Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.

The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.

Photobiomodulation Mediates Neuroprotection against Blue Light Induced Retinal Photoreceptor Degeneration.

Potent neuroprotective effects of photobiomodulation with 670 nm red light (RL) have been demonstrated in several models of retinal disease. RL improves mitochondrial metabolism, reduces retinal inflammation and oxidative cell stress, showing its ability to enhance visual function. However, the current knowledge is limited to the main hypothesis that the respiratory chain complex IV, cytochrome c oxidase, serves as the primary target of RL. Here, we demonstrate a comprehensive cellular, molecular, and functional characterization of neuroprotective effects of 670 nm RL and 810 nm near-infrared light (NIRL) on blue light damaged murine primary photoreceptors. We show that respiratory chain complexes I and II are additional PBM targets, besides complex IV, leading to enhanced mitochondrial energy metabolism. Accordingly, our study identified mitochondria related RL- and NIRL-triggered defense mechanisms promoting photoreceptor neuroprotection. The observed improvement of mitochondrial and extramitochondrial respiration in both inner and outer segments is linked with reduced oxidative stress including its cellular consequences and reduced mitochondria-induced apoptosis. Analysis of regulatory mechanisms using gene expression analysis identified upregulation α-crystallins that indicate enhanced production of proteins with protective functions that point to the rescued mitochondrial function. The results support the hypothesis that energy metabolism is a major target for retinal light therapy.

Association between maternal omega-3 polyunsaturated fatty acids supplementation and preterm delivery: A proteomic study.

Maternal nutrition during pregnancy influences offspring health. Dietary supplementation of pregnant women with (n-3) long-chain polyunsaturated fatty acids (PUFA) was shown to exert beneficial effects on offspring, through yet unknown mechanisms. Here, we conducted a dietary intervention study on a cohort of 10 women diagnosed with threatened preterm labor with a nutritional integration with eicosapentaenoic and docosahexaenoic acids. Microvesicles (MV) isolated form arterial cord blood of the treated cohort offspring and also of a randomized selection of 10 untreated preterm and 12 term newborns, were characterized by dynamic light scattering and analyzed by proteomic and statistical analysis. Glutathione synthetase was the protein bearing the highest discrimination ability between cohorts. ELISA assay showed that glutathione synthetase was more abundant in cord blood from untreated preterm compared to the other conditions. Assay of free SH-groups showed that serum of preterm subjects was oxidized. Data suggest that preterm suffer from oxidative stress, which was lower in the treated cohort. This study confirms that MV are a representative sample of the individual status and the efficacy of dietary intervention with PUFA in human pregnancy in terms of lowered inflammatory status, increased gestational age and weight at birth.

The novel diterpene 7ß-acetoxy-20-hydroxy-19,20-epoxyroyleanone from Salvia corrugata shows complex cytotoxic activities against human breast epithelial cells.

AIMS: The aim of this study was the characterization of the in vitro cytotoxic properties of a recently isolated diterpene compound, 7ß-acetoxy-20-hydroxy-19,20-epoxyroyleanone (compound 1), extracted from Salvia corrugata, versus human cell lines. MAIN METHODS: We used as model study immortalized breast epithelial cells MCF10A and two ERBB2+ breast cancer (BCa) cell lines, SKBR-3 and BT474. Compound 1 was isolated by methanolic extraction from regenerated shoots of Salvia corrugata Vahl, and purified by high pressure liquid chromatography (HPLC). Flow cytometry (FCM) was employed for cell cycle, apoptosis and reactive oxygen species (ROS) analysis. Cell morphology was assessed by immunofluorescence and transmission electron microscopy (TEM). KEY FINDINGS: Compound 1 inhibited cell survival of all breast cell lines. In particular, compound 1 promoted cell cycle arrest in the G0/G1 phase and apoptosis along with impairment of the mitochondrial function, which was reflected in a gross alteration of the mitochondrial network structure. Furthermore, we also detected a potent activation of the ERK1/2 kinase, which suggested the induction of reactive oxygen species (ROS). Partial rescue of survival obtained with n-acetylcysteine (NAC) when coadminstered with compound 1 further supported a contribution of ROS mediated mechanisms to the growth-arrest and proapoptotic activity of compound 1 in both BCa cell lines. ROS production was indeed confirmed in SKBR-3. SIGNIFICANCE: Our findings show that compound 1 has a cytotoxic activity against both human normal and cancer cell lines derived from breast epithelia, which is mediated by ROS generation and mitochondrial damage.

Photobiomodulation with 808-nm diode laser light promotes wound healing of human endothelial cells through increased reactive oxygen species production stimulating mitochondrial oxidative phosphorylation.

Photobiomodulation of cells using near-infrared (NIR) monochromatic light can affect cell functions such as proliferation, viability, and metabolism in a range of cell types. Evidence for the effects of near-infrared light on endothelial cells has been reported, but the studies were mainly performed using VIS light emitted by low-energy lasers, because NIR wavelengths seemed negatively stimulate these cells. Cell viability, free radical-induced oxidative stress, NF-κB activation, nitric oxide release, mitochondrial respiration, and wound healing repair were assessed in human endothelial cells (HECV) irradiated with 808-nm diode laser light (laser setup = 1 W/cm2, 60 s, 60 J/cm2, CW vs measured energy parameter = 0.95 W/cm2, 60 s, 57 J/cm2, mode CW) emitted by an handpiece with flat-top profile. No difference in viability was detected between controls and HECV cells irradiated with 808-nm diode laser light for 60 s. Irradiated cells demonstrated higher proliferation rate and increased migration ability associated to moderate increase in ROS production without a significant increase in oxidative stress and oxidative stress-activated processes. Near-infrared light stimulated mitochondrial oxygen consumption and ATP synthesis in HECV cells. Short near-infrared irradiation did not affect viability of HECV cells, rather led to a stimulation of wound healing rate, likely sustained by ROS-mediated stimulation of mitochondrial activity. Our results demonstrating that near-infrared led to a shift from anaerobic to aerobic metabolism provide new insight into the possible molecular mechanisms by which photobiomodulation with 808-nm diode laser light protects against inflammation-induced endothelial dysfunction, seemingly promising to enhance their therapeutic properties.

Short-pulse neodymium:yttrium-aluminium garnet (Nd:YAG 1064nm) laser irradiation photobiomodulates mitochondria activity and cellular multiplication of Paramecium primaurelia (Protozoa).

Few studies exist to explore the potential photobiomodulation (PBM) effect of neodymium:yttrium-aluminium garnet (Nd:YAG) laser irradiation using a flat-top handpiece delivery system. In this study, we explored the photobiomodulation effect of that laser, on Paramecium primaurelia. The parameters for the different study groups were: 0.50W, 10Hz, 100msp, 30J/cm2; 0.75W, 10Hz, 100msp, 45J/cm2; 1.00W, 10Hz, 100msp, 60J/cm2; 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2. Our results suggest that only the parameter 0.5W, 10Hz, 100msp, 30J/cm2 positively photobiomodulates the Paramecium cells inducing an increment in oxygen consumption, endogenous ATP synthesis and fission rate rhythm. Applying the laser energy with parameters of 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2, induce adverse effect on the Paramecium cells, which protect themselves through the increase in Heat Shock Protein-70 (HSP70). The data presented in our work support our assumption that, when using appropriate parameters of irradiation, the 1064nm Nd:YAG laser with flat-top handpiece could be a valuable aid for effective clinical application of PBM.

An 808-nm Diode Laser with a Flat-Top Handpiece Positively Photobiomodulates Mitochondria Activities.

OBJECTIVE: Photobiomodulation is proposed as a non-linear process. Only the action of light at a low intensity and fluence is assumed to have stimulation on cells; whereas a higher light intensity and fluence generates negative effects, exhausting the cell's energy reserve as a consequence of a too strong stimulation. In our work, we detected the photobiomodulatory effect of an 808-nm higher-fluence diode laser [64 J/cm2-1 W, continuous wave (CW)] irradiated by a flat-top handpiece on mitochondria activities, such as oxygen consumption, activity of mitochondria complexes I, II, III, and IV, and cytochrome c as well as ATP synthesis. MATERIALS AND METHODS: The experiments are performed by standard procedure on mitochondria purified from bovine liver. RESULTS: Our higher-fluence diode laser positively photobiomodulates the mitochondria oxygen consumption, the activity of the complexes III and IV, and the ATP production, with a P/O = 2.6. The other activities are not influenced. CONCLUSION: Our data show for the first time that even the higher fluences (64 J/cm2-1 W), similar to the low fluences, can photobiostimulate the mitochondria respiratory chain without uncoupling them and can induce an increment in the ATP production. These results suggest that the negative effects of higher fluences observed to date are not unequivocally due to higher fluence per se but might be a consequence of the irradiation carried by handpieces with a Gaussian profile.

808-nm laser therapy with a flat-top handpiece photobiomodulates mitochondria activities of Paramecium primaurelia (Protozoa).

Photobiomodulation is proposed as a non-linear process, and only low-level laser therapy (LLLT) is assumed to stimulate exposed cells, whereas high powered laser and fluences can cause negative effects, exhausting the cell's energy reserve as a consequence of excessive photon-based stimulation. In our work, we investigated and compared the effects of 808-nm diode laser (CW) with a new flat-top handpiece. To this purpose, we tested the photobiomodulation effects of 1 and 3 J/cm(2) fluence, both generated by 100 mW or 1 W of laser power and of 64 J/cm(2) of fluence generated by 100 mW, 1 W, 1.5 W or 2 W, as expressed through oxygen consumption and ATP synthesis of Paramecium. Data collected indicates the incremental consumption of oxygen through irradiation with 3 J/cm(2)-100 mW or 64 J/cm(2)-1 W correlates with an increase in Paramecium ATP synthesis. The Paramecium respiration was inhibited by fluences 64 J/cm(2)-100 mW or 64 J/cm(2)-2 W and was followed by a decrease in the endogenous ATP concentration. The 1 J/cm(2)-100 mW or 1 W and 3 J/cm(2)-1 W did not affect mitochondrial activity. The results show that the fluence of 64 J/cm(2)-1 W more than the 3 J/cm(2)-100 mW causes greater efficiency in Paramecium mitochondria respiratory chain activity. Our results suggest that thanks to flat-top handpiece we used, high fluences by high-powered laser have to be reconsidered as an effective and non-invasive therapy. Possible associated benefits of deeper tissue penetration would increase treatment effectiveness and reduced irradiation time.

The protozoan, Paramecium primaurelia, as a non-sentient model to test laser light irradiation: The effects of an 808nm infrared laser diode on cellular respiration.

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm²) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly (p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more expensive mammalian cellular cultures.

Treatment of FANCA cells with resveratrol and N-acetylcysteine: a comparative study.

Fanconi anemia (FA) is a genetic disorder characterised by chromosome instability, cytokine ipersensibility, bone marrow failure and abnormal haematopoiesis associated with acute myelogenous leukemia. Recent reports are contributing to characterize the peculiar FA metabolism. Central to these considerations appears that cells from complementation group A (FANCA) display an altered red-ox metabolism. Consequently the possibility to improve FA phenotypical conditions with antioxidants is considered. We have characterized from the structural and biochemical point of view the response of FANCA lymphocytes to N-acetyl-cysteine (NAC) and resveratrol (RV). Surprisingly both NAC and RV failed to revert all the characteristic of FA phenotype and moreover their effects are not super imposable. Our data suggest that we must be aware of the biological effects coming from antioxidant treatment.

New findings in ATP supply in rod outer segments: insights for retinopathies.

BACKGROUND INFORMATION: The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks. RESULTS: OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F1F0-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O2) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections. CONCLUSIONS: Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O2. The ability of OS to manipulate O2 may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.

Inhibition of hemorragic snake venom components: old and new approaches.

Snake venoms are complex toxin mixtures. Viperidae and Crotalidae venoms, which are hemotoxic, are responsible for most of the envenomations around the world. Administration of antivenins aimed at the neutralization of toxins in humans is prone to potential risks. Neutralization of snake venom toxins has been achieved through different approaches: plant extracts have been utilized in etnomedicine. Direct electric current from low voltage showed neutralizing properties against venom phospholipase A2 and metalloproteases. This mini-review summarizes new achievements in venom key component inhibition. A deeper knowledge of alternative ways to inhibit venom toxins may provide supplemental treatments to serum therapy.