[Effect of antioxidants on human primary and metastatic colon cancer cells at hypoxia and normoxia].

THE AIM: Evaluation of some antioxidants on human colon cancer cells viability and proliferation at various oxygen levels. MATERIAL AND METHODS: Human primary (SW480) and metastatic (SW620) colon cancer cells were cultured at hypoxia (1% oxygen), tissues (10% oxygen) and atmospheric (21% oxygen) normoxia with quercetin, epigallocatechin gallate, lipoic acid, hydroxycitric acid, their mixture, and without studied compounds (control). Antioxidants were used at physiological concentrations. The cell viability was determined by trypan blue dye exclusion and proliferation by MTT assay. RESULTS: The viability of each line ranged from 80% to 97%, and it was independent on the compound and oxygen availability. At hypoxia the cell count of both lines was lower than for the controls in the presence of each studied compound. At tissue normoxia the cell count of primary cancer cells was decreased only with epigallocatechin gallate, whereas metastatic cells were sensitive for each antioxidant. CONCLUSIONS: Our results indicated, that the studied antioxidants were not cytotoxic at physiological levels for both pirmary and metastatic colon cancer. Their cytostatic effect depend on the type of cell, oxygen availability and antioxidant concentration.

Targeting the expression of glutathione- and sulfate-dependent detoxification enzymes in HepG2 cells by oxygen in minimal and amino acid enriched medium.

Cancer cells exhibit specific metabolism allowing them to survive and proliferate in various oxygen conditions and nutrients' availability. Hepatocytes are highly active metabolically and thus very sensitive to hypoxia. The purpose of the study was to investigate the effect of oxygen on the expression of phase II detoxification enzymes in hepatocellular carcinoma cells (HepG2) cultured in minimal and rich media (with nonessential amino acids and GSH). The cells were cultured at 1% hypoxia, 10% tissue normoxia, and 21% atmospheric normoxia. The total cell count was determined by trypan blue exclusion dye and the expression on mRNA level by RT-PCR. The result indicated that the expression of glutathione-dependent enzymes (GSTA, M, P, and GPX2) was sensitive to oxygen and medium type. At 1% hypoxia the enzyme expression (with the exception of GSTA) was higher in minimal compared to rich medium, whereas at 10% normoxia it was higher in the rich medium. The expression was oxygen-dependent in both types of medium. Among phenol sulfotransferase SULT1A1 was not sensitive to studied factors, whereas the expression of SULT1A3 was depended on oxygen only in minimal medium. It can be concluded that in HepG2 cells, the detoxification by conjugation with glutathione and, to a lower extent with sulfate, may be affected by hypoxia and/or limited nutrients' availability. Besides, because the data obtained at 10% oxygen significantly differ from those at 21%, the comparative studies on hypoxia should be performed in relation to 10% but not 21% oxygen.

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia.

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L-Ser and/or L-Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1-mRNA and AST2-mRNA by RT-PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L-Asp. AST1-mRNA expression was independent on oxygen and cell line, but AST2-mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L-Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.

Alteration of Motor Protein Expression Involved in Bidirectional Transport in Peripheral Blood Mononuclear Cells of Patients with Amyotrophic Lateral Sclerosis.

BACKGROUND: Sporadic amyotrophic lateral sclerosis (SALS) is a fatal motor neuron degenerative disease of unclear pathogenesis. Disturbances of intracellular transport are possible causes of the disease. OBJECTIVE: We evaluated the expression of motor proteins involved in the anterograde (kinesins KIF1B, KIF5C) and retrograde (KIFC3, dynactin subunits DCTN1 and DCTN3) intracellular transport in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: PBMCs were obtained from 74 SALS patients with different clinical phenotypes, 65 blood donors (healthy control I), and 29 cases with other neurological diseases (disease control II) divided into subgroups IIA (atypical parkinsonism) and IIB (ALS-mimicking disorders). mRNA expression was studied by real-time qPCR, and protein level by Western blotting. RESULTS: In SALS, KIF5C and KIFC3 expression was significantly lower and DCTN1 higher than in control I, and dependent of age. KIF1B expression was significantly higher in SALS than in subgroup IIB, whereas DCTN1 and DCTN3 were higher in SALS than in subgroup IIA. All changes in the studied proteins were statistically significant in classic ALS but not in progressive muscular atrophy. CONCLUSION: In SALS, and especially in classic ALS, the changes in motor protein expression may alter bidirectional intracellular transport in PBMCs. More studies are needed to find out whether the levels of KIF5C and DCTN1 may be useful in ALS diagnosis, and whether KIF1B expression may discriminate ALS from ALS-mimicking disorders.

Altered L-arginine metabolism in children with controlled asthma.

Decreased level of L-arginine may lead to airway hyperresponsiveness, inflammation, and airway remodeling. Changes in L-arginine metabolism were observed earlier in adult asthmatic patients. Studies on L-arginine metabolism in children with bronchial asthma are limited. Because biosynthesis of L-arginine is insufficient in growing children, its potential metabolic alterations may have important clinical implications. This study was designed to evaluate L-arginine metabolism in children with well-controlled asthma. The studies were conducted on blood serum of 30 asthmatic and 20 healthy children (control group). Levels of L-arginine and its metabolic products, L-citrulline and L-ornithine, were measured by HPLC. Arginase activity was determined spectrophotometrically. Disease severity was evaluated by the asthma control test (ACT) and the level of nitric oxide (NO) in exhaled air. In asthmatic children L-arginine concentration was significantly lowered, whereas arginase activity was unchanged when compared with the healthy group. However, L-ornithine and L-citrulline levels were significantly increased. There was no correlation between arginase activity, amino acids levels, ACT scores, and exhaled NO. In children with chronic, well-controlled asthma L-arginine metabolism is altered. Given that L-arginine is absolutely essential for children, our findings may be of particular importance for the management of children with non-exacerbated asthma. They may also help to develop new therapeutic strategies targeted at L-arginine metabolism in the future.

Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration.

Dynactin is a complex motor protein involved in the retrograde axonal transport disturbances of which may lead to amyotrophic lateral sclerosis (ALS). Mice with hSOD1G93A mutation develop ALS-like symptoms and are used as a model for the disease studies. Similar symptoms demonstrate Cra1 mice, with Dync1h1 mutation. Dynactin heavy (DCTN1) and light (DCTN3) subunits were studied in the CNS of humans with sporadic ALS (SALS), mice with hSOD1G93A (SOD1/+), Dync1h1 (Cra1/+), and double (Cra1/SOD1) mutation at presymptomatic and symptomatic stages. In SALS subjects, in contrast to control cases, expression of DCTN1-mRNA but not DCTN3-mRNA in the motor cortex was higher than in the sensory cortex. However, the mean levels of DCTN1-mRNA and protein were lower in both SALS cortexes and in the spinal cord than in control structures. DCTN3 was unchanged in brain cortexes but decreased in the spinal cord on both mRNA and protein levels. In all SALS tissues immunohistochemical analyses revealed degeneration and loss of neuronal cells, and poor expression of dynactin subunits. In SOD1/+ mice both subunits expression was significantly lower in the frontal cortex, spinal cord and hippocampus than in wild-type controls, especially at presymptomatic stage. Fewer changes occurred in Cra1/SOD1 and Cra1/+ mice.It can be concluded that in sporadic and SOD1-related ALS the impairment of axonal retrograde transport may be due to dynactin subunits deficiency and subsequent disturbances of the whole dynein/dynactin complex structure and function. The Dync1h1 mutation itself has slight negative effect on dynactin expression and it alleviates the changes caused by SOD1G93A mutation.

Kinesin expression in the central nervous system of humans and transgenic hSOD1G93A mice with amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis is a fatal motor neuron degenerative disease. Most cases are sporadic (SALS), and approximately 10% are familial (FALS) among which over 20% are linked to the SOD1 mutation. Both SALS and FALS have been associated with retrograde axonal transport defects. Kinesins (KIFs) are motor proteins involved mainly in anterograde transport; however, some also participate in retrograde transport. OBJECTIVE: The purpose of the study was to investigate and compare the expression of kinesins involved in anterograde (KIF5A, 5C) and retrograde (KIFC3/C2) axonal transport in SALS in humans and FALS in mice with the hSOD1G93A mutation. METHODS: The studies were conducted on various parts of the CNS from autopsy specimens of SALS patients, and transgenic mice at presymptomatic and symptomatic stages using real-time quantitative PCR and reverse-transcription PCR. RESULTS: All KIF expression in the motor cortex of individual SALS subjects was higher than in the adjacent sensory cortex, in contrast to the expression in control brains. It was also significantly higher in the frontal cortex of symptomatic but not presymptomatic mice compared to wild-type controls. However, the mean KIF expression in the SALS motor and sensory cortexes was lower than in control cortexes. To a lesser extent the decrease in KIF mean expression also occurred in human but not in mouse ALS spinal cords and in both human and mouse cerebella. CONCLUSION: Disturbances in kinesin expression in the CNS may dysregulate both anterograde and retrograde axonal transports leading to motor neuron degeneration.

Mice with mutation in dynein heavy chain 1 do not share the same tau expression pattern with mice with SOD1-related motor neuron disease.

Due to controversy about the involvement of Dync1h1 mutation in pathogenesis of motor neuron disease, we investigated expression of tau protein in transgenic hybrid mice with Dync1h1 (so-called Cra1/+), SOD1G93A (SOD1/+), double (Cra1/SOD1) mutations and wild-type controls. Total tau-mRNA and isoforms 0, 1 and 2 N expression was studied in frontal cortex, hippocampus, spinal cord and cerebellum of presymptomatic and symptomatic animals (age 70, 140 and 365 days). The most significant differences were found in brain cortex and cerebellum, but not in hippocampus and spinal cord. There were less changes in Cra1/SOD1 double heterozygotes compared to mice harboring single mutations. The differences in total tau expression and in profile of its isoforms between Cra1/+ and SOD1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

[Intracellular transport proteins: classification, structure and function of kinesins]. / Bialka transportu wewnatrzkomórkowego: klasyfikacja, budowa i funkcje kinezyn.

Correct cell functioning, division and morphogenesis rely on efficient intracellular transport. Apart from dyneins and myosins, kinesins are the main proteins responsible for intracellular movement. Kinesins are a large, diverse group of motor proteins, which based on phylogenetic similarity were classified into fourteen families. Among these families, due to the location of their motor domains, three groups have been characterized: N-, C- and M-kinesin. As molecular motors, kinesins transport various molecules and vesicles mainly towards the microtubule plus end (from the cell body) participating in anterograde transport, although there are also kinesins involved in retrograde transport (C-kinesins). Kinesins are also involved in spindle formation, chromosome segregation, and spermatogenesis. Because of their great importance for the correct functioning of cells, mutations in kinesin coding genes may lead to such neurodegenerative diseases as dominant hereditary spastic paraplegia or Charcot-Marie-Tooth disease.

[Evaluation of beta2-microglobuline expression level in gastrointestinal tract tumors due to usefulness as a reference gene in PCR method]. / Ocena poziomu ekspresji beta2-mikroglobuliny w nowotworach przewodu pokarmowego pod wzgledem przydatnosci jako genu referencyjnego w metodzie PCR.

UNLABELLED: Quantitative and semi-quantitative determination of gene expression by PCR plays an important role in studying of tumors initiation and progression mechanisms. Selection of appropriate reference gene is a critical factor influencing the results of gene expression analysis. One of the most commonly used reference genes in PCR is beta2-microglobuline (beta2-M). Recent studies showed however that expression of some common reference genes might be unstable, therefore it is necessary to verify again their usefulness. The aim of the study was to determine the level of beta2-M mRNA in normal and tumor tissues of gastrointestinal tract due to adequate selection of reference gene in gene expression studies. MATERIAL AND METHODS: Samples were taken from 253 patients operated on for gastrointestinal tract tumors: 22 with oral cavity cancer, 12 with benign and 50 with malignant liver tumors, 86 with colorectal cancer, and 83 with metachronous metastases to liver. Also 56 patients with liver cirrhosis were studied, which was treated as pre-tumor state. Together 309 patients were studied. RNA was isolated from tissues by Chomczynski method. The expression level of 12-M was determined by reverse transcriptase PCR (RT-PCR) and given in terms of optical density values. RESULTS: Expression of beta2-M was observed in all studied tissues. There were no differences between normal and tumor tissue. The level of expression of beta2-M was different due to type of studied tissue (oral cavity, liver, colon). CONCLUSIONS: The lack of significant differences in beta2-M expression level in normal and tumor tissues indicated that beta2-M can be used as reference gene in studies of gene expression in gastrointestinal tract tumors. On the other hand differences of beta2-M expression level in different types of tissues point to its tissue specificity and suggest application in PCR of more than one reference gene.

Dyslipidemia in patients with amyotrophic lateral sclerosis - a case control retrospective study.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder leading to quadriplegia and aphagia. While swallowing difficulties and increased energy demand lead to malnutrition, increased lipid concentration may correlate with survival and respiratory functions. Objective: To analyze the frequency and type of dyslipidemias in a large population of clinically characterized ALS patients (PALS). Methods: The retrospective study included clinical and laboratory data of 650 consecutive PALS fulfilling the El Escorial criteria and 365 age- and gender-matched hospital controls. Results: 65% of PALS suffered from dyslipidemia independently of concomitant metabolic diseases. The most frequent lipid disorder was hypercholesterolemia (35% PALS, 25% controls), followed by mixed dyslipidemia (24.6%, 14%), with rare cases of hypertriglyceridemia and atherogenic dyslipidemia. Triacylglycerols (TAG) and LDL/HDL correlated with BMI, while LDL/HDL and total cholesterol (TCh) with disease duration. Among PALS with concomitant metabolic diseases, TCh correlated with disease duration and ALSFRS-R, while TAG with respiratory functions (FVC) in patients without metabolic diseases. The highest median concentration of TCh, LDL and LDL/HDL was found in classic ALS and PMA and the lowest in PBP. Conclusion: Dyslipidemia occurs more frequently in PALS compared to controls and independently of concomitant metabolic diseases. Similar to the general population, the most frequent lipid disturbance is hypercholesterolemia, followed by mixed dyslipidemia. Although particular lipid parameters correlate with BMI and disease duration, they do not show strong correlations with disease progression rate. There is a need of randomized control trials assessing the risk and benefits of the use of lipid lowering agents in ALS.

The analysis of selected neurotransmitter concentrations in serum of patients with Tourette syndrome.

BACKGROUND AND PURPOSE: Metabolic disturbances of excitatory and inhibitory neurotransmitters are implicated in pathogenesis of Tourette syndrome (TS). The aim of the study was to measure serum concentrations of glutamic acid, g-aminobutyric acid (GABA) and glycine in TS patients and evaluate any correlation between neurotransmitter levels and age at onset, actual age, gender, tic severity, duration of the disease and concomitant psychiatric disorders. MATERIAL AND METHODS: Sixty-seven TS patients, aged 16-59, and 57 healthy controls, aged 19-37, were enrolled in the study. Information regarding medical history and physical investigation was collected using a short questionnaire. Sixty-seven percent of patients were medication-free at the time of examination and the rest had withheld treatment for 24 hours before. Blood samples were taken after a 12-hour fasting period. HPLC technique was used. RESULTS: The TS group had higher glutamic acid and lower GABA levels. Glycine concentrations were comparable. No differences regarding neurotransmitter concentrations between treated and non-treated patients were found. Patients with concomitant obsessive-compulsive disorder and severe tics had higher glutamate levels. Glutamate concentrations correlated positively with the number of comorbid psychiatric disorders and GABA concentrations correlated negatively with the number of behavioural problems in patients with comorbidities. There was no correlation between analysed neurochemicals and age, gender, age at onset or disease duration. CONCLUSIONS: Imbalance between excitatory and inhibitory systems in the brains of TS patients may be reflected by glutamate and GABA serum level changes. Glutamate and GABA may be biomarkers of the disease and high concentration of glutamate may indicate more severe course of TS.

Changes in arginase isoenzymes pattern in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide affecting preferentially patients with liver cirrhosis. The studies were performed on tissues obtained during surgery from 50 patients with HCC, 40 with liver cirrhosis and 40 control livers. It was found that arginase activity in HCC was nearly 5- and 15-fold lower than in cirrhotic and normal livers, respectively. Isoenzymes AI (so-called liver-type arginase) and AII (extrahepatic arginase) were identified by Western blotting in all studied tissues, however the amount of AI, as well as the expression of AI-mRNA were lower in HCC, in comparison with normal liver, and those of AII were significantly higher. Since HCC is arginine-dependent, and arginine is essential for cells growth, the decrease of AI may preserve this amino acid within tumor cells. Concurrently, the rise of AII can increase the level of polyamines, compounds crucial for cells proliferation. Thus, both arginase isoenzymes seem to participate in liver cancerogenesis.

[New insights into arginase. Part I. Structure and characteristics]. / Nowe oblicza arginazy. Czesc I. Struktura i wlasciwosci.

Arginase (amidinohydrolase, EC 3.5.3.1) is present in all living organisms, i.e. bacteria, yeasts, plants, invertebrates, and vertebrates. In ureolitic organisms, arginase expresses the highest activity in the liver, where it takes part in ammonia detoxifi cation. Arginase activity is much lower in extrahepatic tissues and its physiological function is still poorly understood; however, it seems to be involved in L-arginine metabolism. Arginase is a homotrimer consisting of 20- to 40-kDa subunits acting at a pH of 10 and in the presence of manganese ions. Proline, ornithine, and NG-hydroxy-L-arginine, an intermediate in the biosynthesis of NO, are known as competitive arginase inhibitors. Two arginase isoenzymes, AI (the so-called "hepatic arginase") and AII ("extrahepatic arginase") are present in mammalian tissues. There are signifi cant differences between the isoenzymes regarding their subcellular localization, isoelectric point, substrate affinity, and immunological cross-reactivity. Arginase isoenzymes AI and AII have high substrate specifi city, but the affi nity to L-arginine is higher for isoenzyme AI than AII. Both isoenzymes exist in most tissues and their expressions change depending on the functional state and metabosynlic requirements. Besides differences in the amino-acid content of the arginase isoforms within one or different species, they have highly conserved regions responsible for the structure and catalytic properties of arginase.

[New insights into arginase. Part II. Role in physiology and pathology]. / Nowe oblicza arginazy. Czesc II. Rola w fizjologii i patologii.

Arginase (amidinohydrolase, EC 3.5.3.1) is known as the last enzyme in the urea cycle in the liver, but it is also present in extrahepatic tissues. Arginase hydrolyzes L-arginine to L-ornithine and urea and its biochemical and physiological role varies depending on the organism and tissue. Besides its participation in ammonia detoxification, arginase is involved in the synthesis of polyamines, crucial for the proper course of many metabolic processes, proline, the main connective tissues protein, and glutamates, amino acids which take part in nitric metabolism, important in the nervous system and also a substrate for protein synthesis. The competition of arginase with nitric oxide synthase (NOS) for the common substrate L-arginine indicates its participation in the regulation of nitric oxide (NO) synthesis. The physiological role of arginase and its common occurrence indicate its engagement in many pathologies. Due to its competition with NOS for arginine and its participation in proline synthesis, arginase plays an important role in such diseases as cerebral stroke, trauma, inflammation, and depression, whereas its participation in polyamine synthesis indicates arginase's engagement in the development of neoplastic diseases in the human organism.

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation.

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.

[Expression of glutathione S-transferase isoenzymes in tongue and floor of mouth squamous cell carcinomas]. / Ekspresja izoenzymów transferazy S-glutationowej w plaskonablonkowym raku jezyka i dna jamy ustnej.

UNLABELLED: Glutathione S-transferase (GST, EC 2.5.1.18) is one of the most important enzymes protecting cells against toxic, electrophilic compounds. It occurs in isoenzymes, expression of which changes in various pathological conditions including neoplasia. The aim of the present study was to analyze the expression pattern of GST pi, mi4 and alpha in human tongue and floor of mouth squamous cell carcinomas. MATERIAL AND METHODS: The studies were conducted on tissue samples obtained from surgery. RT-PCR was used to determine the GSTs-mRNA expression, and Western blotting to study GST pi protein expression. RESULTS: Indicated statistically significant differences in GST pi mRNA expression in tumor and control tissues. GST pi expression was raised in tumors on both mRNA and protein levels, whereas mRNA-GSTmi4 and alfa expression was diverse. CONCLUSION: Increased level of GST pi expression indicates its role in carcinogenesis.

[Serum arginase activity in patients with liver cirrhosis and hepatocellular carcinoma]. / Arginaza w surowicy krwi chorych z marskoscia i rakiem watrobowokomórkowym.

Preoperative and postoperative arginase activity was determined in blood serum of 25 patients with liver cirrhosis and 25 patients with hepatocellular carcinoma. The rise of serum arginase activity was observed in the majority of patients before the surgery and the decrease after tumor resection or liver transplantation. The preoperative values of serum arginase activity were similar in both groups of patients. A presence of additional, anionic arginase isoform (All) was demonstrated in serum of studied patients, which was absent in healthy subjects. Thus, our results indicate that the arginase activity cannot differentiate liver cirrhosis and hepatocellular carcinoma. However, arginase isoform All seems to be specific for studied liver diseases.

Validation of qPCR reference genes in lymphocytes from patients with amyotrophic lateral sclerosis.

Quantitative polymerase chain reaction (qPCR) is the most specific and reliable method for determination of mRNA gene expression. Crucial point for its accurate normalization is the choice of appropriate internal control genes (ICGs). In the present work we determined and compare the expression of eight commonly used ICGs in lymphocytes from 26 patients with amyotrophic lateral sclerosis (ALS) and 30 control subjects. Peripheral blood mononuclear cells (PBMCs) before and after immortalization by EBV transfection (lymphoblast cell lines-LCLs) were used for qPCR analysis. LCLs were studied before and after liquid nitrogen cryopreservation and culturing (groups LCL1 and LCL2, respectively). qPCR data of 8 ICGs expression was analyzed by BestKeeper, NormFinder and geNorm methods. All studied genes (18SRNA, ACTB, B2M, GUSB,GAPDH, HPRT1, MT-ATP6 and RPS17) were expressed in PBMCs, whereas only first four in LCLs. LCLs cryopreservation had no effect on ICGs expression. Comprehensive ranking indicated RPS17 with MT-ATP6 as the best ICGs for qPCR in PBMCs of control and ALS subjects, and RPS17 with 18RNA or MT-ATP6 in LCLs from ALS. In PBMCs 18RNA shouldn't be used as ICG.

Activity and expression of glutathione S-transferase pi in patients with amyotrophic lateral sclerosis.

Glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme responsible for inactivation of a large variety of toxic, electrophilic compounds and organic peroxides. GST activity and GST pi expression were studied in patients with amyotrophic lateral sclerosis (ALS). Studies were conducted on cerebrospinal fluid (CSF), blood serum and peripheral blood mononuclear cells (PBMC) obtained from 40 ALS patients. CSF from 30 subjects without neurological diseases and blood from 40 healthy blood donors were used as controls. GST activity assayed with 1-chloro-2,4-dinitrobenzene (substrate for transferase activity) and cumene peroxide (substrate for peroxidase activity) was significantly decreased in PBMC of ALS patients, as well as the GST pi expression on both mRNA and protein level. The mean peroxidase activity was however significantly increased in CSF and serum of ALS patients with the specificity of 80% and 73%, and the sensitivity of 78% and 85%, respectively. It can thus be concluded that the protective barrier formed by GST is originally affected in peripheral blood of ALS patients, and may increase their vulnerability to toxic effects of electrophilic compounds and organic peroxides. Studies on a larger group are needed to answer the question whether GSH-Px determination may be implicated in the diagnosis of ALS.