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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.

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.

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.

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.

Arginase from extracts of human heart was purified about 1500-fold. In polyacrylamide-gel electrophoresis the enzyme migrated to the cathode at pH 5.5, showing very low mobility at pH 8.9. Molecular weight determined by gel filtration was 120 000. The Km for L-arginine was 5 mM. L-Ornithine and L-lysine were competitive inhibitors. The enzyme was completely inactivated by treatment with EDTA, and dissociated into subunits with mol.wt. of about 30 000. Addition of Mn2+ ions to the inactive subunits resulted in reappearance of the enzyme activity; the molecular weight of the reactivated enzyme corresponded to that of the native form.

1. The predominant form of rat liver arginase, A1, and that of kidney, A4, were isolated and partially purified. 2. It was found that arginase A4, similarly as A1, has oligomeric structure. Either of the enzymes on EDTA treatment dissociates into inactive subunits of molecular weight 30 000 daltons. Addition of Mn2+ ions restores the activity and causes reassociation of subunits to the native form of 120 000 mol. wt. 3. The subunits of A4 differ considerably in electrophoretic mobility from subunits of A4, which probably is the reason why the native forms of the enzyme from kidney and liver differ in electrophoretic behaviour.

3'-Phosphoadenosine-5'-phosphosulphate-dependent enzymes that catalyse sulphation of p-nitrophenol have been purified from rat kidney and stomach mucosa by affinity chromatography on the p-hydroxyphenylacetic acid-agarose conjugate, by chromatography on DEAE-cellulose and Sephadex G-100. The phenol sulphotransferase (PST) from rat kidney had Mr of 69 000 and that of the stomach enzyme was 32 000. With p-nitrophenol as the sulphate acceptor, the pH optima were 6.4 for the stomach PST and 5.4 and 6.6 for the kidney enzyme. Both enzymes were inhibited by 2,6-dichloro-4-nitrophenol and phenylglyoxal, an arginine specific modifying reagent. Both enzymes readily sulphated p-nitrophenol, 2-naphthol, 1-naphthol and salicylamide and did not act on biogenic amines (e.g. epinephrine, norepinephrine, dopamine, serotonin), acid metabolites of catecholamines (e.g. 3,4-dihydroxyphenylacetic acid, homovanillic acid), and O-methylated metabolites of catecholamines. Only the stomach enzyme sulphated such catecholamine metabolites as homovanillic alcohol and 3-methoxy-4-hydroxyphenylglycol. In contrast to the brain enzyme, but similarly to the liver enzyme, the kidney and stomach phenol sulphotransferases appear to sulphate exogenous phenolic substrates in preference to potential endogenous substrates.