm. The results indicate that the studied cyanobacterium, Anabaena sp. may protect itself from deleterious short wavelength solar radiation by its ability to synthesize a mycosporine-like amino acid in response to UV-B radiation and thereby screen the negative effects of UV-B.     

Scanning near-field IR microscopy of proteins in lipid bilayers

We use infrared near-field microscopy to chemically map the morphology of biological matrices. The investigated sample is built up from surface-tethered membrane proteins (cytochrome c oxidase) reconstituted in a lipid bilayer. We have carried out infrared near-field measurements in the frequency range between 1600 and 1800 cm(-1). By simultaneously recording the topography and chemical fingerprint of the protein-tethered lipid bilayer with a lateral resolution of 80 nm × 80 nm, we were able to probe locally the chemical signature of this membrane and to provide a local map of its surface morphology.     

PHOTOBIOLOGIE DU MESOCOTYLE D'AVOINE–IV. SPECTRES D'ACTION ET EFFICACITE QUANTIQUE DE LA LUMIERE

Abstract— The wavelengths most active in influencing the growth of segments of oat mesocotyl are 435 nm (blue), 665 nm (red) and 730 nm (far red). Using a high-energy source (1.5 × 10¹⁸ quanta/cm²), an inhibition in all areas of the spectrum except the far red was obtained. The efficiency of the different radiations was studied: blue light is the most active, red and far red have about the same efficiency; green is a thousand times less active. The effect induced by blue light cannot be directly attributed to phytochrome, because the wavelength of maximum effect does not correspond with that of maximum absorption of the pigment (there is 55 nm difference); also, blue light is more active than the red radiation.     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

Assignment of conformation-sensitive near-infrared bands in high-spin ferrous haemoproteins. Low-temperature magnetic circular dichroism data

An assignment of the near-infrared bands in the 600–800 nm spectral region observed in magnetic circular dichroism (MCD) spectra of high-spin ferrous haemoproteins is presented. The assignment is based on a relative energy level scheme for iron d-electrons, a comparison of predicted and measured temperature dependences of MCD intensity, a sign of MCD bands and a group theoretical analysis of allowed transitions. The proposed assignment is consistent with the ∼15-nm red shift of the ∼760 nm band on breakage of the Fe-His bond in deoxy-myoglobin at low pH, with low-temperature photolysis experiments available for CO complexes of several haemoproteins. In accordance with the observations, the intensity of the MCD bands for proteins with a sulphur anion of cysteine as proximal haemligand (cytochrome P450 and chloroperoxidase) is predicted to be diminished by at least one order of magnitude compared to that for proteins with an imidazole of a histidine as a protein-derived haemligand (i.e. myoglobin, haemoglobin and horseradish peroxidase). Received: 4 February 1997 / Accepted: 1 May 1997     

AUTOFLUORESCENCE SPECTROSCOPY OF OPTICALLY TRAPPED CELLS

Abstract— Cellular autofluorescence spectra were monitored in a single-beam gradient force optical trap ("optical tweezers") in order to probe the physiological effects of near infrared and UVA (320–400 nm) microirradiation. Prior to trapping, Chinese hamster ovary cells exhibited weak UVA-excited autofluorescence with maxima at 455 nm characteristic of β-nicotinamide adenine dinucleotide (phosphate) emission. No strong effect of a 1064 nm NIR microbeam on fluorescence intensity and spectral characteristics was found during trapping, even for power densities up to 70 MW/cm² and radiant exposures of 100 GJ/cm². In contrast to the 1064 nm trap, a 760 nm trapping beam caused a two-fold autofluorescence increase within 5 min (about 20 GJ/cm²). Exposure to 365 nm UVA (1 W/cm²) during 1064 nm trapping significantly altered cellular autofluorescence, causing, within 10 min, a five-fold increase and a 6 nm red shift versus initial levels. We conclude that 1064 nm microbeams can be applied for an extended period without producing autofluorescence changes characteristic of alterations in the cellular redox state. However, 760 nm effects may occur via a two-photon absorption mechanism, which, in a manner similar to UVA exposure, alters the redox balance and places the cell in a state of oxidative stress.     

Ultrafast energy transfer in light-harvesting chlorosomes from the green sulfur bacterium Chlorobium tepidum

Two independent pump-probe techniques were used to study the antenna energy transfer kinetics of intact chlorosomes from the green sulfur bacterium Chlorobium tepidum with femtosecond resolution. The isotropic kinetics revealed by one-color experiments in the BChl c antenna were inhomogeneous with respect to wavelength. Multiexponential analyses of the photobleaching/stimulated emission (PB/SE) decay profiles typically yielded (apart from a approximately 10 fs component that may stem from the initial coherent oscillation) components with lifetimes 1-2 ps and several tens of ps. The largest amplitudes for the latter component occur at 810 nm, the longest wavelength studied. Analyses of most two-color pump-probe profiles with the probe wavelength red-shifted from the pump wavelength yielded no PB/SE rise components. PB/SE components with approximately 1 ps risetime were found in 790 --> 810 and 790 --> 820 nm profiles, in which the probe wavelength is situated well into the BChl a absorption region. A 760 --> 740 nm uphill two-color experiment yielded a PB/SE component with 4-6 ps risetime. Broadband absorption difference spectra of chlorosomes excited at 720 nm (in the blue edge of the 746 nm BChl c Qy band) exhibit approximately 15 nm red-shifting of the PB/SE peak wavelength during the first several hundred fs. Analogous spectra excited at 760 nm (at the red edge) show little dynamic spectral shifting. Our results suggest that inhomogeneous broadening and spectral equilibration play a larger role in the early BChl c antenna kinetics in chlorosomes from C. tepidum than in those from C. aurantiacus, a system studied previously. As in C. aurantiacus, the initial one-color anisotropies r(0) for most BChl c wavelengths are close to 0.4. The corresponding residual anisotropies r(infinity) are typically 0.19-0.25, which is much lower than found in C. aurantiacus (> or = 0.35); the transition moment organization is appreciably less collinear in the BChl c antenna of C. tepidum. However, the final one-color anisotropies at 789 and 801 nm are approximately 0 and 0.09 respectively, and the final anisotropy in time 780 --> 800 nm experiment is approximately -0.1. These facts indicate that the BChI a transition moments themselves exhibit some order, and are directed at an angle > 54.7 degrees on the average from the BChl c moments. The one-color profiles exhibit coherent oscillations at most wavelengths, including 800 nm; Fourier analyses of these oscillations frequently yield components with frequencies 70-80 and 130-140 cm-1.     

INDUCTION OF DIRECT AND INDIRECT SINGLE-STRAND BREAKS IN HUMAN CELL DNA BY FAR- AND NEAR-ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single-strand breaks (SSBs, frank breaks plus alkali-labile sites) in human P3 teratoma cells in culture by monochromatic 254-, 270-, 290-, 313-, 334-, 365-, and 405-nm radiation is described. The cells were held at +0.5C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D₂O environment (compared with the normal H₂O environment) did not alter the rate of induction of SSBs by 290-nm radiation, whereas the D₂O environment enhanced the induction of SSBs by 365- and 405-nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs . wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far-UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near-UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365-nm radiation.