Light-induced modulation of the mitochondrial respiratory chain activity: possibilities and limitations.

Biological effects of high fluence low-power (HFLP) lasers have been reported for some time, yet the molecular mechanisms procuring cellular responses remain obscure. A better understanding of the effects of HFLP lasers on living cells will be instrumental for the development of new experimental and therapeutic strategies. Therefore, we investigated sub-cellular mechanisms involved in the laser interaction with human hepatic cell lines. We show that mitochondria serve as sub-cellular "sensor" and "effector" of laser light non-specific interactions with cells. We demonstrated that despite blue and red laser irradiation results in similar apoptotic death, cellular signaling and kinetic of biochemical responses are distinct. Based on our data, we concluded that blue laser irradiation inhibited cytochrome c oxidase activity in electron transport chain of mitochondria. Contrary, red laser triggered cytochrome c oxidase excessive activation. Moreover, we showed that Bcl-2 protein inhibited laser-induced toxicity by stabilizing mitochondria membrane potential. Thus, cells that either overexpress or have elevated levels of Bcl-2 are protected from laser-induced cytotoxicity. Our findings reveal the mechanism how HFLP laser irradiation interfere with cell homeostasis and underscore that such laser irradiation permits remote control of mitochondrial function in the absence of chemical or biological agents.

Effects of photobiomodulation on mitochondria of brain, muscle, and C6 astroglioma cells.

The purpose of this study was to investigate the effect of different doses of photobiomodulation (PBM) on mitochondrial respiratory complexes and oxidative cellular energy metabolic enzymes in the mitochondria of brain, muscle, and C6 glioma cells after different time intervals. C6 cells were irradiated with an AlGaInP laser at 10, 30, and 60 J/cm2 for 20, 60, and 120 s, respectively. After irradiation, the cells were maintained in serum-free Dulbecco's Modified Eagle's medium for 24 h, and biochemical measurements were made subsequently. Mitochondrial suspensions from adult rat skeletal muscles/brains were irradiated with an AlGaInP laser at the abovementioned doses. In one group, the reaction was stopped 5 min after irradiation and in the other 60 min after irradiation. Both the C6 cells that received the doses of 10 and 30 J/cm² showed increased complex I activity; the cells that were irradiated at 30 J/cm2 showed increased hexokinase activity. Five minutes after the introduction of PBM of the muscle mitochondria (at 30 and 60 J/cm2), the activity of complex I increased, while the activity of complex IV increased only at 60 J/cm2. One hour after the laser session, complex II activity increased in the cells treated with 10 and 60 J/cm²; however, complex IV activity showed an increase in all PBM groups. In brain mitochondria, 5 min after irradiation only the activity of complex IV increased in all PBM groups. One hour after the laser session, complex II activity increased at 60 J/cm2, and complex IV activity increased for all PBM groups when compared to controls. PBM could increase the activity of respiratory chain complexes in an apparently dose- and time-dependent manner.

Unexpected dose response of HaCaT to UVB irradiation.

Application of high-dosage UVB irradiation in phototherapeutic dermatological treatments present health concerns attributed to UV-exposure. In assessing UV-induced photobiological damage, we investigated dose-dependent effects of UVB irradiation on human keratinocyte cells (HaCaT). Our study implemented survival and apoptosis assays and revealed an unexpected dose response wherein higher UVB-dosage induced higher viability. Established inhibitors, such as AKT- (LY294002), PKC- (Gö6976, and Rottlerin), ERK- (PD98059), P38 MAPK- (SB203580), and JNK- (SP600125), were assessed to investigate UV-induced apoptotic pathways. Despite unobvious contributions of known signaling pathways in dose-response mediation, microarray analysis identified transcriptional expression of UVB-response genes related to the respiratory-chain. Observed correlation of ROS-production with UVB irradiation potentiated ROS as the underlying mechanism for observed dose responses. Inability of established pathways to explain such responses suggests the complex nature underlying UVB-phototherapy response.

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain.

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley (Hordeum vulgare). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b6f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue.

(Re)-wiring a brain with light: Clinical and visual processing findings after application of specific coloured glasses in patients with symptoms of a visual processing disorder (CVPD): Challenge of a possible new perspective?

The present investigation examined whether changes of electrophysiological late event related potential pattern could be used to reflect clinical changes from therapeutic intervention with coloured glasses in a group of patients with symptoms of central visual processing disorder. Subjects consisted of 13 patients with average age 16years (range 6-51years) with attention problems and learning disability, respectively. These patients were provided with specified coloured glasses which were required to be used during day time. Results indicated that specified coloured glasses significantly improved attention performance. Furthermore electrophysiological parameters revealed a significant change in the late event related potential distribution pattern (latency, amplitudes). This reflects a synchronization of together firing wired neural assemblies responsible for visual processing, suggesting an accelerated neuromaturation process when using coloured glasses. Our results suggest that the visual event related potentials measures are sensitive to changes in clinical development of patients with deficits of visual processing wearing appropriate coloured glasses. It will be discussed whether such a device might be useful for a clinical improvement of distraction symptoms caused by visual processing deficits. A model is presented explaining these effects by inducing the respiratory chain of the mitochondria such increasing the low energy levels of ATP of our patients.

Cancer phototherapy via selective photoinactivation of respiratory chain oxidase to trigger a fatal superoxide anion burst.

AIMS: Here, we develop a novel cancer treatment modality using mitochondria-targeting, high-fluence, low-power laser irradiation (HF-LPLI) in mouse tumor models and explore the mechanism of mitochondrial injury by HF-LPLI. RESULTS: We demonstrated that the initial reaction after photon absorption was photosensitization of cytochrome c oxidase (COX), to inhibit enzymatic activity of COX in situ and cause respiratory chain superoxide anion (O2(-•)) burst. We also found that HF-LPLI exerted its main tumor killing effect through mitochondrial O2(-•) burst via electron transport chain (ETC). These phenomena were completely absent in the respiration-deficient cells and COX knockdown cells. With a carefully selected irradiation protocol, HF-LPLI could efficaciously destroy tumors. The inhibition of enzymatic activity of COX and generation of O2(-•) by HF-LPLI in vivo were also detected. INNOVATION: It is the first time that the mechanism involved in the interaction between light and its photoacceptor under HF-LPLI treatment is clarified. Our results clearly indicate that HF-LPLI initiates its effects via targeted COX photoinactivation and that the tumor-killing efficacy is dependent of the subsequent mitochondrial O2(-•) burst via ETC. CONCLUSION: Based on both in vitro and in vivo results, we conclude that HF-LPLI can selectively photoinactivate respiratory chain oxidase to trigger a fatal mitochondrial O2(-•) burst, producing oxidative damage on cancer cells. This study opens up the possibilities of applications of HF-LPLI as a mitochondria-targeting cancer phototherapy.

Comparative physiological responses of Solanum nigrum and Solanum torvum to cadmium stress.

• Under cadmium (Cd) stress, Solanum nigrum accumulated threefold more Cd in its leaves and was tolerant to Cd, whereas its low Cd-accumulating relative, Solanum torvum, suffered reduced growth and marked oxidative damage. However, the physiological mechanisms that are responsible for differential Cd accumulation and tolerance between the two Solanum species are largely unknown. • Here, the involvement of antioxidative capacity and the accumulation of organic and amino acids in response to Cd stress in the two Solanum species were assessed. • Solanum nigrum contains higher antioxidative capacity than does S. torvum under Cd toxicity. Metabolomics analysis indicated that Cd treatment also markedly increased the production of several organic and amino acids in S. nigrum. Pretreatment with proline and histidine increased Cd accumulation; moreover, pretreatment with citric acid increased Cd accumulation in leaves but decreased Cd accumulation in roots, which indicates that its biosynthesis could be linked to Cd long-distance transport and accumulation in leaves. • Our data provide novel metabolite evidence regarding the enhancement of citric acid and amino acid biosynthesis in Cd-treated S. nigrum, support the role of these metabolites in improving Cd tolerance and accumulation, and may help to provide a better understanding of stress adaptation in other Solanum species.

Relationships between the photochemical reflectance index (PRI) and chlorophyll fluorescence parameters and plant pigment indices at different leaf growth stages.

This study aimed to evaluate the photochemical reflectance index (PRI) for assessing plant photosynthetic performance throughout the plant life cycle. The relationships between PRI, chlorophyll fluorescence parameters, and leaf pigment indices in Solanum melongena L. (aubergine; eggplant) were studied using photosynthetic induction curves both in short-term (diurnal) and long-term (seasonal) periods under different light intensities. We found good correlations between PRI/non-photochemical quenching (NPQ) and PRI/electron transport rate (ETR) in the short term at the same site of a single leaf but these relationships did not hold throughout the life of the plant. In general, changes in PRI owing to NPQ or ETR variations in the short term were <20 % of those that occurred with leaf aging. Results also showed that PRI was highly correlated to plant pigments, especially chlorophyll indices measured by spectral reflectance. Moreover, relationships of steady-state PRI/ETR and steady-state PRI/photochemical yield of photosystem II (Φ(PSII)) measured at uniform light intensity at different life stages proved that overall photosynthesis capacity and steady-state PRI were better correlated through chlorophyll content than NPQ and xanthophylls. The calibrated PRI index accommodated these pigments effects and gave better correlation with NPQ and ETR than PRI. Further studies of PRI indices based on pigments other than xanthophylls, and studies on PRI mechanisms in different species are recommended.

Combined effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101: a mechanistic view.

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO(2) partial pressure (pCO(2)) with higher N(2) fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO(2) (150 and 900 microatm) and light (50 and 200 micromol photons m(-2) s(-1)) on Trichodesmium IMS101. We expand on a complementary study that demonstrated that while elevated pCO(2) enhanced N(2) fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO(2) and light controlled the operation of the CO(2)-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO(2) and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N(2) fixation and growth at elevated pCO(2) and light. We suggest that changes in the redox state of the photosynthetic electron transport chain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enable Trichodesmium to flourish in future surface oceans characterized by elevated pCO(2), higher temperatures, and high light.

Photoinduced electron transfer in porphyrin- and fullerene/porphyrin-based rotaxanes as studied by time-resolved EPR spectroscopy.

Photoinduced intramolecular electron-transfer (ET) and energy-transfer (EnT) processes in two rotaxanes, one containing both zinc porphyrin and C(60) fullerene moieties incorporated around the Cu(I) bisphenanthroline core [(ZnP)(2)-Cu(I)(phen)(2)-C(60)] and a second complex lacking the fullerene [(ZnP)(2)-Cu(I)(phen)(2)], were studied by time-resolved electron paramagnetic resonance (TREPR) spectroscopy at 9.5 GHz (X-band) combined with a selective photoexcitation of the rotaxane moieties. The experiments were carried out in isotropic toluene and ethanol and in anisotropic nematic liquid-crystal (E-7) media over a wide range of temperatures corresponding to the different states of the solvents. The TREPR results are compared with those obtained previously by optical methods in dichloromethane at room temperature. It is demonstrated that the efficiencies and pathways of the light-driven ET and EnT processes in both rotaxanes strongly depend on the properties of their microenvironment, resulting in the formation of distinct charge-separated states under different experimental conditions. The complementary results revealed by the optical and TREPR techniques are attributed to the relatively high conformational mobility of the mechanically interlocked rotaxane systems. Because of the solute-solvent interactions, the rotaxanes are able to change conformation in different microenvironments, which affects the parameters of the photoinduced processes occurring in these systems.

Evaluation of mitochondrial respiratory chain activity in muscle healing by low-level laser therapy.

BACKGROUND: Recent studies demonstrate that low-level laser therapy (LLLT) modulates many biochemical processes, especially the decrease of muscle injures, the increase in mitochondrial respiration and ATP synthesis for accelerating the healing process. OBJECTIVE: In this work, we evaluated mitochondrial respiratory chain complexes I, II, III and IV and succinate dehydrogenase activities after traumatic muscular injury. METHODS: Male Wistar rats were randomly divided into three groups (n=6): sham (uninjured muscle), muscle injury without treatment, muscle injury with LLLT (AsGa) 5J/cm(2). Gastrocnemius injury was induced by a single blunt-impact trauma. LLLT was used 2, 12, 24, 48, 72, 96, and 120 hours after muscle-trauma. RESULTS: Our results showed that the activities of complex II and succinate dehydrogenase after 5days of muscular lesion were significantly increased when compared to the control group. Moreover, our results showed that LLLT significantly increased the activities of complexes I, II, III, IV and succinate dehydrogenase, when compared to the group of injured muscle without treatment. CONCLUSION: These results suggest that the treatment with low-level laser may induce an increase in ATP synthesis, and that this may accelerate the muscle healing process.

Low-intensity light therapy: exploring the role of redox mechanisms.

Low-intensity light therapy (LILT) appears to be working through newly recognized photoacceptor systems. The mitochondrial electron transport chain has been shown to be photosensitive to red and near-infrared (NIR) light. Although the underlying mechanisms have not yet been clearly elucidated, mitochondrial photostimulation has been shown to increase ATP production and cause transient increases in reactive oxygen species (ROS). In some cells, this process appears to participate in reduction/oxidation (redox) signaling. Redox mechanisms are known to be involved in cellular homeostasis and proliferative control. In plants, photostimulation of the analogous photosynthetic electron transport chain leads to redox signaling known to be integral to cellular function. In gene therapy research, ultraviolet lasers are being used to photostimulate cells through a process that also appears to involve redox signaling. It seems that visible and near visible low-intensity light can be used to modulate cellular physiology in some nonphotosynthetic cells, acting through existing redox mechanisms of cellular physiology. In this manner, LILT may act to promote proliferation and/or cellular homeostasis. Understanding the role of redox state and signaling in LILT may be useful in guiding future therapies, particularly in conditions associated with pro-oxidant conditions.

Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones.

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, QA and QB, play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from QA to QB takes place in the time range of 5-200 micros. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which QB- formation precedes QA- oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between QA and QB has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the QA to QB electron transfer is slowed 8-fold compared to that in native RCs. This IR signature has been reported as a difference spectrum involving states X+, X, QA, and QA- [Hermes, S., et al. (2006) Biochemistry 45, 13741-13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either QA or QB measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the QA-/QA and QB-/QB difference spectra. The calculated QA- QB/QA QB- double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported QA- X+/QA X spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from QB and QB-, respectively. Further comparison of the QA- QB/QA QB- double-difference spectra in native and DN(L210) RCs also allows the possibility that QB- formation precedes QA- reoxidation to be ruled out for native RCs.

Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy.

Laser therapy is used in many biomedical sciences to promote tissue regeneration. Many studies involving low-level laser therapy have shown that the healing process is enhanced by such therapy. In this work, we evaluated mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase activities in wounds after irradiation with low-level laser. The animals were divided into two groups: group 1, the animals had no local nor systemic treatment and were considered as control wounds; group 2, the wounds were treated immediately after they were made and every day after with a low-level laser (AsGa, wavelength of 904 nm) for 10 days. The results showed that low-level laser therapy improved wound healing. Besides, our results showed that low-level laser therapy significantly increased the activities of complexes II and IV but did not affect succinate dehydrogenase activity. These findings are in accordance to other works, where cytochrome c oxidase (complex IV) seems to be activated by low-level laser therapy. Besides, we showed, for the first time, that complex II activity was also activated. More studies are being carried out in order to evaluate other mitochondrial enzymes activities after different doses and irradiation time of low-level laser.

[The responses of photosynthesis to strong light in the medicinal plants Anisodus tanguticus (Maxim.) Pascher and Rheum tanguticum Maxim. on the Qinghai-Tibet Plateau].

Photosynthetic electron transport and light energy allocation were studied in the alpine plants Anisodus tanguticus (Maxim.) Pascher and Rheum tanguticum Maxim. ex Balf on the Qinghai-Tibet Plateau by using gas exchange and chlorophyll fluorescence. The results indicated that apparent quantum yield (AQY) of leaves of A. tanguticus was marginally higher than that of R. tanguticum although it had a lower maximum net photosynthetic rate (Pmax). The net photosynthetic rate (P(n)) of A. tanguticus was higher than R. tanguticum within the range of middle photosynthetic photon flux density (PPFD). However, the P(n) in R. tanguticum increased concomitantly with PPFD and did not appear to show light saturation of P(n) even under 2000 micromol m(-2) s(-1) which is similar to full light in summer (Fig.1). Increasing the PPFD to 1200 micromol m(-2) s(-1) decreased the ratio of carboxylation rate to total photosynthetic electron flow rate (J(C)/J(F)) although increased the ratio of photorespiration (J(O)/J(F)) for both species. Both J(C)/J(F) and J(O)/J(F) stabilized with a PPFD of more than 1200 micromol m(-2) s(-1) (Fig.2). The changes in the ratios of Rubisco oxygenation to carboxylation (V(O)/V(C)) were similar to changes to J(O)/J(F) (Fig.3). The increase of thermal energy dissipation (D) in A. tanguticus was higher than R. tanguticum with increased PPFD (Fig.4). It can be concluded that the two species adopt different mechanisms to cope with increased solar radiation. Increasing the fractions of PSII thermal energy dissipation and electron transport through photorespiration were the main adaptations in A. tanguticus. Enhancement of photosynthetic capacity with increased PPFD to balance the higher light energy absorbed by leaves is considered the main adaptation for R. tanguticum.

Influence of UV-B exclusion and selenium treatment on photochemical efficiency of photosystem II, yield and respiratory potential in pumpkins (Cucurbita pepo L.).

The influence of ambient and filtered solar ultraviolet-B (UV)-B radiation and of selenium treatment was determined on photochemical efficiency, respiratory potential measured by electron transport system (ETS) activity, and yield in pumpkins, Cucurbita pepo L. Yield and ETS activity were higher when solar UV-B was filtered out. The results suggested that the decreased yield was related to the UV-B impaired flow of electrons in the respiratory chain. Selenium increased yield under ambient radiation conditions. However, no significant effect of excluding UV-B radiation or of treatment with selenium was observed on the photochemical efficiency of photosystem II (PSII).

Light-dependent gene expression for proteins in the respiratory chain of potato leaves.

Expression of genes for respiratory chain dehydrogenases was investigated in potato (Solanum tuberosum L. cv. Desiree) leaves. The recently characterized nda1 and ndb1 genes, homologues to genes encoding the non-proton pumping respiratory chain NADH-dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). As leaves develop from young to mature, the nda1 transcript level increases, accompanied by an elevation in immunodetected NDA protein and internal rotenone-insensitive NADH oxidation. The other investigated transcripts, proteins and NAD(P)H oxidation activities were essentially unchanged. A variation in transcript level, specific for nda1, is seen at different times of the day with highest expression in the morning. This variation also influences the apparent developmental induction. Further, the nda1 mRNA in leaves specifically and completely disappears during dark treatment, with a rapid re-induction when plants are returned to light. Corresponding immunodetected NDA protein is specifically decreased in mitochondria isolated from dark-treated plants, accompanied by a lower capacity for internal rotenone-insensitive NADH oxidation. Complete light dependence and diurnal changes in expression have previously not been reported for genes encoding respiratory chain proteins. Qualitatively similar to NDA, the alternative oxidase showed developmental induction and light dependence. In addition to the specific change in nda1, a general, slower down-regulation in darkness was seen for the other NAD(P)H dehydrogenase genes. The nda1 expression during development, and in response to light, indicates a specific role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.

Destruction of vitamin K1 of cultured carrot cells by ultraviolet radiation and its effect on plasma membrane electron transport reactions.

The effect of ultraviolet radiation on plasma membrane electron transport reactions was studied in cultured carrot cells. It was found that a 90 min treatment inhibited transmembrane hexacyanoferrate reduction greater than 50%. Extraction of lipophilic quinones from irradiated cells showed that vitamin K1 and coenzyme Q were totally destroyed, while control unirradiated cells showed the presence of 0.4 mumole vitamin K1 g dry wt.-1. The addition of exogenous vitamin K1 in concentrations of 1-10 microM partially restored plasma membrane electron transport with impermeable hexacyanoferrate as the electron acceptor. Total restoration of activity was given by growing irradiated cells in vitamin K1 supplemented growth media for 6 days. This shows that vitamin K1 may function as a member of the transplasma membrane electron transport chain in cultured carrot cells.