Single Light Flashes Research Articles

Effect of flash photoreactivation onEscherichia coli recAinduction by ultraviolet light

Excision-deficient Escherichia coli, carrying the gene for the photolyase on a multicopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps-) mutant MT-GS18 carrying deletions of the genes for cytochrome c 1 and b of the bc 1 complex and for cytochrome c 2. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc 1 complex and cyt. c 2 still leaves several haems of c- and b-type with Em7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b 415 and cyt. b 275 are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c 2 and bc 1 complex might involve high-potential membrane bound haems of b- and c-type.

Mutants of Arabidopsis thaliana with decreased amplitude in their phototropic response.

Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in first positive' phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable to the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism.

The electroretinogram of the little owl ( Athene noctua)

Electroretinographic responses (ERGs) have been recorded from the cornea of the little owl ( Athene noctua) in response to single light flashes and to alternating sinusoidal gratings (pattern) at different levels of light adaptation. Both flash- and pattern-evoked ERGs show scotopic as well as photopic components. The pattern evoked ERG is spatially tuned with tuning functions which shift towards lower frequencies by reducing the mean luminance. The retinal acuity is about 6c/deg at 2.3 log cd/m 2 and decreases progressively by reducing the mean luminance. No pattern ERG can be recorded beyond −6. 7 log cd/m 2 at any spatial frequency. The pattern ERG amplitude decreases progressively by reducing the contrast. The extrapolated contrast threshold is about 1%. Acuity and contrast sensitivity ERG values are in the range of those obtained by operant techniques in other species with duplex retinae such as owls and cats.

Electron spin resonance study of light-induced conformational changes in nitroxide labelled bovine rhodopsin

Bovine rhodopsin was isolated in the unbleached form as a retinal disc membrane suspension and spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Both conventional and saturation transfer electron spin resonance methods were used to investigate the sensitivity of the spin-label to conformational changes of rhodopsin induced by both transient and long-term exposure to light. The results indicate that the ESR methods do display sensitivity to such changes. An exponential decay curve with a time constant of 10 s was obtained by following the height of a single peak in the saturation transfer electron spin resonance spectrum in response to a single light flash.

Photoreactivation in phr mutants of Escherichia coli k-12: Cloning from the gal-attλ interval increases the photoreactivable response in phrA strains

In this report we have cloned restriction fragments from the gal-attλ region obtained from a purified preparation of λ dgal transducing 'phage DNA, and demonstrate the appearance of a photoreactivable response in a photoreactivation-deficient phrA phrB strain. We also show that when this plasmid is transduced into a Δ phrA strain there is an increase in the photoreactivable response after a single high intensity light flash and after continuous illumination. These data have been discussed in relation to the hypothesis of the presence of multiple photolyase molecules in Escherichia coli.

Electrophysiological analysis of the action of nifedipine and nicardipine on myocardial fibers.

The effects of nifedipine and nicardipine, 2 dihydropyridines (DHP) used in the treatment of cardiovascular disorders, were compared in frog atrial fibers. Rapid photolysis of nifedipine with a single UV flash (1-ms duration) reversed the block, allowing comparison of effects of both drugs on the same preparation, and manipulation of the calcium channel on a millisecond timescale. The results show that inhibition of the action potential (AP) and slow inward current (Isi) is more pronounced with nifedipine than with nicardipine. Concentration-effect relationships confirm that nicardipine (IC50 = 1 microM) is less potent than nifedipine (IC50 = 0.2 microM) in blocking cardiac calcium channels. Both DHP block calcium channels in the closed state at the resting potential, inducing a large tonic block (in the absence of stimulation). An additional phasic block can be observed with nifedipine and nicardipine. A slight voltage dependence to the block is observed for both DHP, their effects being enhanced depolarization holding potentials. Rapid unblocking of calcium channels by a single light flash, presented during the decay phase of Isi, reveals a higher affinity of DHP for inactivated channels. This effect is most pronounced when inactivation is slowed by using Ba++, Sr++, or Na+ ions as the current carriers. Open channel block is also suggested. The mechanism of DHP action on calcium channels can be described according to the "modulated receptor hypothesis". These DHP exhibit an additional nonspecific effect on potassium channels. It is concluded that nicardipine is a less potent Ca++ antagonist than nifedipine in atrial fibers and that the reduction of delayed potassium current, which occurs in a similar range of concentrations to the blockade of Isi, could also be involved in its therapeutic effects.

Demonstration of a collisional interaction of ubiquinol with the ubiquinol-cytochrome c2 oxidoreductase complex in chromatophores from Rhodobacter sphaeroides

(1) Ubiquinone-10 can be extracted from lyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. (2) The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the E h range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative E h's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. (3) In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to E h values more positive than in controls. This effect is saturated when the size of the UQ pool is about 2–3-times larger than the native one. (4) The reduction of Cyt b-561 always occurs a short time after the flash is fired; also the duration of this lag is dependent on E h and on the size of the UQ pool. A decrease or an increase in the pool size causes a displacement of the titration curve of the lag to more negative or to more positive E h's, respectively. Similarly, the lag becomes E h independent and markedly longer than in controls when the pool is completely extracted. (5) These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b- c 1 complex depends on the actual concentration of ubiquinol present in the membrane and that ubiquinol from the pool is oxidized at this site with a collisional mechanism. Kinetic analysis of the data indicates that this reaction obeys a Michaelis-Menten type equation, with a K m of 3–5 ubiquinol molecules per reaction center.

Electroretinograms in Carriers of Blue Cone Monochromatism

We recorded full-field electroretinograms from seven female obligate carriers of X-linked blue cone monochromatism and eight daughters of obligate carriers. We observed that all obligate carriers had one or more of the following abnormalities: delayed cone b-wave implicit times to 30-Hz white flicker, loss of the a1 oscillation in responses to single flashes of white light under dark-adapted conditions, subnormal b-wave amplitudes to single flashes of white light under dark-adapted conditions, and subnormal cone responses to 30-Hz white flicker. All had normal rod responses to blue light. Three of eight daughters of obligate carriers had abnormal electroretinograms comparable to those recorded from obligate carriers. These obligate carriers have a partial but comparable deficiency of red and green cone function.

The optical cross section and absolute size of a photosynthetic unit

The concepts of a photosynthetic unit (PSU) and of an optical cross section are defined. The various estimates of sizes of photosynthetic units are described, and it is shown how an unambiguous measurement of the size of a unit can be obtained by measurement of its optical cross section via the saturation response to a single turnover light flash. The Emerson-Arnold unit must be divided by the quantum requirement for oxygen to obtain the true size of the unit. The size so obtained is the average number of chlorophylls per trap or reaction center. The effects of escape from open and closed traps are considered and it is shown that when these escape probabilities are equal, their effect on the saturation curve vanishes, leaving the simple cumulative one hit Poisson distribution.

Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli.

The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvr A6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp+ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp+ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7-2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvr A6 background.

Uncoupling of detectable O 2 evolution from the apparent S-state transitions in Photosystem II by lauroylcholine chloride: Possible implications in the photosynthetic water-splitting mechanism

Recently we have introduced the use of choline / fatty acid derived compounds, in particular lauroylcholine chloride (LCC), to probe selectively Photosystem II (PS II) structure and function (Wydrzynski, T. and Huggins, B.J. (1983) in The Oxygen-Evolving System of Photosynthesis (Inoue, Y., Crofts, A.R., Govindjee, Murata, N., Renger, G. and Satoh, K., eds.), pp. 265–272, Academic Press Tokyo, Japan). In this paper we report an unusual condition in thylakoid membrane samples at relatively low amounts of LCC in which detectable O 2 evolution cannot be measured, yet electron flow through PS II is near normal without added electron donors. LCC does not appear to interfere with the O 2 yield measurements directly nor act as an electron donor itself after the Tris block. Under this condition, steady state and flash O 2 yield measurements show no O 2 release or uptake, while steady-state ferricyanide photoreduction and the variable component of the chlorophyll a fluorescence transient remains at more than 50% of the control. The photoreduction of the primary quinone acceptor, Q A, measured by microsecond range chlorophyll a fluorescence continues for a minimum of 200 single turnover excitation light flashes. Most importantly, the yield of the 35 |gms component of the chlorophyll a delayed fluorescence remains at approx. 65% of the control and oscillates with a normal period four over two cycles, indicating the normal cycling of the S-state transitions in PS II. Thus, it appears that PS II can operate normally without detectable O 2 evolution. The question remains as to whether water is still being photooxidized under this condition without the release of the dioxygen product, or whether there is another source of electrons. The results are interpreted in terms of the possible existence of an additional water binding component (termed ‘H’) in PS II and a concerted oxidation reaction mechanism for photosynthetic water splitting.

Crystallization and dopant activation of amorphous silicon films by light annealing

Amorphous-silicon (a-Si) thin films prepared by three different methods, namely, glow discharge, low-pressure chemical-vapour deposition (LPCVD), and electron-beam evaporation, were crystallized using an incoherent white-light source. The light source used was a plasma arc with a duration of ~10 μs with an incident light intensity of ~5 J/cm2. The crystal-growth mechanism in a thin film is briefly discussed. The films were characterized by measurements of electrical conductivity and its temperature dependence, optical microscopy, x-ray diffraction, and Raman spectroscopy. The dark conductivity of the a-Si films differed greatly depending on the method of preparation and ranged from 10−11 to 10−6 Ω−1∙cm−1. After crystallization by a single light flash, they all had approximately the same dark conductivity in the range 10−5–10−4 Ω−1∙cm−1. Crystallization of doped LPCVD films by the light source resulted in a six orders of magnitude increase in their electrical conductivity, clearly indicating dopant activation.

A near infrared electronic transition associated with conversion between S‐states of the photosynthetic O2‐evolving complex

A new electronic transition is observable in O2‐evolving photosynthetic membranes of spinach enriched in Photosystem II. The transition occurs predominantly in the oxidation state created after a single flash of light (S2 state). It oscillates in yield with a period of flashes when exposed to a train of short light flashes. Conditions which abolish O2 evolution and remove functional manganese destroy this effect. The spectrum and absorptivity of the oscillating component are similar to those found in synthetic manganese dimers poised in the mixed‐valent oxidation state Mn2(III,IV). A similar structural unit is thus implicated for the manganese site involved in O2 evolution.

Light-induced proton gradients and internal volumes in chromatophores of Rhodopseudomonas sphaeroides

To test the predictions of the chemiosmotic hypothesis, it is essential to have sensitive and accurate measures of the aqueous volume and pH within membrane compartments. One unique feature of the present investigation is the application of electron spin resonance probes to determine internal aqueous volume and pH changes in bacterial chromatophores under virtually identical conditions. Volumes of the chromatophores ranged from 6 to 16 μl/mg bacteriochlorophyll among different preparations, and were sensitive to the osmolarity of the suspending buffer. pH gradients reached two units in illuminated chromatophores as determined with ESR methods, and increased when KCl and valinomycin were added to the assay. Measurements with the fluorescent dye 9-amino-acridine yielded similar pH gradients, provided that an operational vesicle volume, which corrected for the binding of the dye to the membrane, was used in the calculation. The sensitivity of the ESR method allowed the measurement of pH gradients resulting from only a few light flashes. A plot of pH gradients versus number of flashes was linear up to about 30 flashes, and intercepted the origin. This result is consistent with proton release into the bulk aqueous phase after only a single light flash. This ability to measure small pH gradients offers new opportunities for the study of energy-transducing mechanisms.

Differences in visual sensitivity among mindfulness meditators and non-meditators.

Tachistoscopic presentation of light flashes was used to test for differences in visual sensitivity among 3 groups of practitioners of Buddhist mindfulness meditation and non-meditator-controls. Meditation practitioners were able to detect light flashes of shorter duration than the non-meditators. There were no differences among the meditator groups. There were no differences among the groups in ability to discriminate between closely spaced successive light flashes. The lower detection threshold for single light flashes for the meditators may reflect an enduring increase in sensitivity, perhaps the long-term effects of the practice of mindfulness meditation on certain perceptual habit patterns. The lack of significant differences in the discrimination of successive light flashes probably reflects the resistance of other perceptual habit patterns to modification. The results support the statements found in Buddhist texts on meditation concerning the changes in perception encountered during the practice of mindfulness.

Ongoing compound field potentials from octopus brain are labile and vertebrate-like

Ongoing electrical activity was recorded from the brain of the virtually intact, semirestrained, unanesthetized octopus by semimicroelectrodes thrust through the cartilage into the optic, vertical or basal lobe. With flexible lead-in wires such electrodes were carried by and moved with the head without causing movement artifacts. Controls suggest that the activity reported comes from the brain; it is reversibly flattened by doses of urethane that do not embarrass respiration. Muscle potentials are only troublesome on occasion. Seen through a wideband filter, neuronal spikes are usually small or below noise level under these conditions; slow waves (1–70 Hz) dominate, with a maximum < 25 Hz, usually < 10 Hz and no consistent sharp peaks. The fall in power above ca. 25 Hz is usually slower than in the typical vertebrate EEG but the average power spectrum is much more like those of vertebrate brains than of cerebral ganglia of other invertebrates (insect, crustacean, gastropod). Variance among sample epochs is large, e.g. short spells may have relatively much more low frequency (< 25 Hz) or more ‘hashy’ high frequency (> 50 Hz) energy; there may be runs of spikes. Fluctuation in the composition of ongoing activity is graphically shown by writing out in parallel the outputs of several narrow band (one octave) filters; this shows irregular low frequency waxing and waning of the amplitude in each band. The envelopes were computed and their peak power is around 1 Hz or lower; the waxing and waning in the several bands is sometimes strongly correlated, especially when the envelope amplitude is large and slow. Optic lobe activity tends usually to be faster, with more small spikey hash than in the vertical lobe. The described electrical activity of the brain is strikingly episodic; it is recorded during seconds or minutes separated by long intervals of nearly electrical silence (10–40 dB lower power; the difference larger at frequencies > 20 Hz). Active and quite periods are usually not correlated with differences in visible behavior. Under my conditions the animal is usually not moving except for quiet ventilation and occasional local writhing of an arm. Potentials evoked by single flashes of light (0.2–1/sec) are conspicuous in the optic but not in the vertical lobe. They form a sequence of large, slow waves; the first peak may be at about 40 msec, others up to at least 400 msec. In a few attempts no evoked potentials were found for weak electric fields (100 μV/cm; 100 msec) such as electroreceptive fish respond to strongly. It is concluded that the compound field potential of the brain is not primarily the sum of spikes but of synaptic and other forms of graded potentials — as in vertebrate higher centers more than invertebrates.

Behavioral responses of the coastal copepod Acartia hudsonica (Pinhey) to simulated dinoflagellate bioluminescence

Light pulses were used to mimic dinoflagellate bioluminescence and test its effects on the swimming behavior of Acartia hudsonica (Pinhey). The horizontal swimming patterns of the copepod were tracked and described using a video-computer system. Single flashes of light of 60 ms duration, with a wavelength of peak emission of 475 nm and an intensity of 2 μE · m −2 · s −1 caused a “startle” response consisting of a short burst of high speed swimming. A series of these flashes repeated every 5 s resulted in higher average swimming speed, more swimming speed bursts, and straighter paths. These behavioral changes are similar to those previously found for A. hudsonica in the presence of bioluminescent dinoflagellates. The effects of altering the intensity, duration, and color of the simulated dinoflagellate flash were also tested. Our results support the hypothesis that dinoflagellate bioluminescence is a highly evolved adaptation for repelling nocturnal grazers.

Binary flash-induced oscillations of [ 14C]DCMU binding to the photosystem II acceptor complex

Binding of radioactively-labelled DCMU to spinach chloroplast membranes is determined following pre-illumination by single turnover saturating light flashes. When binding is assayed rapidly following inhibitor addition (< 40 s) distinct binary oscillations of [ 14C]DCMU binding can be observed. By dark-adapted samples or those, following an even number of flashes, more inhibitor is bound than after one or an odd number of flashes. During longer incubation times (2 min), [ 14C]DCMU binding becomes independent of flash preillumination. Comparison of the [ 14C]DCMU binding kinetics following one flash and the kinetics of the DCMU-induced increase of chlorophyll fluorescence rise reveals a substantial amount of binding which is not accompanied by a corresponding fluorescence rise. These data are discussed within the framework of the ‘inhibitor-plastoquinone competition model’ [FEBS Lett. (1981) 126, 277–281] and with reference to DCMU binding data derived from fluorescence measurements [Biochim. Biophys. Acta (1982) 682, 245–253].

The effect of temperature on the formation and decay of the multiline EPR signal species associated with photosynthetic oxygen evolution

We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S 2 state gives rise to this multiline signal; we find that the S 1 state can be fully advanced to the S 2 state at temperatures as low as 160 K. The S 2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.