S. Passarella

A peptide containing residues 26-44 of tau protein impairs mitochondrial oxidative phosphorylation acting at the level of the adenine nucleotide translocator.

Having confirmed that adenovirus-mediated overexpression of NH(2)-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH(2)-derived tau peptides, i.e. NH(2)-26-44 and NH(2)-1-25 fragments, affect mitochondrial function. We tested both fragments on each step of the processes leading to ATP synthesis via oxidative phosphorylation: i) electron flow via the respiratory chain from physiological substrates to oxygen with the activity of each individual complex of the respiratory chain investigated in some detail, ii) membrane potential generation arising from externally added succinate and iii) the activity of both the adenine nucleotide translocator and iv) ATP synthase. Oxidative phosphorylation is not affected by NH(2)-1-25 tau fragment, but dramatically impaired by NH(2)-26-44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH(2)-26-44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH(2)-26-44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria.

The irradiation of rabbit sperm cells with He-Ne laser prevents their in vitro liquid storage dependent damage

The aim of the study was to investigate the effects of different energy doses of helium-neon (He-Ne) laser irradiation on both mitochondrial bioenergetics functions and functional quality of rabbit spermatozoa during 48 h of in vitro liquid storage at 15 degrees C. 11 rabbit semen pools were each divided into four aliquots: three of them were irradiated with He-Ne laser with different energy doses (3.96, 6.12 and 9.00 J/cm(2)) being the last control kept under the same experimental conditions without irradiation. Sperm motility, viability and acrosome integrity were monitored together with cytochrome c oxidase (COX) activity and the cell energy charge (EC) at 0, 24 and 48 h of storage. Irradiated samples stored for 24 and 48 h better maintained motility (P < 0.01), acrosome integrity (P < 0.01) and viability (P < 0.05) with respect to the control, particularly with the energy dose of 6.12 J/cm(2) that showed the most intense biostimulative effect. COX activity and EC were immediately increased by irradiation particularly in the treatments 6.12 and 9.00 J/cm(2) (P < 0.05), that maintained their levels higher with respect to the control after 48 h of storage (P < 0.01). COX activity of rabbit sperm cells was positively correlated with EC (P < 0.05), viability (P < 0.01) and acrosome integrity (P < 0.05) parameters. These results indicate that the effects of He-Ne laser irradiation on sperm cells are mediated through the stimulation of the sperm mitochondrial respiratory chain and that this effect plays a significant role in the augmentation of the rabbit sperm cells capability to survive during liquid storage conditions.

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

The mitochondrial L-lactate dehydrogenase affair

The existence of a mitochondrial L-lactate dehydrogenase (m-L-LDH) suggested by Dianzani (1951), was shown by Baba and Sharma (1971) with the enzyme located in the mitochondrial matrix; later Brooks et al. (1999) proposed the intracellular lactate shuttle and in the third millennium the existence of m-L-LDH was definitively been confirmed in mammalian, plant and yeast mitochondria as reviewed by Schurr (2006), Passarella et al. (2008), and Brooks (2009), being its existence finally recognized by inclusion of m-L-LDH in the Mitocarta (http://www.broadinstitute.org/pubs/MitoCarta/index.html). The experimental strategy to be used to show whether and how L-lactate can enter mitochondria to be metabolized is well-established and has been applied to a variety of mitochondria including heart (Brooks et al., 1999; Valenti et al., 2002), liver (Brooks et al., 1999; de Bari et al., 2004), skeletal muscle (Dubouchaud et al., 2000; de Bari et al., 2008; Passarella et al., 2008) plant (Paventi et al., 2007), brain (Schurr, 2006; Atlante et al., 2007; Schurr and Payne, 2007; Hashimoto et al., 2008), and cancer cells (de Bari et al., 2010a; Pizzuto et al., 2012). Thus, it is a matter for considerable surprise that the overwhelming evidence for an m-L-LDH located inside mitochondria is not by now universally accepted (Rasmussen et al., 2002; Sahlin et al., 2002; Ponsot et al., 2005; Gladden, 2007; Yoshida et al., 2007; Elustondo et al., 2013).

The post-thaw irradiation of avian spermatozoa with He-Ne laser differently affects chicken, pheasant and turkey sperm quality

The effects of post-thaw Helium-Neon (He-Ne) laser irradiation on mobility and functional integrity of frozen/thawed chicken, pheasant and turkey spermatozoa were investigated. Cytochrome C oxidase (COX) activity was also determined as a measure of the effect of irradiation on mitochondrial bioenergetics. Semen samples from each species were collected, processed and frozen according to the pellet procedure. After thawing, each semen sample was divided into two subsamples: the first one was the control; the second one was irradiated with a single mode continuous He-Ne laser wave (wavelength 632.8 nm; 6 mW; 3.96 J/cm(2)). Then the samples were assessed for sperm mobility (Accudenz(®) swim-down test), viability (SYBR-14/PI staining), osmotic-resistance (HOS test) and COX activity. The irradiation was effective P<0.05 increasing sperm motility in the turkey semen (0.228 ± 0.01 compared with 0.294 ± 0.02). The irradiation also caused an increase (P<0.05) of the COX activity in pheasant (+135 ± 4%) and turkey (+116 ± 4%) sperm, without affecting viability and osmotic-resistance. The COX was positively correlated (P<0.05) with the viability of chicken sperm, however no significant interactions were found between mobility and COX activity in the three avian species. Due to the difference in energetic metabolism among avian species used in this study, the He-Ne laser irradiation has a differential action on bio-stimulation of turkey, chicken and pheasant spermatozoa. The present results are the first to elucidate the possibility for restoration of motility of cryopreserved avian spermatozoa by bio-stimulation provided via He-Ne laser irradiation.

Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality

This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9xa0J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (nxa0=xa07) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12xa0J/cm(2), mass sperm motility, progressive motility and viability increased (Pxa0<xa00.05), with no significant changes observed in the other investigated properties. In parallel, an increase (Pxa0<xa00.05) in ATP content was detected in the 6.12xa0J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12xa0J/cm(2) (Pxa0<xa00.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions.

The He-Ne laser irradiation improves the quality of stored rabbit semen

Abstract The aim of the study was to investigate the effect of different energy doses of Helium-Neon (He-Ne) laser irradiation on rabbit semen quality during 48 h of storage. From 30 commercial hybrid bucks semen was collected by artificial vagina. The ejaculates of bucks were pooled (3-4 ejaculates/pool) and in total 8 semen pools were used. Each pool was divided into four aliquots. One sample was not irradiated (control), the other ones were irradiated with a single mode continuous He-Ne laser wave (wavelenght 632,8) and exposed to the following energy doses: 3.96, 6.12 and 9 J/cm2 respectively. Samples were kept at 15° C during laser irradiation. They were subsequently stored again at the same temperature for a further 48 h. Samples were evaluated for sperm motility (Accudenz procedure), sperm viability (SyBr-PI procedure) and acrosome integrity (PSA-FITC procedure) at 3,24 and 48h of storage. Laser irradiation at all energy doses utilized significantly improved motility (P>0.01), viability (P>0.01) and acrosome integrity (P>0.05) than in control either at 24 and 48 h of storage. Moreover, the energy dose of 6.12J/cm2 recorded a better semen quality during storage compared to the 3,96 and 9J/cm2 ones respectively. No significant differences for measured seminal quality parameters were found at 3h; this might be due to the fact that irradiation treatment requires a longer period of time to determine modification on semen quality. The present study shows that laser irradiation, as shown in a previous research for turkey semen, seems to improve the quality of rabbit semen stored for up 48h even if higher energy doses were utilized. Therefore, the irradiation laser might be a useful technique to enhance quality of stored rabbit semen, although further studies are need.