Is stallion epididymal fluid phosphoproteome affected by the equine reproductive season?

Phosphorylation and dephosphorylation of proteins are considered to be the most important processes in sperm maturation during epididymal transit. The main aim of this study was to isolate and identify phosphoproteins from the epididymal milieu obtained from reproductively mature stallions during and out of the breeding season. With the use of 1D-PAGE and nanoLC-MS/MS, we identified phosphoproteins that fulfil various functions: regulatory, transport, motility, ubiquitination, chaperone, antioxidant, apoptotic and enzymatic. Moreover, we characterized tyrosine, serine and threonine phosphorylation patterns, taking into consideration the seasonal and epididymal segment variables. The intensity of phosphorylation and profiles of phosphoproteins varied in subsequent regions of the epididymis. With the use of western and immunoblot tests, we demonstrated that fourteen proteins underwent phosphorylation both during and out of the breeding season. However, significant differences (p≤0.05) in the phosphorylation status were demonstrated in the case of 44 kDa (glutamine synthetase), 38 kDa (malate dehydrogenase), 34 kDa (clusterin/inorganic pyrophosphatase), 31 kDa (clusterin/ /ubiquitin thioesterase), 29 kDa (14-3-3 protein/purine nucleotide phosphorylase) for the season factor and 55 (Rab GDP dissociation inhibitor alpha) and 31 kDa ((clusterin/ubiquitin thioesterase) proteins for the segment factor. The occurrence of the other phosphoproteins was spontaneous among individuals and in both seasons.

Mitochondrial alterations triggered by repeated exposure to fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) fractions of ambient particulate matter.

Nowadays ambient particulate matter (PM) levels still regularly exceed the guideline values established by World Health Organization in most urban areas. Numerous experimental studies have already demonstrated the airway toxicity of the fine fraction of PM (FP), mainly triggered by oxidative stress-induced airway inflammation. However, only few studies have actually paid close attention to the ultrafine fraction of PM (UFP), which is likely to be more easily internalized in cells and more biologically reactive. Mitochondria are major endogenous sources of reactive oxygen species (ROS) through oxidative metabolism, and coordinate many critical cellular signaling processes. Mitochondria have been often studied in the context of PM toxicity and generally associated with apoptosis activation. However, little is known about the underlying adaptation mechanisms that could occur following exposure at sub-apoptotic doses of ambient PM. Here, normal human bronchial epithelial BEAS-2B cells were acutely or repeatedly exposed to relatively low doses (5 µg.cm-2) of FP (PM2.5-0.18) or quasi-UFP (Q-UFP; PM0.18) to better access the critical changes in mitochondrial morphology, functions, and dynamics. No significant cytotoxicity nor increase of apoptotic events were reported for any exposure. Mitochondrial membrane potential (ΔΨm) and intracellular ATP content were also not significantly impaired. After cell exposure to sub-apoptotic doses of FP and notably Q-UFP, oxidative phosphorylation was increased as well as mitochondrial mass, resulting in increased production of mitochondrial superoxide anion. Given this oxidative boost, the NRF2-ARE signaling pathway was significantly activated. However, mitochondrial dynamic alterations in favor of accentuated fission process were observed, in particular after Q-UFP vs FP, and repeated vs acute exposure. Taken together, these results supported mitochondrial quality control and metabolism dysfunction as an early lung underlying mechanism of toxicity, thereby leading to accumulation of defective mitochondria and enhanced endogenous ROS generation. Therefore, these features might play a key role in maintaining PM-induced oxidative stress and inflammation within lung cells, which could dramatically contribute to the exacerbation of inflammatory chronic lung diseases. The prospective findings of this work could also offer new insights into the physiopathology of lung toxicity, arguably initiate and/or exacerbate by acutely and rather repeated exposure to ambient FP and mostly Q-UFP.

Air pollution-derived PM2.5 impairs mitochondrial function in healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells.

In order to clarify whether the mitochondrial dysfunction is closely related to the cell homeostasis maintenance after particulate matter (PM2.5) exposure, oxidative, inflammatory, apoptotic and mitochondrial endpoints were carefully studied in human bronchial epithelial BEAS-2B, normal human bronchial epithelial (NHBE) and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells acutely or repeatedly exposed to air pollution-derived PM2.5. Some modifications of the mitochondrial morphology were observed within all these cell models repeatedly exposed to the highest dose of PM2.5. Dose- and exposure-dependent oxidative damages were reported in BEAS-2B, NHBE and particularly COPD-DHBE cells acutely or repeatedly exposed to PM2.5. Nuclear factor erythroid 2-p45 related factor 2 (NRF2) gene expression and binding activity, together with the mRNA levels of some NRF2 target genes, were directly related to the number of exposures for the lowest PM2.5 dose (i.e., 2 µg/cm2), but, surprisingly, inversely related to the number of exposures for the highest dose (i.e., 10 µg/cm2). There were dose- and exposure-dependent increases of both nuclear factor kappa-B (NF-κB) binding activity and NF-κB target cytokine secretion in BEAS-2B, NHBE and particularly COPD-DHBE cells exposed to PM2.5. Mitochondrial ROS production, membrane potential depolarization, oxidative phosphorylation, and ATP production were significantly altered in all the cell models repeatedly exposed to the highest dose of PM2.5. Collectively, our results indicate a cytosolic ROS overproduction, inducing oxidative damage and activating oxygen sensitive NRF2 and NF-kB signaling pathways for all the cell models acutely or repeatedly exposed to PM2.5. However, one of the important highlight of our findings is that the prolonged and repeated exposure in BEAS-2B, NHBE and in particular sensible COPD-DHBE cells further caused an oxidative boost able to partially inactivate the NRF2 signaling pathway and to critically impair mitochondrial redox homeostasis, thereby producing a persistent mitochondrial dysfunction and a lowering cell energy supply.

Deregulation of the hypoxia inducible factor-1α pathway in monocytes from sporadic amyotrophic lateral sclerosis patients.

The clinical course of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) is closely related to hypoxia. The normal response to hypoxia involves two pathways in particular: the hypoxia inducible factor 1α (HIF-1α) pathway (which notably controls the synthesis of vascular endothelial growth factor (VEGF)) and the nuclear factor kappa B (NF-κb) pathway (responsible for the production of inflammatory mediators, including prostaglandin E2 (PGE2)). Defects in VEGF gene expression are known to cause motor neuron degeneration in animal models. Circulating monocytes are precursors of the microglia, which are involved in the pathogenesis of ALS. To establish whether the HIF-1 and/or NF-κB pathways are deregulated during hypoxia in early-stage, sporadic ALS, we analyzed the response to acute (1 h) and prolonged (24 h) hypoxia in monocytes from ALS and healthy controls. We measured protein expression and mRNA transcription for VEGF, HIF-1, HIF-2, prolyl hydroxylases 1 and 2 (PHD-1 and -2, part of the HIF proteasome-dependent degradation pathway) and their modulation by PGE2. Our results showed that (i) the HIF-1 (but not HIF-2) and VEGF production induced by acute and prolonged hypoxia was selectively and markedly altered in ALS patients and (ii) this defect was not compensated for by PGE2 addition. Moreover, altered HIF-1α activation was associated with low levels of proteolysis by PHD-2 in cells from sporadic ALS patients (relative to controls). For the first time, we have demonstrated clinical and functional abnormalities in the HIF-1 pathway during hypoxia in monocytes from sporadic ALS patients.

Overcoming chemoresistance of non-small cell lung carcinoma through restoration of an AIF-dependent apoptotic pathway.

Non-small cell lung carcinomas (NSCLCs) are typically resistant against apoptosis induced by standard chemotherapy. We evaluated the effects of the two potential antitumor agents of the lamellarin class on a highly apoptosis-resistant NSCLC cell line. Both the marine alkaloid lamellarin-D and its synthetic amino derivative PM031379 induced the activation of Bax, the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), as well as the activation of caspase-3. However, only PM031379 triggered cell death and sign of nuclear apoptosis coupled to the nuclear translocation of AIF. Depletion of AIF with small interfering RNA or microinjection of a neutralizing anti-AIF antibody largely prevented PM031379-induced cytotoxicity, underscoring the essential contribution of AIF to NSCLC killing. Using NSCLC cells lacking mitochondrial DNA, we showed that the generation of mitochondrial reactive oxygen species (ROS) was crucial for the PM031379-induced translocation of AIF to the nucleus and subsequently cell death. Pretreatment of NSCLC cells with menadione, a mitochondrial ROS generator, was able to restore the deficient chemotherapy-induced apoptosis of NSCLC cells. Altogether, these data suggest that mitochondrial ROS generation is crucial for overriding the chemoresistance of NSCLC cells. Moreover, this study delineates the unique mechanism of action of lamellarins as potential anticancer agents.

Gel permeation chromatography of dextrans in parenteral solutions: calibration procedure development and method validation.

We describe development and validation of a gel permeation chromatographic (GPC) method for dextrans in parenteral solutions. The GPC method was adopted from USP monographs on Dextran 40 and Dextran 70 raw materials. The method was optimized with a mobile phase flow rate of 1 mL/min and column temperature of 40 degrees C, to sharpen dextran and dextrose peaks. An easy-to-use, curve-fitting program capable of non-linear regression was developed in-house, using Microsoft Excel and its Solver add-in to successfully meet the GPC calibration requirements for dextrans and dextrose, i.e., the experimental molecular weights within 100+/-5% of the known molecular weights for dextrans and molecular weight of dextrose within 180+/-2 Da. The GPC method was validated in terms of its stability indicating nature, robustness (column temperature of 40+/-3 degrees C), accuracy (lack of effects of pH and concentration of dextrans or matrix components), and precision (repeatability and intermediate). Molecular weight distribution of dextrans were unchanged when the dextran containing test solutions were subjected to forced degradation using heat, light (daylight and UV light), extreme alkaline conditions or oxidative conditions. The method was capable of detecting changes in molecular weight distribution caused by degradation under extreme acidic conditions and heat, thereby confirming the stability indicating nature of the method. The concentration of Dextran 40 and Dextran 70 (75-125% of the nominal assay concentration), matrix components (108-111% of their nominal concentrations), and solution pH (pH 3-7 for Dextran 40 solutions and pH 4-7 for Dextran 70 solutions) did not affect the measured molecular weight distribution of Dextran 40 or Dextran 70. The method was precise with %R.S.D. of less than 1% for M (W) values of Dextran 40 or Dextran 70.

Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS-103.

TAS-103 is a DNA intercalating indeno-quinoline derivative that stimulates DNA cleavage by topoisomerases. This synthetic drug has a broad spectrum of antitumor activity against many human solid tumor xenografts and is currently undergoing clinical trials. We investigated the induction of apoptosis in human promyelocytic leukemia cells treated with TAS-103. The treatment of proliferating human leukemia cells for 24 h with various concentrations of the drug induces significant variations in the mitochondrial transmembrane potential (delta(psi)mt) measured by flow cytometry using the fluorochromes 3,3-dihexyloxacarbocyanine iodide, Mitotracker Red, and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide. The collapse of delta(psi)mt is accompanied by a marked decrease of the intracellular pH. Cleavage experiments with the substrates N-acetyl-Asp-Glu-Val-Asp-pNA, poly(ADP-ribose) polymerase, and pro-caspase-3 reveal unambiguously that caspase-3 is a key mediator of the apoptotic pathway induced by TAS-103. Caspase-8 is also cleaved, and the bcl-2 oncoprotein is underexpressed. Drug-induced internucleosomal DNA fragmentation and the externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane were also characterized. The cell cycle perturbations produced by TAS-103 can be connected with the changes in deltapsi(mt). At low concentrations (2-25 nM), the drug induces a marked G2 arrest and concomitantly provokes an increase in the potential of mitochondrial membranes. In contrast, treatment of the HL-60 cells with higher drug concentrations (50 nM to 1 microM) triggers massive apoptosis and a collapse of deltaP(mt) that is a signature for the opening of the mitochondrial permeability transition pores. The discovery of a correlation between the G2 arrest and changes in mitochondrial membrane potential provides an important mechanistic insight into the action of TAS-103.

Relationship between cell cycle changes and variations of the mitochondrial membrane potential induced by etoposide.

Etoposide, a clinically useful anticancer drug, is a potent inhibitor of topoisomerase II. The DNA strand breaks caused by this epipodophyllotoxin lead to apoptotic death of tumor cells. Flow cytometry was used to investigate the relationship between the effects of the drug on the cell cycle of human leukemia HL-60 cells and the variations of the mitochondrial transmembrane potential (DeltaPsi(mt)). Three cationic fluorescent probes, DiOC(6), JC-1, and TMRM, were used to measure drug-induced changes of DeltaPsi(mt). In all three cases, we found that the arrest in the G2/M phase of the cells treated with 0.5 microM etoposide is associated with an increase in the potential of mitochondrial membranes whereas treatment with a tenfold higher drug concentration trigger massive apoptosis and a collapse of DeltaPsi(mt). DNA fragmentation (TUNEL assay) and externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane (annexin V binding) were measured to characterize the apoptotic cell population.