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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.

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.

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.

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.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3-5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease. METHODS: Affinity proteomics in the form of antibody suspension bead arrays were applied to profile plasma samples from 367 ALS patients and 101 controls. The plasma protein content was directly labeled and protein profiles obtained using 352 antibodies from the Human Protein Atlas targeting 278 proteins. A focused bead array was then built to further profile eight selected protein targets in all available samples. RESULTS: Disease-associated significant differences were observed and replicated for profiles from antibodies targeting the proteins: neurofilament medium polypeptide (NEFM), solute carrier family 25 (SLC25A20), and regulator of G-protein signaling 18 (RGS18). INTERPRETATION: Upon further validation in several independent cohorts with inclusion of a broad range of other neurological disorders as controls, the alterations of these three protein profiles in plasma could potentially provide new molecular markers of disease that contribute to the quest of understanding ALS pathology. QC 20150115

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.

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, γ-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.

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.

Glutathione S-transferase pi (GSTP1) is a crucial enzyme in detoxification of electrophilic compounds and organic peroxides. Together with Se-dependent glutathione peroxidase (Se-GSHPx) it protects cells against oxidative stress which may be a primary factor implicated in motor neuron disease (MND) pathogenesis. We investigated GSTP1 polymorphisms and their relationship with GST and Se-GSTPx activities in a cohort of Polish patients with MND. Results were correlated with clinical phenotypes. The frequency of genetic variants for GSTP1 exon 5 (I105V) and exon 6 (A114V) was studied in 104 patients and 100 healthy controls using real-time polymerase chain reaction. GST transferase activity was determined in serum with 1-chloro-2,4-dinitrobenzene, its peroxidase activity with cumene hydroperoxide, and Se-GSHPx activity with hydrogen peroxide. There were no differences in the prevalence of GSTP1 polymorphism I105V and A114V between MND and controls, however the occurrence of CT variant in codon 114 was associated with a higher risk for MND. GSTP1 polymorphisms were less frequent in classic ALS than in progressive bulbar palsy. In classic ALS C* (heterozygous I /V and A /V) all studied activities were significantly lower than in classic ALS A* (homozygous I /I and A/A). GST peroxidase activity and Se-GSHPx activity were lower in classic ALS C* than in control C*, but in classic ALS A* Se-GSHPx activity was significantly higher than in control A*. It can be concluded that the presence of GSTP1 A114V but not I105V variant increases the risk of MND, and combined GSTP1 polymorphisms in codon 105 and 114 may result in lower protection of MND patients against the toxicity of electrophilic compounds, organic and inorganic hydroperoxides.

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.

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.

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

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin--derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine--derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.

The mean arginase activity in breast cancers (n = 80) was significantly higher than in control tissues and it accounted for 0.31 +/- 0.23 U/g wet tissue and 0.083 +/- 0.061 U/g (P < 0.05), respectively. With the cutoff value of 0.1 U/g wet tissue, raised arginase activity was observed in 74% of tumors. The preoperative arginase activity in blood serum from women with breast cancer was 11.2 +/- 7.9 U/l (n = 115), and it was significantly higher than in 70 healthy controls, where it was 5.7 +/- 2.4 U/l (P < 0.05). With the cutoff value for normal serum arginase activity above 8.0 U/l, the activity was raised in 10% of control individuals, and in 63% of women with breast cancer. The sensitivity and specificity of the arginase test in blood serum were 63% and 60%, respectively. Two isoforms immunologically identical to human kidney arginases (L-arginine amidinohydrolase) were found in both normal and cancerous breast tissues. The level of anionic form was similar in control and cancerous tissues, whereas the cationic isoform predominated in breast cancer. The cationic isoform was the only one present in serum of both ill and healthy women, and its level was higher in patients with breast cancer. Thus, it can be concluded that the cationic isoform is responsible for the increase of arginase activity in serum of patients with breast cancer.

Photodynamic therapy (PDT), a promising therapeutic modality for the management of solid tumors, is a two-phase treatment consisting of a photosensitizer and visible light. Increasing evidence indicates that tumor cells in regions exposed to sublethal doses of PDT can respond by rescue responses that lead to insufficient cell death. We decided to examine the role of superoxide dismutases (SODs) in the effectiveness of PDT and to investigate whether 2-methoxyestradiol (2-MeOE(2)), an inhibitor of SODs, is capable of potentiating the antitumor effects of this treatment regimen. In the initial experiment we observed that PDT induced the expression of MnSOD but not Cu,Zn-SOD in cancer cells. Pretreatment of cancer cells with a cell-permeable SOD mimetic, Mn(II)-tetrakis(4-benzoic acid)porphyrin chloride, and transient transfection with the MnSOD gene resulted in a decreased effectiveness of PDT. Inhibition of SOD activity in tumor cells by preincubation with 2-MeOE(2) produced synergistic antitumor effects when combined with PDT in 3 murine and 5 human tumor cell lines. The combination treatment was also effective in vivo producing retardation of the tumor growth and prolongation of the survival of tumor-bearing mice. We conclude that inhibition of MnSOD activity by 2-MeOE(2) is an effective treatment modality capable of potentiating the antitumor effectiveness of PDT.

BACKGROUND Colorectal carcinoma (CRC) is one of the most common malignancies. In the current work, the role of arginase as a diagnostic marker in patients with recurrent CRC and colorectal liver metastases (CRCLM) was studied. METHODS Arginase activity was monitored in serum from 40 patients with primary CRC and from 100 patients with CRCLM. Blood was taken before and after patients underwent tumor resection. Studies were conducted for 3 years. RESULTS Preoperative arginase activity in serum from patients with CRC and CRCLM was much greater compared with the arginase activity in serum from healthy control blood donors. One and two cut-off levels of increased arginase activity were observed in patients with CRC and CRCLM, respectively. After patients underwent tumor resection, the arginase activity decreased to normal values in both patients with CRC and patients with CRCLM. Activity levels remained low in patients who did not develop recurrent CRC or CRCLM (first or second). In patients who developed subsequent recurrences or metastases that appeared after surgery, during 3 years of surveillance, a significant rise in serum arginase activity was observed. The clinical prognosis for patients was worst when the postoperative serum arginase activity was very high, because those patients more often developed second liver metastases or died. CONCLUSIONS The authors conclude that the determination of serum arginase activity may be a complementary test to confirm the occurrence of CRC and may be useful for the early diagnosis of patients who develop recurrent CRC and/or CRCLM. Cancer 2002;94:2930–4. © 2002 American Cancer Society. DOI 10.1002/cncr.10563

Objective To assess the utility of arginase activity and expression in diagnosis of liver diseases. Design and methods Arginase activity, sensitivity and specificity were determined in serum of 140 patients including 50 with HCC, 60 with LC, 30 with choledocholithiasis (CDL) and 90 healthy controls. In HCC and LC arginase activity in serum was studied before and after tumor resection or liver transplantation. Arginase sensitivity in HCC was compared to that of alpha-fetoprotein (AFP) and aminotransferases (AST, ALT). In LC the activity was determined also in bile before and after transplantation. The expression of arginase isoenzymes in serum was studied by Western blotting. Results In HCC and LC the preoperative arginase activity was significantly higher compared to controls, and it decreased after surgery. The sensitivity of arginase in HCC was much higher than that of AFP, AST and ALT (96, 40, 20 and 18%, respectively). In HCC it was higher than in LC (93%) and CDL (33%). The specificity of arginase was above 80%. In bile of cirrhotic patients the highest activity was immediately after liver transplantation. It decreased with time but increased dramatically at the time of the graft rejection. Arginase AII was present in serum of HCC and LC but not the control cases. Conclusions The increase of arginase activity in serum accompanied by the presence of isoenzyme AII can be useful in HCC and LC diagnosis. The determination of arginase activity in bile may be helpful in monitoring liver graft recipients.

The isoelectric point and substrate specificity of the main isoform of glutathione-S-transferase (GST, EC 2.5.1.18) isolated from brain stem, hippocampus and parietal cortex of pig brain were determined. The effect of serotonin, its precursors (Try, 5-HTry), physiologically active derivative (melatonin) and final metabolite (5-HIAA) on the activity of this form was examined. Investigation indicated that serotonin did not affect the activity of GST in all studied regions of brain. The inhibitory effect of Try was stronger than that of 5-HTry, but weaker than the one expressed by melatonin and especially by 5-HIAA. Studies on the type of inhibition showed that Try, melatonin and 5-HIAA can compete for the active site with the electrophilic substrate but not with glutathione. Therefore precursors and endogenous derivatives of serotonin but not serotonin itself may affect the detoxification function of brain glutathione-S-transferase and increase the exposure of brain to toxic electrophiles.

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 (Dync1h1, 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.

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, alpha-methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (alpha-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated alpha-methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.

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.

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.

Objective The usefulness of simultaneous l -arginine and arginase determination in diagnosis of primary and metastatic colorectal cancer. Methods 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). Results 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%. Conclusion Simultaneous determination of l -arginine and arginase increases the value of arginase itself in diagnosis and follow up of patients with CRC and CRCLM.

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.

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.

Arginase (EC 3.5.3.1) activity was determined in 54 colorectal tissues obtained from patients with primary colorectal adenocarcinoma as well as in serum of 45 patients and 65 healthy individuals. In patients, the preoperative values of the mean serum arginase activity and the activity in colorectal tumors were much higher than in serum of healthy subjects and control tissues. Two isoforms of arginase, anionic and cationic, were identified in colorectal tissues (normal and cancerogenous), and only one, the cationic form, in serum. These arginases were different from the main human liver cationic arginase (pI 9.3). The anionic colorectal arginase was identical with the human liver anionic isoform (pI 7.7), and the cationic arginase from colorectal tissues and blood serum with the human kidney cationic enzyme (pI 8.9). The total activity and the level of protein of the cationic arginase in colorectal cancer was higher than in control tissue, and it was also higher in serum of patients with colorectal cancer than in healthy subjects. Thus, it can be concluded that the increased arginase activity in blood serum and colorectal cancer in studied patients was due to the raised level of the cationic arginase and this isoform seems to be a discriminating parameter for assessing the presence of colorectal cancer.

Male reproductive organs are extremely sensitive to the negative influence of toxic environmental factors as well as drugs, and until now not many attempts have been made at studying the detoxication enzymes and the relationship between the activity of those enzymes and spermatozoa fertility. In the present work we studied cytosolic glutathione-S-transferases (GST, EC 2.5.1.18) from different parts (head, corpus and tail) of bull and boar epididymis. We isolated two molecular forms of GST from each part of epididymis, characterized their biochemical properties and examined the mechanism of the catalyzed reaction. On the basis of their substrate specificity and isoelectric point, the isoforms were found to belong to the near neutral GST class mi. All examined GST forms exhibited higher affinity towards GSH than towards 1-chloro-2,4-dinitrobenzene (CDNB) and bull epididymis GST forms showed biphasic Lineweaver-Burk double reciprocal curves in the presence of GSH as a variable substrate. Boar epididymis anionic GST had the -SH groups both in the GSH and the CDNB binding place, whereas the cationic GST form--arginine residues in the CDNB binding place. Bull epididymis GST forms contained neither thiol nor arginine residues essential for catalytic activity.

Phenol sulfotransferases (PST) from bovine lung and small intestine were purified about 1000-fold. PST from bovine small intestine, similarly as the bovine lung enzyme, catalyzes sulfation of only exogenous phenols. A single thermostable form of PST, active with high concentrations of phenol (Km = 1.43 mM) was found in the small intestine. The effect of divalent cations on the activity of the two phenol sulfotransferases was determined. The molecular weight of the native enzymes was estimated as about 69,000 and subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis as 35,000. In double immunodiffusion tests the bovine lung PST showed antigenic identity with the bovine small intestine enzyme but complete immunological incompatibility with rat liver sulfotransferase.

Le but est de comparer la sulfotransferase d'enterocytes d'ileon humain avec celle de poumon humain, celles d'autres tissus humains et avec celle du tractus gastrointestinal d'autres especes. Les resultats permettent de caracteriser le sulfotransferase d'intestin grele et montre une mobilite electrophoretique differente de celle d'ileon de bœuf

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.

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.

Phenol sulfotransferases (PST) from bull and boar testis were partially purified and characterized. A single form of PST adsorbed on DEAE-cellulose was found in the bull testis, whereas from boar testis two different peaks of PST activity were separated. The bull testis PST and both boar testis enzymes were active with p-nitrophenol and adrenalin. They all showed higher affinity to pNP than to adrenalin and were inhibited by these substrates at higher concentrations. Their optimal pH was at 8.5. Bull testis PST and boar PST II which were adsorbed on DEAE-cellulose were thermostable, whereas boar PST I was thermolabile. Those three PST forms differed in sensitivity to 2,6-dichloro-4-nitrophenol (DCNP), N-ethyl maleimide (NEM), lodoacetamide (IAA) and phenylglyoxal (PG). Bull and boar PST II were more rapidly inactivated in the presence of DCNP than boar PST I. In the presence of NEM, the —SH groups reagent, the bull phenol sulfotransferase and boar PST I lost their activity, whereas the activity of boar PST increased. Also iodoacetamide, another —SH group modificator, raised boar PST II activity and decreased boar PST I activity. DTT, which protects thiol groups, had an opposite effect on the enzymes studied than NEM. Phenylglyoxal, a reagent specific for arginine residues inhibited bull testis PST and both boar phenol sulfotransferases. Substrate protection experiments were also performed to determine the localization of reactive groups in bull and boar testis phenol sulfotransferases.

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.

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.