Blue light induces mitochondrial DNA damage and free radical production in epithelial cells

Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390-550 nm at 2.8 milliwatts/cm2) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis.     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.     

Photosynthetic functions and thylakoid membrane polypeptide composition in light-sensitive mutants of Chlamydomonas reinhardii

In this paper we compared the pigment composition, photochemical activity, chloroplast ultrastructure, thylakoid membrane polypeptide composition and ribosomal content of wild-type and seven light-sensitive mutants of Chlamydomonas reinhardii.All the mutants had low chlorophyll and carotenoid content compared to wild-type. Mutants lts-30 and lts-135 were also characterized by a complete absence of visible carotenoids, while mutant lts-19 was fully deficient in chlorophylls.In most mutants, the chloroplast fragment could not carry out any DCIP photoreduction and O₂ evolution was also blocked. The PSI/P₇₀₀/activity was decreased in most cases.The mutant strains contained mostly single lamellae in their plastids, that is the stacking capacity of the thylakoid membranes was very decreased or fully absent. In most cases the number of lamellae was also very low.The relative amounts of 70 S ribosomes were decreased in all of the mutants. The thylakoid membranes showed anomalies in the region of 24 000–30 000 dalton polypeptides. The common characteristic for them was the relatively higher amount of the 30 000 dalton polypeptide and considerably decreased level of the 27 000 and 24 000 dalton polypeptides relative to the wild-type. These polypeptides were probably constituents of the chlorophyll-protein complex II which has been suggested to be the light harvesting pigment complex for PSII. The polypeptide of 30 000 daltons is the precursor for the LHCP apoprotein (24 000 dalton protein). It may be that the lighstimulated conversion of this precursor into LHCP apoprotein was blocked in our pigment-deficient mutants.Abbreviations CPI Chlorophyll-protein complex I - PSI Photosystem I - PSII Photosystem II - LHCP Light-harvesting pigment complex - DCIP 2,6-dichlorophenolindophenol - RuDPC-ase Ribulose-1,5-biphosphate-carboxylase - SDS Sodium dodecyl sulfate - LIDS Lithium dodecyl sulfate - PAG Polyacrylamide gel - TKM buffer 25 mM Tris-HCl, pH 7.S; 25 mM KCl; 25 mM Mg acetate     

Lipid peroxidation induces cholesterol domain formation in model membranes

Numerous reports have established that lipid peroxidation contributes to cell injury by altering the basic physical properties and structural organization of membrane components. Oxidative modification of polyunsaturated phospholipids has been shown, in particular, to alter the intermolecular packing, thermodynamic, and phase parameters of the membrane bilayer. In this study, the effects of oxidative stress on membrane phospholipid and sterol organization were measured using small angle x-ray diffraction approaches. Model membranes enriched in dilinoleoylphosphatidylcholine were prepared at various concentrations of cholesterol and subjected to lipid peroxidation at physiologic conditions. At cholesterol-to-phospholipid mole ratios (C/P) as low as 0.4, lipid peroxidation induced the formation of discrete, membrane-restricted cholesterol domains having a unit cell periodicity or d-space value of 34 A. The formation of cholesterol domains correlated directly with lipid hydroperoxide levels and was inhibited by treatment with vitamin E. In the absence of oxidative stress, similar cholesterol domains were observed only at C/P ratios of 1.0 or higher. In addition to changes in sterol organization, lipid peroxidation also caused reproducible changes in overall membrane structure, including a 10 A reduction in the width of the surrounding, sterol-poor membrane bilayer. These data provided direct evidence that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.     

Bupivacaine hydrochloride induces muscle fiber necrosis and hydroxyl radical formation-dimethyl sulphoxide reduces hydroxyl radical formation

We induced acute skeletal muscle necrosis in rats using bupivacaine hydrochloride and found that both 2,5- and 2,3-dihydroxybenzoic acid significantly increased in skeletal muscle. A single administration of dimethyl sulphoxide, a free radical scavenger, significantly lowered concentrations of 2,5- and 2,3-dihydroxybenzoic acid. These results suggest that dimethyl sulphoxide is an effective hydroxyl radical scavenger and may be useful in the treatment of myopathy.     

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.     

Radical formation in single crystals of aromatic amines and radical transformation by light

Single crystals of the four aromatic bioamine salts phenylethylamine hydrochloride, tyramine hydrochloride, tryptamine hydrochloride, and histamine dihydrochloride were grown in various states of deuteration. Free radicals were produced by exposure to X-rays between 77 and 300 K and investigated by electron spin resonance spectroscopy. Dissociation of atomic hydrogen from C beta of the aliphatic chain occurs in all compounds studied except tryptamine. However deamination as usually present in the analogous amino acids is not found. The C beta-radical is characterized by an anisotropic H alpha-splitting and two isotropic H beta-splittings. The latter splittings depend strongly on temperature in tyramine. In comparison to the analogous amino acids, radical formation in the aromatic residues is favoured. Among the seven different aromatic radicals found only one is identified in histamine but two in each of the three other bioamines. Two of these are characterized by hydrogen dissociation which occurs in phenylethylamine and tyramine. One hydrogen addition radical is found in each of the three compounds phenylethylamine, tyramine and histamine. In tryptamine two different addition radicals are detected. One of the two products can be converted into the other by visible light. The reverse process is induced by heat, thus permitting the switching of the radical site reversibly between two different structures.     

Tyrosine 740 phosphorylation of discoidin domain receptor 2 by Src stimulates intramolecular autophosphorylation and Shc signaling complex formation

DDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes.     

Regulation of WNK1 by an autoinhibitory domain and autophosphorylation

WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.     

Blue light induces stomatal transpiration in wheat seedlings with chlorophyll deficiency caused by SAN 9789

Wheat seedlings ( Truicum aestivum L. cv. Starke II, Weibull) grown in a solution of the herbicide SAN 9789 axe deficient in chlorophyll and carotenoids. Such seedlings were used in order to isolate a blue light response of stomata, not mediated by photosynthesis. When illuminated with blue light (430-505 nm) SAN-grown seedlings showed a considerable transpiration response, whereas red light (>590 nm) gave no response whatsoever with the intensities used. The transpiration was measured with hygrometers in an open air-flow system. Furthermore, blue light had a superior effect, relative to red, in causing a transpiration response in untreated green seedlings. The transpiration level after two hours of illumination was higher in blue than in red light, although the blue light had its major effect immediately after the light was switched on. The difference between the effects of blue and red light was most pronounced at low light intensities. This, together with the high sensitivity of SAN-grown seedlings to low ntensity blue light, led to the conclusion that blue light is of special importance at low quantum fluxes. Because of the very low carotenoid content in the SAN-grown seedlings, the role of the carotenoids as photoreceptors for the blue light response of stomata was excluded.     

Blue Light Induction of Carbonic Anhydrase Activity in Chlamydomonas reinhardtii

Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m². The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. ¹On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)     

Trifluoperazine induces domain formation in zwitterionic phosphatidylcholine but not in charged phosphatidylglycerol bilayers

The interaction of trifluoperazine with the zwitterionic lipids dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine and with anionic dimyristoylphosphatidylglycerol was studied by means of microcalorimetry and fluorescence spectroscopy. Intercalation of drug molecules into the lipid bilayers was confirmed by the observed differential scanning calorimetry peak broadening and the decrease in chain-melting temperatures. For trifluoperazine:lipid mole ratios higher than 0.4 and 0.6 (for dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, respectively) the deconvolution of transition profiles into two Gaussian components was possible, which suggests phase separation in the studied mixtures. Deconvolution of the thermograms was not possible for any of the drug:dimyristoylphosphatidylglycerol mole ratios studied. To confirm the existence of phase separation in trifluoperazine-phosphatidylcholine mixtures fluorescence spectroscopy experiments were performed using Laurdan as a probe. From the generalised polarisation versus excitation wavelength dependences, recorded at different temperatures, we conclude that a phase separation occurs in the gel state of the studied trifluoperazine-phosphatidylcholine mixtures. We attribute the existence of domains in the bilayer to the dissimilar interactions of two protonation forms of trifluoperazine with phosphatidylcholine molecules. Structural defects present at domain boundaries could be related to the trifluoperazine induced increase of membrane permeability and fluidity. This may partially explain the mechanism of multidrug resistance modulation by trifluoperazine.     

Blue light and the regeneration of human rhodopsin in situ

Hubbard has found that the photoisomerization of retinene was important for the regeneration of rhodopsin in vitro, and the object of the present investigation was to find whether this was also true for regeneration in the living human eye. In the Appendix is described a device which permits the rhodopsin density to be measured by analysing the light reflected from the fundus oculi in an ophthalmoscopic arrangement, the measurement taking about 5 seconds. Now if a blue and a yellow light viewed scotopically are adjusted in intensity so as to appear identical, they must bleach rhodopsin equally, but the blue will be more than 10 times as effective in isomerizing retinene. Therefore if retinene isomerization is important for rhodopsin regeneration, blue light should cause a more rapid regeneration after bleaching, and during bleaching the equilibrium level attained should be less profound. But, as the figures show, the course of bleaching and regeneration is identical for the matched yellow or blue bleaching lights, therefore isomerization of retinene is not important for rhodopsin regeneration in the living human eye.     

The circadian clock, light, and cryptochrome regulate feeding and metabolism in Drosophila

Recent studies in mammals have demonstrated a central role for the circadian clock in maintaining metabolic homeostasis. In spite of these advances, however, little is known about how these complex pathways are coordinated. Here, we show that fundamental aspects of the circadian control of metabolism are conserved in the fruit fly Drosophila. We assay feeding behavior and basic metabolite levels in individual flies and show that, like mammals, Drosophila display a rapid increase in circulating sugar following a meal, which is subsequently stored in the form of glycogen. These daily rhythms in carbohydrate levels are disrupted in clock mutants, demonstrating a critical role for the circadian clock in the postprandial response to feeding. We also show that basic metabolite levels are coordinated in a clock-dependent manner and that clock function is required to maintain lipid homeostasis. By examining feeding behavior, we show that flies feed primarily during the first 4 hours of the day and that light suppresses a late day feeding bout through the cryptochrome photoreceptor. These studies demonstrate that central aspects of feeding and metabolism are dependent on the circadian clock in Drosophila. Our work also uncovers novel roles for light and cryptochrome on both feeding behavior and metabolism.     

Interplay between kinase domain autophosphorylation and F-actin binding domain in regulating imatinib sensitivity and nuclear import of BCR-ABL

Background

The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood.

Methodology/Principal Findings

By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib.

Conclusions/Significance

Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML.     

Regulation of the Chlamydomonas cell cycle by light and dark

By growing cells in alternating periods of light and darkness, we have found that the synchronization of phototrophically grown Chlamydomonas populations is regulated at two specific points in the cell cycle: the primary arrest (A) point, located in early G1, and the transition (T) point, located in mid-G1. At the A point, cell cycle progression becomes light dependent. At the T point, completion of the cycle becomes independent of light. Cells transferred from light to dark at cell cycle position between the two regulatory points enter a reversible resting state in which they remain viable and metabolically active, but do not progress through their cycles. The photosystem II inhibitor dichlorophenyldimethylurea (DCMU) mimics the A point block induced by darkness. This finding indicates that the A point block is mediated by a signal that operates through photosynthetic electron transport. Cells short of the T point will arrest in darkness although they contain considerable carbohydrate reserves. After the T point, a sharp increase occurs in starch degradation and in the endogenous respiration rate, indicating that some internal block to the availability of stored energy reserves has now been released, permitting cell cycle progression.     

New retinal light damage QTL in mice with the light-sensitive RPE65 LEU variant

The purpose of this study was to determine the QTL that influence acute, light-induced retinal degeneration differences between the BALB/cByJ and 129S1/SvImJ mouse strains. Five- to 6-week-old F₂ progeny of an intercross between the two strains were exposed to 15,000 LUX of white light for 1 h after their pupils were dilated, placed in the dark for 16 h, and kept for 10–12 days in dim cyclic light before retinal rhodopsin was measured spectrophotometrically. This was used as the quantitative trait for retinal degeneration. Neither gender nor pigmentation had a significant influence on the amount of rhodopsin after light exposure in the F₂ progeny. For genetic study, DNAs of the 27–36 F₂ progeny with the highest and 27–36 F₂ with the lowest levels of rhodopsin after light exposure were genotyped with 71 dinucleotide repeat markers spanning the genome. Any marker with a 95% probability of being associated with phenotype was tested in all 289 F₂ progeny. Data were analyzed with Map Manager QTX. Significant QTL were found on mouse Chrs 1 and 4, and suggestive QTL on Chrs 6 and 2. The four QTL together equal an estimated 78% of the total genetic effect, and each of the QTL represents a gene with BALB/c susceptible alleles. The Chr 6 QTL is in the same region as a highly significant age-related retinal degeneration QTL found previously. Identification of these QTL is a first step toward identifying the modifier genes/alleles they represent, and identification of the modifiers may provide important information for human retinal diseases that are accelerated by light exposure.