Oxidative stress parameters and their relation to motor subtype of Parkinson's disease and levodopa treatment status.

Parkinson's disease (PD) is an oxidative stress-linked neurodegenerative disorder, with the highest prevalence among seniors. The objective of this study were: (1) to analyse levels of following oxidative stress parameters: total antioxidant capacity (TAC), uric acid (UA), total glutathione (tGSH), bilirubin (Bil) and albumin (Alb), in blood of PD patients and healthy controls; (2) to find possible associations of examined oxidative stress parameters with PD subtypes and levodopa treatment status; and (3) to evaluate power and relevance of the aforementioned oxidative stress parameter for the prediction of onset and progression of PD by utilizing Random Forest machine learning (RFML). Oxidative stress parameters were determined in 125 PD patients and 55 healthy controls. Evaluated with frequentist statistics, our data revealed that UA is the only oxidative stress parameter associated with PD. However, when the PD cohort was divided in gender-dependent manner, tGSH and Bil were also significantly associated with PD in subgroup of female patients. RFML rendered no predictive power of any of the tested oxidative stress parameters in respect to PD, its subtypes, and/or status of levodopa treatment. In conclusion, despite the positive association of UA with PD (in complete cohort of PD patients) and of tGSH and Bil with PD but only in female patients, these oxidative stress parameters are of no use in clinical practice due to the lack of the predictive/diagnostic power.

Blood and Brain Metabolites after Cerebral Ischemia.

The study of an organism's response to cerebral ischemia at different levels is essential to understanding the mechanism of the injury and protection. A great interest is devoted to finding the links between quantitative metabolic changes and post-ischemic damage. This work aims to summarize the outcomes of the most studied metabolites in brain tissue-lactate, glutamine, GABA (4-aminobutyric acid), glutamate, and NAA (N-acetyl aspartate)-regarding their biological function in physiological conditions and their role after cerebral ischemia/reperfusion. We focused on ischemic damage and post-ischemic recovery in both experimental-including our results-as well as clinical studies. We discuss the role of blood glucose in view of the diverse impact of hyperglycemia, whether experimentally induced, caused by insulin resistance, or developed as a stress response to the cerebral ischemic event. Additionally, based on our and other studies, we analyze and critically discuss post-ischemic alterations in energy metabolites and the elevation of blood ketone bodies observed in the studies on rodents. To complete the schema, we discuss alterations in blood plasma circulating amino acids after cerebral ischemia. So far, no fundamental brain or blood metabolite(s) has been recognized as a relevant biological marker with the feasibility to determine the post-ischemic outcome or extent of ischemic damage. However, studies from our group on rats subjected to protective ischemic preconditioning showed that these animals did not develop post-ischemic hyperglycemia and manifested a decreased metabolic infringement and faster metabolomic recovery. The metabolomic approach is an additional tool for understanding damaging and/or restorative processes within the affected brain region reflected in the blood to uncover the response of the whole organism via interorgan metabolic communications to the stressful cerebral ischemic challenge.

Effects of Green Tea Polyphenol Epigallocatechin-3-Gallate on Markers of Inflammation and Fibrosis in a Rat Model of Pulmonary Silicosis.

Inhalation of silica particles causes inflammatory changes leading to fibrotizing silicosis. Considering a lack of effective therapy, and a growing information on the wide actions of green tea polyphenols, particularly epigallocatechin-3-gallate (EGCG), the aim of this study was to evaluate the early effects of EGCG on markers of inflammation and lung fibrosis in silicotic rats. The silicosis model was induced by a single transoral intratracheal instillation of silica (50 mg/mL/animal), while controls received an equivalent volume of saline. The treatment with intraperitoneal EGCG (20 mg/kg, or saline in controls) was initiated the next day after silica instillation and was given twice a week. Animals were euthanized 14 or 28 days after the treatment onset, and the total and differential counts of leukocytes in the blood and bronchoalveolar lavage fluid (BALF), wet/dry lung weight ratio, and markers of inflammation, oxidative stress, and fibrosis in the lung were determined. The presence of collagen and smooth muscle mass in the walls of bronchioles and lung vessels was investigated immunohistochemically. Early treatment with EGCG showed some potential to alleviate inflammation, and a trend to decrease oxidative stress-induced changes, including apoptosis, and a prevention of fibrotic changes in the bronchioles and pulmonary vessels. However, further investigations should be undertaken to elucidate the effects of EGCG in the lung silicosis model in more detail. In addition, because of insufficient data from EGCG delivery in silicosis, the positive and eventual adverse effects of this herbal compound should be carefully studied before any preventive use or therapy with EGCG may be recommended.

Endoplasmic reticulum stress induces mitochondrial dysfunction but not mitochondrial unfolded protein response in SH-SY5Y cells.

In the present study we have shown that treatment of SH-SY5Y cells with either thapsigargin or tunicamycin is associated with a significant decrease in ROUTINE and ATP-coupled mitochondrial respiration as well as a decrease in spare and maximal respiratory capacity. We have also shown that treating cells with either thapsigargin or tunicamycin is associated with significant changes in mitochondrial membrane potential (ΔΨm) generation, which is mainly associated with the reversal of the succinyl-CoA ligase reaction and a decreased activity of complex II. Despite the induction of endoplasmic reticulum (ER) specific unfolded protein response (UPR), as documented by increased expression of HRD1, ER stress did not induce mitochondrial UPR since the expression of both mitochondrial protease LONP1 and mitochondrial chaperone HSP60 was not significantly altered. Inhibition of IRE1α ribonuclease with STF-083010 did not protect the SH-SY5Y cells from ER stress-induced mitochondrial dysfunction. STF-083010 itself had significant impact on both mitochondrial respiration and generation of ΔΨm, which has mainly been associated with the uncoupling of respiratory chain from ATP synthesis.

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45 µmol/g of body weight) twice a day for a period of 2 weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca2+-handling proteins, Ca2+-ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy.

Magnesium Extravaganza: A Critical Compendium of Current Research into Cellular Mg2+ Transporters Other than TRPM6/7.

Magnesium research has boomed within the last 20 years. The real breakthrough came at the start of the new millennium with the discovery of a plethora of possible Mg homeostatic factors that, in particular, included putative Mg2+ transporters. Until that point, Mg research was limited to biochemical and physiological work, as no target molecular entities were known that could be used to explore the molecular biology of Mg homeostasis at the level of the cell, tissue, organ, or organism and to translate such knowledge into the field of clinical medicine and pharmacology. Because of the aforementioned, Mg2+ and Mg homeostasis, both of which had been heavily marginalized within the biomedical field in the twentieth century, have become overnight a focal point of many studies ranging from primary biomedical research to translational medicine.The amount of literature concerning cellular Mg2+ transport and cellular Mg homeostasis is increasing, together with a certain amount of confusion, especially about the function(s) of the newly discovered and, in the majority of instances, still only putative Mg2+ transporters/Mg2+ homeostatic factors. Newcomers to the field of Mg research will thus find it particularly difficult to orient themselves.Here, we briefly but critically summarize the status quo of the current understanding of the molecular entities behind cellular Mg2+ homeostasis in mammalian/human cells other than TRPM6/7 chanzymes, which have been universally accepted as being unspecific cation channel kinases allowing the flux of Mg2+ while constituting the major gateway for Mg2+ to enter the cell.

Differential profiling of prostate tumors versus benign prostatic tissues by using a 2DE-MALDI-TOF-based proteomic approach.

Oncoproteomic technologies offer a complementary approach to the understanding of cancer proteins' function and the translation of molecular knowledge into clinical practice. Our aim was to compare the proteomic profiles of prostate tumors versus benign prostatic hyperplasia (BPH) tissues in order to identify modulated proteins as the potential biomarkers for prostate cancer. Proteins extracted from twenty prostate cancer tissue specimens and ten BPH tissues were analyzed by two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. Western blot and quantitative real-time PCR (RT-PCR) were performed to confirm the different amounts of protein biomarkers revealed by 2DE combined with MALDI mass spectrometry. We found 42 spots whose expression in the prostate was altered more than 1.5-fold compared with BPH tissue (p<0.05). These spots represented ten different proteins that were identified by a database search after mass spectrometry: they comprised proteins involved in the regulation of actin dynamics, the cytoskeleton, and cell motility (ACTG2, ACTA2, TPM1, DES, VIM, FLNA, and TAGLN), heat shock protein-27 (Hsp27), and proteins with other functions (TR and RANBP3). Subsequent western blot and RT-PCR assays for DES, VIM, TAGLN, and Hsp27 in prostate tumor tissues and BPH tissues confirmed the observations obtained by proteomic analysis. The cytoskeletal and cytoskeleton-associated proteins identified by this approach might be useful molecular targets for prostate cancer diagnostics and may contribute to novel therapies for prostate cancer.

Homocysteine and Mitochondria in Cardiovascular and Cerebrovascular Systems.

Elevated concentration of homocysteine (Hcy) in the blood plasma, hyperhomocysteinemia (HHcy), has been implicated in various disorders, including cardiovascular and neurodegenerative diseases. Accumulating evidence indicates that pathophysiology of these diseases is linked with mitochondrial dysfunction. In this review, we discuss the current knowledge concerning the effects of HHcy on mitochondrial homeostasis, including energy metabolism, mitochondrial apoptotic pathway, and mitochondrial dynamics. The recent studies suggest that the interaction between Hcy and mitochondria is complex, and reactive oxygen species (ROS) are possible mediators of Hcy effects. We focus on mechanisms contributing to HHcy-associated oxidative stress, such as sources of ROS generation and alterations in antioxidant defense resulting from altered gene expression and post-translational modifications of proteins. Moreover, we discuss some recent findings suggesting that HHcy may have beneficial effects on mitochondrial ROS homeostasis and antioxidant defense. A better understanding of complex mechanisms through which Hcy affects mitochondrial functions could contribute to the development of more specific therapeutic strategies targeted at HHcy-associated disorders.

Dietary Mg2+ Intake and the Na+/Mg2+ Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes.

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na+/Mg2+ exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI-CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and Slc41a1-/- mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the Slc41a1 genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of Slc41a1-/- genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of Slc41a1 genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria.

A comparison of albumin removal procedures for proteomic analysis of blood plasma.

Blood biomarkers are usually present in low concentration and can be masked by the high-abundance proteins, of which albumin is the predominant one. The purpose of this study was to compare four different albumin removal methods compatible with in-gel based proteomics, applicable for plasma, without requiring specific techniques and high financial input. Plasma underwent albumin depletion with ultrafiltration device Amicon Ultra, commercial ProteoPrep Blue Albumin and IgG Depletion Kit, acetonitrile precipitation method and precipitation with acetonitrile-methanol protocol. All samples were evaluated by 1-D and 2-D gel electrophoresis with subsequent mass spectrometry protein identification. Two of the tested methods (ProteoPrep BlueKit and acetonitrile-methanol precipitation) maintained sufficient protein content for further in-gel analyses. Their 2-D protein profiles were distinctively separated and overlapped with protein profile of crude plasma. Protein spot count showed significant increase in protein spots, compared to crude plasma, only with acetonitrile-methanol precipitation method. Precipitation with acetonitrile-methanol method significantly increased number of protein spots on 2-D protein profile and improved score of mass spectrometry identification. However, albumin was still present and found in number of protein spots.

SNPs rs11240569, rs708727, and rs823156 in SLC41A1 Do Not Discriminate Between Slovak Patients with Idiopathic Parkinson's Disease and Healthy Controls: Statistics and Machine-Learning Evidence.

Gene SLC41A1 (A1) is localized within Parkinson's disease-(PD)-susceptibility locus PARK16 and encodes for the Na+/Mg2+-exchanger. The association of several A1 SNPs with PD has been studied. Two, rs11240569 and rs823156, have been associated with reduced PD-susceptibility primarily in Asian populations. Here, we examined the association of rs11240569, rs708727, and rs823156 with PD in the Slovak population and their power to discriminate between PD patients and healthy controls. The study included 150 PD patients and 120 controls. Genotyping was performed with the TaqMan® approach. Data were analyzed by conventional statistics and Random Forest machine-learning (ML) algorithm. Individually, none of the three SNPs is associated with an altered risk for PD-onset in Slovaks. However, a combination of genotypes of SNP-triplet GG(rs11240569)/AG(rs708727)/AA(rs823156) is significantly (p < 0.05) more frequent in the PD (13.3%) than in the control (5%) cohort. ML identified the power of the tested SNPs in isolation or of their singlets (joined), duplets and triplets to discriminate between PD-patients and healthy controls as zero. Our data further substantiate differences between diverse populations regarding the association of A1 polymorphisms with PD-susceptibility. Lack of power of the tested SNPs to discriminate between PD and healthy cases render their clinical/diagnostic relevance in the Slovak population negligible.

Association of Induced Hyperhomocysteinemia with Alzheimer's Disease-Like Neurodegeneration in Rat Cortical Neurons After Global Ischemia-Reperfusion Injury.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder that results in massive hippocampal and neocortical neuronal loss leading to dementia and eventual death. The exact cause of Alzheimer's disease is not fully explored, although a number of risk factors have been recognized, including high plasma concentration of homocysteine (Hcy). Hyperhomocysteinemia (hHcy) is considered a strong, independent risk factor for stroke and dementia. However, the molecular background underlying these mechanisms linked with hHcy and ischemic stroke is not fully understood. Paper describes rat model of global forebrain ischemia combined with the experimentally induced hHcy. Global ischemia-reperfusion injury (IRI) was developed by 4-vessels occlusion lasting for 15 min followed by reperfusion period of 72 h. hHcy was induced by subcutaneous injection of 0.45 µmol/g of Hcy in duration of 14 days. The results showed remarkable neural cell death induced by hHcy in the brain cortex and neurodegeneration is further aggravated by global IRI. We demonstrated degeneration of cortical neurons, alterations in number and morphology of tissue astrocytes and dysregulation of oxidative balance with increased membrane protein oxidation. Complementary to, an immunohistochemical analysis of tau protein and ß-amyloid peptide showed that combination of hHcy with the IRI might lead to the progression of AD-like pathological features. Conclusively, these findings suggest that combination of risk factor hHcy with IRI aggravates neurodegeneration processes and leads to development of AD-like pathology in cerebral cortex.

Effect of Hyperhomocysteinemia on Redox Balance and Redox Defence Enzymes in Ischemia-Reperfusion Injury and/or After Ischemic Preconditioning in Rats.

Increased level of homocysteine (hHcy) in plasma is an accompanying phenomenon of many diseases, including a brain stroke. This study determines whether hyperhomocysteinemia (which is a risk factor of brain ischemia) itself or in combination with ischemic preconditioning affects the ischemia-induced neurodegenerative changes, generation of reactive oxygen species (ROS), lipoperoxidation, protein oxidation, and activity of antioxidant enzymes in the rat brain cortex. The hHcy was induced by subcutaneous administration of homocysteine (0.45 µmol/g body weight) twice a day in 8 h intervals for 14 days. Rats were preconditioned by 5 min ischemia. Two days later, 15 min of global forebrain ischemia was induced by four vessel's occlusion. The study demonstrates that in the cerebral cortex, hHcy alone induces progressive neuronal cell death and morphological changes. Neuronal damage was associated with the pro-oxidative effect of hHcy, which leads to increased ROS formation, peroxidation of lipids and oxidative alterations of cortical proteins. Ischemic reperfusion injury activates degeneration processes and de-regulates redox balance which is aggravated under hHcy conditions and leads to the augmented lipoperoxidation and protein oxidation. If combined with hHcy, ischemic preconditioning could preserve the neuronal tissue from lethal ischemic effect and initiates suppression of lipoperoxidation, protein oxidation, and alterations of redox enzymes with the most significant effect observed after prolonged reperfusion. Increased prevalence of hyperhomocysteinemia in the Western population and crucial role of elevated Hcy level in the pathogenesis of neuronal disorders makes this amino acid as an interesting target for future research. Understanding the multiple etiological mechanisms and recognition of the co-morbid risk factors that lead to the ischemic/reperfusion injury and ischemic tolerance is therefore important for developing therapeutic strategies in human brain stroke associated with the elevated level of Hcy.

Proteomic analysis of mitochondrial proteins in the guinea pig heart following long-term normobaric hyperoxia.

Normobaric hyperoxia is applied for the treatment of a wide variety of diseases and clinical conditions related to ischemia or hypoxia, but it can increase the risk of tissue damage and its efficiency is controversial. In the present study, we analyzed cardiac mitochondrial proteome derived from guinea pigs after 60 h exposure to 100% molecular oxygen (NBO) or O2 enriched with oxygen cation (NBO+). Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry identified twenty-two different proteins (among them ten nonmitochondrial) that were overexpressed in NBO and/or NBO+ group. Identified proteins were mainly involved in cellular energy metabolism (tricarboxylic acid cycle, oxidative phosphorylation, glycolysis), cardioprotection against stress, control of mitochondrial function, muscle contraction, and oxygen transport. These findings support the viewpoint that hyperoxia is associated with cellular stress and suggest complex adaptive responses which probably contribute to maintain or improve intracellular ATP levels and contractile function of cardiomyocytes. In addition, the results suggest that hyperoxia-induced cellular stress may be partially attenuated by utilization of NBO+ treatment.

The role of CYP17A1 in prostate cancer development: structure, function, mechanism of action, genetic variations and its inhibition.

Androgens play an important role during the development of both normal prostate epithelium and prostate cancer and variants of genes involved in androgen metabolism may be related to an increased risk of prostate disease. Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a key regulatory enzyme in the steroidogenic pathway; it catalyses both 17α-hydroxylase and 17,20-lyase activities and is essential for the production of both androgens and glucocorticoids. In this review, we focus on the structure and enzymatic activity of CYP17A1 and the mechanism of modulation of CYP17A1 activities. We discuss the relationship between common genetic variations in CYP17A1 gene and prostate cancer risk and the main effects of these variations on the prediction of susceptibility and clinical outcomes of prostate cancer patients. The mechanism of action, the efficacy and the clinical potential of CYP17A1 inhibitors in prostate cancer are also summarized.

The Effect of Aging on Mitochondrial Complex I and the Extent of Oxidative Stress in the Rat Brain Cortex.

One of the characteristic features of the aging is dysfunction of mitochondria. Its role in the regulation of metabolism and apoptosis suggests a possible link between these cellular processes. This study investigates the relationship of respiratory complex I with aging-related oxidative stress in the cerebral mitochondria. Deterioration of complex I seen in senescent (26-months old) mitochondria was accompanied by decline in total thiol group content, increase of HNE and HNE-protein adducts as well as decreased content of complex I subunits, GRIM-19 and NDUFV2. On the other hand, decline of complex I might be related with the mitochondrial apoptosis through increased Bax/Bcl-2 cascade in 15-month old animal brains. Higher amount of Bcl-2, Bcl-xL with the lower content of GRIM-19 could maintain to some extent elevated oxidative stress in mitochondria as seen in the senescent group. In the cortical M1 region increased presence of TUNEL+ cells and more than 20-times higher density of Fluoro-Jade C+ cells in 26-months old was observed, suggesting significant neurodegenerative effect of aging in the neuronal cells. Our study supports a scenario in which the age-related decline of complex I might sensitize neurons to the action of death agonists, such as Bax through lipid and protein oxidative stimuli in mitochondria. Although aging is associated with oxidative stress, these changes did not increase progressively with age, as similar extent of lesions was observed in oxidative stress markers of the both aged groups.

Effects of mild hyperhomocysteinemia on electron transport chain complexes, oxidative stress, and protein expression in rat cardiac mitochondria.

Hyperhomocysteinemia (HHcy) is an independent risk factor of cardiovascular disease, but the mechanisms of tissue injury are poorly understood. In the present study, we investigated the effect of HHcy on rat heart function, activities electron transport chain (ETC) complexes, mitochondrial protein expression, and protein oxidative damage. HHcy was induced by subcutaneous injection of Hcy (0.45 µmol/g of body weight) twice a day for a period of 2 weeks. Performance of hearts excised after the Hcy treatment was examined according to the Langendorff method at a constant pressure. Left ventricular developed pressure, as well as maximal rates of contraction (+dP/dt) and relaxation (-dP/dt), was significantly depressed in HHcy rats. HHcy was accompanied by significant inhibition of ETC complexes II-IV, whereas activity of the complex I was unchanged. The decline in ETC activities was not associated with elevated protein oxidative damage, as indicated by unchanged protein carbonyl, thiol, and dityrosine contents. Moreover, the level of protein adducts with 4-hydroxynonenal was decreased in HHcy rats. Additionally, 2D-gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry did not show alterations in contents of inhibited ETC complexes. However, mass spectrometry analyses identified 8 proteins whose expression was significantly increased by HHcy. These proteins are known to play important roles in the cellular stress response, bioenergetics, and redox balance. Altogether, the results suggest that oxidative damage and altered protein expression are not possible causes of ETC dysfunction in HHcy rats. Increased expression of the other mitochondrial proteins indicates a protective response to Hcy-induced myocardial injury.

Nanomaterials - a New and Former Public Health Issue. The Case of Slovakia.

Nanoparticles exist for a long time as both inorganic and organic parts of nature. Recently, massive expansion of nanotechnologies is evidenced, together with intentional production of new nanoparticles which have not been in contact with living organisms until now. Besides obvious positive aspects, potential threats related to their exposure should be taken into consideration. Unique physical-chemical properties of nanoparticles cause a high bioactivity following their intake (through air, ingestion and skin) and unrestricted spread in exposed organs. Primary effects of nanoparticles on cellular level represent oxidative stress and reactions leading to apoptosis, autophagocytosis and necrosis. Number of studies indicating contribution of nanoparticles to numerous disorders has been recently increasing. However, detailed mechanisms of health effects are not well known. Similarly, there is insufficient information on life cycle of nanoparticles in the environment. Research in this field as well as legislation is behind rapid development and use of nanotechnologies. Considering absence of mandatory exposure limits and other protective measures, nanomaterials represent a potential threat for population health. Recommendations and guidelines of international institutions can contribute to deal with situation, however, passing of effective legislation both on national and European level is urgently needed.

Possible contribution of proteins of Bcl-2 family in neuronal death following transient global brain ischemia.

Proteins of Bcl-2 family are crucial regulators of intrinsic (mitochondrial) pathway of apoptosis that is implicated among the mechanisms of ischemic neuronal death. Initiation of mitochondrial apoptosis depends on changes of equilibrium between anti-apoptotic and pro-apoptotic proteins of Bcl-2 family as well as on translocation of pro-apoptotic proteins of Bcl-2 family to mitochondria. The aim of this work was to study the effect of transient global brain ischemia on expression and intracellular distribution of proteins of Bcl-2 family in relation to the ischemia-induced changes of ERK and Akt kinase pathways as well as disturbances in ubiquitin proteasome system. Using four vessel occlusion model of transient global brain ischemia, we have shown that both ischemia in duration of 15 min and the same ischemia followed by 1, 3, 24, and 72 h of reperfusion did not affect the levels of either pro-apoptotic (Bad, PUMA, Bim, Bax, Noxa) or anti-apoptotic (Bcl-2, Bcl-xl, Mcl-1) proteins of Bcl-2 family in total cell extracts from rat hippocampus. However, significantly elevated level of Bad protein in the mitochondria isolated from rat hippocampus was observed already 1 h after ischemia and remained elevated 3 and 24 h after ischemia. We did not observe significant changes of the levels of Puma, Bax, Bcl-2, and Bcl-xl in the mitochondria after ischemia and ischemia followed by reperfusion. Our results might indicate possible involvement of Bad translocation to mitochondria in the mechanisms of neuronal death following transient global brain ischemia.

Mitochondrial Dysfunction in Sporadic Amyotrophic Lateral Sclerosis Patients: Insights from High-Resolution Respirometry.

Amyotrophic lateral sclerosis is a severe neurodegenerative disease whose exact cause is still unclear. Currently, research attention is turning to the mitochondrion as a critical organelle of energy metabolism. Current knowledge is sufficient to confirm the involvement of the mitochondria in the pathophysiology of the disease, since the mitochondria are involved in many processes in the cell; however, the exact mechanism of involvement is still unclear. We used peripheral blood mononuclear cells isolated from whole fresh blood from patients with amyotrophic lateral sclerosis for measurement and matched an age- and sex-matched set of healthy subjects. The group of patients consisted of patients examined and diagnosed at the neurological clinic of the University Hospital Martin. The set of controls consisted of healthy individuals who were actively searched, and controls were selected on the basis of age and sex. The group consisted of 26 patients with sporadic forms of ALS (13 women, 13 men), diagnosed based on the definitive criteria of El Escorial. The average age of patients was 54 years, and the average age of healthy controls was 56 years. We used a high-resolution O2K respirometry method, Oxygraph-2k, to measure mitochondrial respiration. Basal respiration was lower in patients by 29.48%, pyruvate-stimulated respiration (respiratory chain complex I) was lower by 29.26%, and maximal respiratory capacity was lower by 28.15%. The decrease in succinate-stimulated respiration (respiratory chain complex II) was 26.91%. Our data confirm changes in mitochondrial respiration in ALS patients, manifested by the reduced function of complex I and complex II of the respiratory chain. These defects are severe enough to confirm this disease's hypothesized mitochondrial damage. Therefore, research interest in the future should be directed towards a deeper understanding of the involvement of mitochondria and respiratory complexes in the pathophysiology of the disease. This understanding could develop new biomarkers in diagnostics and subsequent therapeutic interventions.