Inactivation of adenosine 5''-triphosphate synthesis and reduced-form nicotinamide adenine dinucleotide dehydrogenase activity in Escherichia coli by near-ultraviolet and violet radiations.

Near-ultraviolet (near-UV) light (300 to 380 nm) is a significant component of sunlight and has a variety of effects on biological systems. The present work is an attempt to identify chromophores (molecular absorbers of light) and targets (critical damaged molecules) for inhibition of adenosine triphosphate (ATP) synthesis in Escherichia coli by near UV. The fluence of 334 nm required for 37% survival of net ATP synthesis (F37) in E. coli AB2463 in succinate medium is 140 kJ/m2. The action spectrum for this inactivation is almost structureless, exhibiting a smooth transition from high efficiency at 313 nm to low efficiency at 405 nm. The action spectrum for inhibition of net ATP synthesis is consistent with the chromophore being either ubiquinone Q-8 or vitamin K2. The fluence required is consistent with ubiquinone Q-8 also being a target molecule. The activity of reduced nicotinamide adenine dinucleotide dehydrogenase in extracts of E. coli B is also inactivated by near UV and shows an F37 of about 40 kJ/m2. The action spectrum for this effect is quite structureless; it shows high efficiency at 313 nm and low efficiency at 435 nm. The data do not suggest a target molecule for this action, although it is possible that ubiquinone Q-8 absorbs the near-UV energy and then passes it on to some other target molecule. The data further indicate that inactivation of the oxidative phosphorylation system is not a primary factor in near-UV-induced growth delay in E. coli.     

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.     

Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum

The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro. In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g. epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity. The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E. coli, having lambda max = 384 nm. However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro. Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield. These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+     

Perithecial Formation in Gelasinospora reticulispora VII: Action Spectra in UV Region for the Photoinduction and the Photoinhibition of Photoinductive Effect Brought by Blue Light

An action spectrum for photoinduction of perithecial formationafter a prior 72 h dark growth period was determined in theUV region with apically growing mycelia of a sordariaceous fungus,Gelasinospora reticulispora. The spectrum exhibited a peak at280 nm. Quantum effectiveness of 280 nm irradiation was ca.1.7 times higher than that of 450 nm light. The number of peritheciainduced by UV radiation was saturated at a lower level as comparedwith blue light. UV radiation having a fluence greater thanthe saturation level decreased the number of induced perithecia.UV radiation that was given after a saturating exposure to inductiveblue light inhibited the inductive effect of blue light. Anaction spectrum for this inhibition exhibited a peak between260 and 270 nm. Monochromatic light beyond 350 nm had no inhibitoryeffect. Inhibitory effects of UV radiation given after inductiveblue light irradiation were observed in the fluence range wherephotoinductive effects of UV radiation became obvious. Therefore,the true height of the UV peak in the photoinduction actionspectrum,when free of distortion from the inhibitory effect, should behigher than the peak obtained in this study. (Received August 20, 1983; Accepted November 4, 1983)     

Action spectrum for the in vitro induction of simian virus 40 by ultraviolet radiation

A line of simian virus 40-transformed hamster kidney cells was exposed to ultraviolet radiation at eleven different wavelengths in the region 238-302 nm. An action spectrum derived from the resulting exposure-response curves for the induction of simian virus 40 from these cells exhibits a broad peak in the region 260-270 nm suggesting DNA as the major chromophore for this response. This conclusion is consistent with results obtained by other investigators who have noted viral induction by a number of DNA-damaging agents.     

The wavelength dependence of 8-methoxypsoralen photosensitization of radiation-enchanced reactivation in a mammalian cell-virus system.

The combined effect of 8-methoxypsoralen (8-MOP) and ultraviolet (UV) radiation on the ability of an irradiated mammalian cell (CV-1) to reactivate UV-irradiated mammalian virus (Herpes simplex) was tested. Prior treatment of cells with 8-MOP was found to increase Radiation-Enhanced Reactivation (RER) at one wavelength (297 nm) in the far ultraviolet but not at others (240-289 nm). This same treatment induced RER in the near UV (302-370 nm) and the visible region (380-400 nm). An action spectrum for the photo-sensitized induction of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum for 8-MOP and the theory that damage to DNA is, at least in part, responsible for Radiation-Enhanced Reactivation.     

Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii

A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm.     

The ultraviolet action spectrum for stomatal opening in broad bean

The ultraviolet action spectrum for stomatal opening was measured using epidermal peels from leaves of broad bean (Vicia faba). The spectrum was calculated from hyperbolic fluence response curves using 11 wavelengths ranging from 275 to 459 nm. The action spectrum exhibits a major peak at approximately 280 nm and a minor peak at approximately 360 nm. The response at 280 nm is about three times greater than the response at 459 nm. Under the conditions utilized (i.e. the absence of saturating red light), stomatal opening saturated at extremely low fluence rates: <0.2 μmol m⁻² s⁻¹ at 280 nm, and approximately 1.0 μmol m⁻² s⁻¹ at 459 nm. The threshold for blue-light-induced stomatal opening was approximately 0.02 μmol m⁻² s⁻¹. In light-mixing experiments, the addition of 280 nm light to saturating 650 nm (red) light caused additional stomatal opening, which is indicative of separate photoreceptors. In contrast, adding 280 nm of light to saturating 459 nm (blue) light did not increase stomatal opening, suggesting that they both excite the same receptor. The results with white light were similar to those with blue light. We infer that ultraviolet light acts via the blue light photoreceptor rather than through photosynthesis. The additional absorbance peak at 360 nm suggests that the chromophore is either a flavin or a cis-carotenoid, both of which exhibit peaks in this region. It is proposed that the chromophore can be excited either directly by blue light or by energy transferred from the protein portion of the protein-pigment complex after it absorbs 280 nm light.     

A study of the functional activity of macrophages of peritoneal exudate of mice exposed to low-intensity laser radiation in vitro and in vivo

The effect of low-intensity laser radiation generated by semiconductor devices in the red (650 nm) and infrared (850 nm) regions of the spectrum in vitro and in vivo on the phagocytic activity and synthesis of proinflammatory cytokines by peritoneal macrophages during the phagocytosis of bacterial cells has been studied. A culture of the clinical strain of the enteropathogenic bacterium Escherichia coli was used as an object. The radiation dose was varied by changing the power and duration of exposure. The results obtained indicate that infrared low-intensity laser radiation has a stimulating effect on the phagocytic activity of macrophages. It was shown that the effect of low-intensity laser radiation on the activity of the phagocytic process, the enhancement of the adhesion of bacteria by macrophages, killing of bacteria, and the production of proinflammatory cytokines is dose-dependent. The exposure to the rays of the red region of the spectrum on phagocytizing macrophages induced a decrease in their activity; as the dose was increased, the destruction of cells was registered.     

Reversible effects of green and orange-red radiation on plant cell elongation

The differential cell elongation of cress (Lepidium sativum) roots that results in geotropic bending can be decreased by green radiation with an action spectrum peaking at 550 nm. This decrease can be negated by prior or simultaneous irradiation by orange-red light with an action spectrum peaking at 620 nm. The green radiation appears to be effective during the cell elongation phase of geotropic response.     

Similar ultraviolet action spectra for the protection by glycerol of transforming DNA against single-strand breaks and inactivation of biological activity

An action spectrum for the protection of purified DNA by glycerol against the induction of single-strand breaks in the DNA by ultraviolet (uv) light is described. Protection was not observed below 300 nm, was maximal between 334 and 365 nm, and decreased at 405 nm. This spectrum closely matched the spectrum for the protection by glycerol against the inactivation of biological transforming activity by near uv, described previously. Also, deviations from the reciprocity rule are similar for inactivation of transforming activity and for induction of DNA breaks by 365-nm radiation. That is, the deviations for the two end points are quantitatively the same, such that high fluence rates are less effective than low fluence rates.     

Control of development in plants and fungi by far-UV radiation

Far-UV (200–320 nm) radiation regulates development in plants and fungi. Some of these responses are controlled by a chromophore which absorbs strongly near 260 nm, possibly a nucleic acid. Other responses are controlled by a chromophore(s). with maximal in vivo sensitivity near 295 nm. In plants. far-UV induces genes in the phenylpropanoid pathway and the synthesis of phytoalexins and flavonoids. Far-UV also regulates growth rate. controls curvature and taxis, and stimulates sexual and asexual morphogenesis of fungi. Some of these developmental responses may prevent damage by far-UV radiation.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. I. The photoreceptor

Broad-band UV-B radiation inhibited hypocotyl elongation in etiolated tomato ( Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings. This inhibition could be elicited by < 3 μmol m⁻² s⁻¹ of UV-B radiation provided against a background of white light (> 620 μmol m⁻² s⁻¹ between 320 and 800 nm), and was similar in wild-type and phytochrome-1-deficient aurea mutant seedlings. These observations suggest that the effect of UV-B radiation is not mediated by phytochrome. An activity spectrum obtained by delivering 1 μmol m⁻² s⁻¹ of monochromatic UV radiation against a while light background (63 μmol m⁻² s⁻¹ showed maximum effectiveness around 300 nm, which suggests that DNA or aromatic residues in proteins are not the chromophores mediating UV-B induced inhibition of elongation. Chemicals that affect the normal (photo)chemistry of flavins and possibly pterins (KI, NaN, and phenylacetic acid) largely abolished the inhibitor) effect of broad-hand UV-B radiation when applied to the root zone before irradiation. KI was effective at concentrations < 10⁻⁴ M , which have been shown in vitro to be effective in quenching the triplet excited stales of flavins but not fluorescence from pterine or singlet states of flavins. Elimination of blue light or reduction of UV-A, two sources of flavin excitation, promoted hypocotyl elongation, but did not affect the inhibition of elongation evened by UV-B. Kl applied after UV-B irradiation had no effect on the inhibition response. Taken together these findings suggest that the chromophore of the photoreceptor system invoked in UV-B perception by tomato seedlings during de-etiolation may be a flavin.     

Optical rotary dispersion of some heptaene antibiotics]

Experimental differences in the curves of the optic rotation dispersion (ORD) of cystrans-heptaenic antibiotics were found. The ORD curves of amphotericin B, mycoheptin, levorin components and isolevorin A2, components of criptomycin and candidin were registered. The curves of the ORD which were smooth had been prepared in dimethylsulphoxide in the spectral range at 450 to 600 nm. In the spectral range at 300 to 420 nm the ORD curves appeared to be anomal with a complex Kotton effect, they were prepared in methyl alcohol. The Kotton effect was probably due to asymmetry of the electron membranes of polyenic chromophore induced by the other part of the polyen molecule. This was evident from the fact that the curve of the Kotton effect was situated in the same spectral range as the absorption bond of the polyenic chromophore. The oscillating structure in the absorption spectrum and the curve of the complex Kotton effect were analogous.     

Ultraviolet radiation inhibits human natural killer activity and lymphocyte proliferation

Exposure to ultraviolet radiation (UVR) has been implicated in the predisposition to certain neoplasms and leads to viral reactivation. Natural killer (NK) activity may play a role in immunosurveillance and response to certain viral infections. We have evaluated the sensitivity to UVR of human NK activity, a nonproliferative function, and the proliferative response to the mitogen phytohemagglutinin (PHA). In vitro exposure to UVR resulted in a dose-dependent inhibition of NK activity and response to PHA. The wavelength dependence for UVR inhibition of NK activity and of the PHA response of lymphocytes were virtually superimposable at wavelengths at or above 300 nm, but NK activity was less sensitive to UVR at 260 and 280 nm. Maximal sensitivity for both functions was found at 260 nm, consistent with a nucleic acid chromophore mediating UVR inhibition of both activities. The DNA-directed drugs mitomycin C, acridine orange, and adriamycin at concentrations that inhibit proliferation are poor inhibitors of NK activity. These results suggest that UVR inhibition of NK activity as well as proliferation may be mediated by a nucleic acid chromophore. NK activity, however, is less sensitive to direct damage of DNA by alkylation, distortion, or oxidation. At 300 nm, the amount of radiation required to inhibit NK activity and proliferation is within the range penetrating to the dermis and capillaries during environmental exposure to sunlight.     

Action spectra for the photoperiodic control of polymorphism in the aphid Megoura viciae

Energy compensated action spectra are given for the photoperiodic control of polymorphism in Megoura. The production of ‘long day’ parthenogenetic virginoparae and ‘short day’ oviparae mainly depend on the night length. Light has three different effects. ‘Early’ interruptions of the dark phase in a long night cycle reverse the time-measuring dark response. The action spectrum for a 1 hr interruption placed 1.5 hr after the onset of darkness (during dark stage 1) shows a relatively narrow band of activity, mainly in the blue (450–470 nm). The threshold is ca. 0.25 μW cm^?2. ‘Late’ interruptions placed 7.5 or 8 hr after dark hour 0 (during dark stage 3) strongly promote the production of virginoparae without causing a reversal of the response. The action spectrum has the same blue maximum but sensitivity extends into the yellow and red spectral regions. The third photosensitive component, the main photoperiod itself, is required for initiating the dark timing response and has an intermediate action spectrum. Time/intensity curves for a single wavelength (471 nm) show that the responses during stages 1 and 3 depart markedly from reciprocity. Short durations cannot be compensated by high intensities. The shape of the reciprocity curve for an ‘early’ interruption suggests that the stage 1 response is complete after ca. 1.25 hr.The action spectra are believed to be compatible with the view that the photoreceptor is a caroteno-protein. It is suggested that all three pigment forms are related and that time measurement is largely a function of spontaneous ‘dark reaction’ changes in the pigment system. Stage 1 may represent the reversible conjugation phase of the protein/chromophore moieties. In Stage 2 the pigment is presumably photorefractory and is transitional to the highly sensitive broad spectrum form of stage 3.     

Absorption spectra of photoactive yellow protein chromophores in vacuum

The absorption spectra of two photoactive yellow protein model chromophores have been measured in vacuum using an electrostatic ion storage ring. The absorption spectrum of the isolated chromophore is an important reference for deducing the influence of the protein environment on the electronic energy levels of the chromophore and separating the intrinsic properties of the chromophore from properties induced by the protein environment. In vacuum the deprotonated trans-thiophenyl-p-coumarate model chromophore has an absorption maximum at 460 nm, whereas the photoactive yellow protein absorbs maximally at 446 nm. The protein environment thus only slightly blue-shifts the absorption. In contrast, the absorption of the model chromophore in aqueous solution is significantly blue-shifted (lambda(max) = 395 nm). A deprotonated trans-p-coumaric acid has also been studied to elucidate the effect of thioester formation and phenol deprotonation. The sum of these two changes on the chromophore induces a red shift both in vacuum and in aqueous solution.     

Growth and morphological responses to different UV wavebands in cucumber (Cucumis sativum) and other dicotyledonous seedlings

We examined the influence of short-term exposure of different UV wavebands on the fine-scale kinetics of hypocotyl growth of dim red light-grown cucumbers (Cucumis sativus L.) and other selected dicotyledonous seedlings to evaluate: (1) whether responses induced by UV-B radiation (280-320 nm) are qualitatively different from those induced by UV-A (320-400 nm) radiation, and (2) whether different wavebands within the UV-B elicit different responses. Responses to brief (30 min) irradiations with 3 different UV wavebands all included transient inhibition of elongation during irradiation followed by wavelength specific responses. Irradiations with proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm) induced inhibition of hypocotyl elongation within 20 min of onset of irradiation, while UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation with a lag of 1-2 h. The response to short wavelength UV-B was persistent for at least 24 h, while the response to long wavelength UV-B lasted only 2-3 h. The UV-A treatment induced reductions in elongation rates of approximately 6-9 h following exposure followed by a continued decline in rates for the following 15-18 h. Short wavelength UV-B also induced positive phototropic curvature in both cucumber and Arabidopsis seedlings, and this response was present in nph-1 mutant Arabidopsis seedlings defective in normal blue light phototropism. Reciprocity was not found for the response to short wavelength UV-B. The short wavelength and long wavelength UV-B responses differed in dose-response relationships and both short wavelength responses (phototropic curvature and elongation inhibition) increased sharply at wavelengths below 300 nm. These results indicate that different photosensory processes are involved in mediating growth and morphological responses to short wavelength UV-B (280-300 nm), long wavelength UV-B (essentially 300-320 nm) and UV-A. The existence of two separate types of hypocotyl inhibition responses to UV-B, with one that depends on the intensity of the light source, provides alternate interpretations to findings in other studies of UV-B induced photomorphogenesis and may explain inconsistencies between action spectra for inhibition of stem growth.     

Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid.

A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wild-type cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.