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ABSTRACT: Dysfunction of fast axonal transport, vital for motor neurons, may lead to neurodegeneration. Anterograde transport is mediated by N-kinesins (KIFs), while retrograde transport by dynein 1 and, to a minor extent, by C-kinesins. In our earlier studies we observed changes in expression of N- and C-kinesins (KIF5A, 5C, C2) in G93ASOD1-linked mouse model of motor neuron degeneration. In the present work we analyze the profile of expression of the same kinesins in mice with a dynein 1 heavy chain mutation (Dync1h, called Cra1), presenting similar clinical symptoms, and in Cra1/SOD1 mice with milder disease progression than SOD1 transgenics. We found significantly higher levels of mRNA for KIF5A and KIF5C but not the KIFC2 in the frontal cortex of symptomatic Cra1/+ mice (aged 365 days) compared to the wild-type controls. No changes in kinesin expression were found in the spinal cord of any age group and only mild changes in the hippocampus. The expression of kinesins in the cerebellum of the presymptomatic and symptomatic mice (aged 140 and 365 days, respectively) was much lower than in age-matched controls. In Cra1/SOD1 mice the changes in KIFs expression were similar or more severe than in the Cra1/+ groups, and they also appeared in the spinal cord. Thus, in mice with the Dync1h1 mutation, which impairs dynein 1-dependent retrograde transport, expression of kinesin mRNA is affected in various structures of the CNS and the changes are similar or milder than in mice with double Dync1h1/hSOD1G93A mutations.
Acta biochimica Polonica 03/2013; · 1.49 Impact Factor
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ABSTRACT: Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.
Acta biochimica Polonica 11/2011; 58(4):621-6. · 1.49 Impact Factor
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ABSTRACT: 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.
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.
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).
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.
Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego 07/2011; 31(181):24-30.
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ABSTRACT: 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.
Neurochemical Research 03/2011; 36(6):978-85. · 2.24 Impact Factor
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ABSTRACT: 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.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 01/2011; 65:588-96.
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ABSTRACT: Heroin is an illicit narcotic abused by millions of people worldwide. In our earlier studies we have shown that heroin intoxication changes the antioxidant status in human brain. In the present work we continued our studies by estimating the effect of heroin abuse on reduced glutathione (GSH) and enzymes related to this cofactor, such as glutathione S-transferase detoxifying electrophilics (GST) and organic peroxides (as Se-independent glutathione peroxidase-GSHPx), and Se-dependent glutathione peroxidase (Se-GSHPx) specific mainly for hydrogen peroxide. Studies were conducted on human brains obtained from autopsy of 9 heroin abusers and 8 controls. The level of GSH and the activity of glutathione-related enzymes were determined spectrophotometrically. The expression of GST pi on mRNA and protein level was studied by RT-PCR and Western blotting, respectively. The results indicated significant increase of GST and GSHPx activities, unchanged Se-GSHPx activity, and decreased level of GSH in frontal, temporal, parietal and occipital cortex, brain stem, hippocampus, and white matter of heroin abusers. GST pi expression was increased on both mRNA and protein levels, however the increase was lower in brain stem than in other regions. Heroin affects all regions of human brain, and especially brain stem. Its intoxication leads to an increase of organic rather then inorganic peroxides in various brain regions. Glutathione S-transferase plays an important role during heroin intoxication, however its protective effect is lower in brain stem than in brain cortex or hippocampus.
Drug and alcohol dependence 01/2011; 113(1):8-12. · 3.60 Impact Factor
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ABSTRACT: Cirrhosis leads to an inability of the liver to perform its biochemical functions. It can also lead to hepatocellular carcinoma in which, as we showed lately, arginase isoenzyme pattern changes. The present work presents our results on arginase isoenzymes and their possible role in liver cirrhosis. The study was performed on tissues obtained during liver transplantation from 60 patients with liver cirrhosis, and on samples of histologically normal liver (control) from 40 patients with benign or colorectal cancer liver metastases removed during surgery, 6-7 cm from the tumor border. Arginase isoenzymes AI (so-called liver-type arginase) and AII (called extrahepatic arginase) were identified by Western blotting and isolated by ion-exchange chromatography. Their expression on mRNA level was studied by RT-PCR. A significant decrease in arginase activity, dependent of the liver clinical stage, was observed in cirrhotic tissue. Arginase AI activity and its mRNA level were significantly decreased in cirrhotic liver, whereas the activity and expression of arginase AII were concurrently raised, as compared to normal liver. Since arginase AI is a key enzyme of the urea cycle, whereas arginase AII most probably takes part in the biosynthesis of ornithine and polyamines, the defective ammonia inactivation and increased collagen biosynthesis observed in cirrhotic liver may be related to the changes in arginase AI and AII levels, respectively.
Acta biochimica Polonica 08/2009; 56(3):465-9. · 1.49 Impact Factor
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ABSTRACT: The usefulness of simultaneous L-arginine and arginase determination in diagnosis of primary and metastatic colorectal cancer.
L-arginine and arginase were determined before and after surgery in serum from 43 patients with colorectal cancer (CRC), 24 with colorectal cancer liver metastasis (CRCLM), and 39 control subjects (10 patients with non-malignant diseases and 29 healthy blood donors).
Preoperative L-arginine concentration in the patient groups was 2-fold higher, whereas arginase activity was over 3- and 6-fold higher in CRC and CRCLM when compared with control. The values of both parameters lowered significantly after surgery. The sensitivity of single parameter in CRC was 79% for L-arginine and 81% for arginase, and in CRCLM it was 83% for each parameter. The combination of L-arginine with arginase improved the sensitivity to 93% and 100% in CRC and CRCLM, respectively. The specificity of L-arginine and arginase calculated for 39 subjects was 87% and 82%.
Simultaneous determination of L-arginine and arginase increases the value of arginase itself in diagnosis and follow up of patients with CRC and CRCLM.
Clinical biochemistry 01/2009; 42(4-5):353-7. · 2.02 Impact Factor
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 10/2008; 377(2):337-40. · 2.48 Impact Factor
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ABSTRACT: 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.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 02/2008; 62:206-13.
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ABSTRACT: 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.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 02/2008; 62:214-21.
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ABSTRACT: 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.
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.
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.
Increased level of GST pi expression indicates its role in carcinogenesis.
Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego 10/2007; 23(135):196-9.
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ABSTRACT: The work is a continuation of studies on tau expression and alternative splicing in the central nervous system of transgenic mice harboring human SOD1 with G93A amyotrophic lateral sclerosis (ALS)-associated mutation. Since age is an important risk factor for ALS, we expanded the studies into younger animals (age 5 and 25 days). We also included cerebellum, a structure not studied in the context of neurodegeneration in ALS. We found decreased total tau-mRNA expression in hippocampus but not in cortex and spinal cord of young transgenics, and a lack of exon 10 in 5-day-old mice. In cerebellum, the total tau-mRNA expression was increased in transgenic animals during the whole period of life, however at the symptomatic stage of ALS (age 120 days) the level of protein was decreased. It can be concluded that the SOD1 G93A mutation causes early alterations of tau expression in cns, which are not exclusively restricted to the upper and lower motor neuron.
Neurochemical Research 04/2007; 32(3):415-21. · 2.24 Impact Factor
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ABSTRACT: 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.
Wiadomości lekarskie (Warsaw, Poland: 1960) 02/2007; 60(5-6):215-8.
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ABSTRACT: Pancreatitis is the most common disease of the pancreas. Difficulties in early recognition of pancreatitic diseases, particularly chronic pancreatitis, are the reason of the search for new diagnostic methods. In our earlier studies we have shown that the determination of arginase activity in serum of patients with pancreatic cancer may be useful test in preoperative diagnosis of this cancer. The aim of this study was to asses the arginase activity in serum of patients with acute and chronic pancreatitis before and after medical treatment.
Arginase activity was studied in serum of 10 patients with acute and 10 patients with chronic pancreatitis obtained before, after and/or during the medical treatment.
The increase of arginase activity was observed in both studied groups before the medical treatment, and a statistically significant decrease after the treatment (p < 0,05). There were not significant differences between arginase activity in acute and chronic pancreatitis.
Arginase activity determination seems to be useful in monitoring the treatment of patients with acute and chronic pancreatitis.
Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego 12/2006; 21(126):522-4.
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ABSTRACT: Tau is a protein involved in regulation of microtubule stability, axonal differentiation and transport. Alteration of retrograde transport may lead to motor neuron degeneration. Thus alternative mRNA splicing and expression of tau isoforms were studied in a transgenic mouse model harboring the human SOD1 G93A mutation. The studies were performed on cortex, hippocampus and spinal cord of 64- and 120-day-old animals (presymptomatic and symptomatic stage) and wild type controls. Exon 10 was found in all studied tissues. The 2N isoform containing exons 2 and 3 (+2+3) and the 1N (+2-3) predominated over the 0N (-2-3) in brain regions of the studied mice. The 2N expression was significantly lower in cortex and hippocampus of symptomatic animals compared to analogue control tissues. The decrease in 2N expression resulted in lower levels of total tau mRNA and tau protein. No changes in tau expression were observed in spinal cord of studied animals.
Neurochemical Research 06/2006; 31(5):597-602. · 2.24 Impact Factor
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ABSTRACT: 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.
Clinica Chimica Acta 03/2006; 364(1-2):217-21. · 2.54 Impact Factor
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ABSTRACT: Heroin is an illicit narcotic abused by millions of people worldwide. In the present work, we estimated peroxyl radical-trapping capacity (PRTC), oxidative stress markers - malondialdehyde and protein carbonyl groups, as well as antioxidant enzymes - superoxide dismutase and catalase, in different regions of brain. Studies conducted on nine brains from heroin abusers and eight from control subjects revealed a decrease in PRTC in each part of heroin intoxicated brains and an increase in lipid peroxidation in brain cortex, brain stem and white matter but not in hippocampus. Protein oxidation was increased in hippocampus and in brain stem, but it was unchanged in gray and white matters. Superoxide dismutase and catalase activities were unchanged in heroin addicts. We conclude that heroin intoxication changes the antioxidant status in human brain by increasing the amount of organic rather then inorganic peroxides. The most severe condition of oxidative stress occur in brain stem.
Environmental Toxicology and Pharmacology 01/2006; 21(1):80-5. · 1.47 Impact Factor
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ABSTRACT: The expression of glutathione S-transferase pi (GST pi), an enzyme responsible for inactivation of a large variety of toxic compounds was studied in spinal cord, motor and sensory brain cortex obtained from patients who died in the course of amyotrophic lateral sclerosis (ALS). The studies were performed on formalin-fixed, paraffin-embedded (FFPE) and freshly frozen tissues. The method of RNA isolation from FFPE was modified. A significant decrease of GST pi-mRNA expression was found in cervical spinal cord and motor brain cortex of ALS subjects comparing to analogue control tissues (P<0.01), as well as in motor cortex of ALS subjects comparing to their sensory cortex (P<0.05). In spinal cords the decrease in GST pi-mRNA expression was accompanied by a decrease of GST pi protein level. Results indicated lowered GST pi expression on both mRNA and protein levels in the regions of nervous system affected by ALS. The non-properly inactivated by GST toxic electrophiles and organic peroxides may thus contribute to motor neurons damage.
Neurochemical Research 09/2005; 30(8):1003-7. · 2.24 Impact Factor
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ABSTRACT: Glutathione S-transferase (GST) is a part of the most important enzymatic systems protecting organisms from toxic, electrophilic compounds. Expression of numerous GST isoenzymes is changed in various pathological conditions, including neoplasia. The aim of present study was to analyze the expression pattern of GST pi, mu (mu4 and mu5) and alpha at the mRNA and protein levels in human primary gliomas. The studies were conducted on tissue samples obtained from surgery. RESULTS: There were no changes in GST pi mRNA expression, determined by RT-PCR, between gliomas and tumor adjacent tissues. However, using Western blotting method, an increase in GST pi protein in tumors was observed, which might be caused by the lower rate in protein degradation. Decrease in the expression of GST mu at mRNA level (mu4), as well as at protein level was shown in gliomas compared to control tissues. mRNA for GST mu5 isoform was demonstrated only in two tumor cases, but not in normal tissues, and for GST alpha, in one sample of control tissue. At the protein level, GST pi expression was increased in gliomas, whereas GST mu was decreased. CONCLUSION: Changes in GST isoenzymes expression can play an important role in the susceptibility of central nervous system to carcinogenesis.
Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego 07/2005; 18(108):676-9.
