* Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil, and Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
1 To whom correspondence should be addressed at Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105900Santa Maria, RS, Brasil. Fax: + (055)- 220 8240. E-mail: jbtrocha{at}yahoo.com.br.
Received July 7, 2004; accepted August 31, 2004
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ABSTRACT |
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Key Words: organochalcogens; methylmercury; intermediate filaments; phosphorylation; neuroprotection.
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INTRODUCTION |
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Selenium is a structural component of several enzymes with physiologically antioxidant properties, including glutathione peroxidases (Flohé et al., 1973). In the last decades, the interest for the development of synthetic organic selenium compounds has increased considerably, since various of these compounds present chemical and biological antioxidant properties (Mugesh and Singh, 2000
; Muller et al., 1984
; Parnham and Sies, 2000
; Sies, 1993
). Of particular importance, the organoselenium compound ebselen has been demonstrated to be neuroprotective in preclinical studies (Davalos, 1999
; Saito et al., 1998
; Yamaguchi et al., 1998
) and on a variety of in vitro and in vivo animal models of neuropathological conditions, including ischemia (Dawson et al., 1995
; Imai et al., 2003
; Porciuncula et al., 2003
; Takasago et al., 1997
), quinolic acid- or glutamate-induced lipoperoxidation (Porciuncula et al., 2001
; Rossato et al., 2002a
,b
), and exposure to methylmercury (Farina et al., 2003
). The antioxidant activity of organoselenides has been tentatively attributed to their glutathione peroxidase-like activity (Muller et al., 1984
; Wendel et al., 1984
). Organochalcogens also display antioxidant activities that are not linked to their glutathione peroxidase-like activity (Rossato et al., 2002b
). However, these organocompounds can have extreme neurotoxic effects on rodents (Nogueira et al., 2003a
).
Protein phosphorylation is established as an important mechanism of cell regulation in the central nervous system (CNS) (Dunkley, 1992; Nestler and Greengard, 1999
; Rodnight and Wofchuk, 1992
; Wallas and Greengard, 1991
). Many types of proteins in the CNS are regulated by phosphorylation, and these include the cytoskeletal proteins. Considering this, the aim of our study was to investigate the effects of diphenyl diselenide, ebselen, and methylmercury on the in vitro phosphorylation of intermediate filament (IF) proteins in cortical slices of young rats. The use of diphenyl diselenide is of particular interest, since this compound shares with ebselen anti-inflammatory, anti-nociceptive, neurochemical, and neuroprotective activities and is acutely less toxic to rodents than ebselen (Ghisleni et al., 2003
; Meotti et al., 2003
; Moretto et al., 2003
; Nogueira et al., 2003b
). In addition, we investigated the protective effect of ebselen and diphenyl diselenide against the effects elicited by methylmercury on the in vitro incorporation of 32P into IF cytoskeletal proteins.
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METHODS |
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Animals.
All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and every effort was made to reduce the number and the suffering of the animals used. 17-day-old Wistar rats were obtained from our breeding stock. The rats were maintained on a 12-h light/12-h dark cycle in a constant temperature (22°C) colony room. On the day of birth the litter size was culled to eight pups. Litters smaller than eight pups were not included in the experiments. Water and a 20% (w/w) protein commercial chow were provided ad libitum.
Preparation and labeling of slices.
Rats were killed by decapitation, and the cerebral cortex was dissected onto Petri dishes placed on ice and cut into 400-µm-thick slices with a McIlwain chopper.
Preincubation.
Tissue slices were initially preincubated at 30°C for 10 min in 124 mM NaCl, 4 mM KCl, 1.2 mM MgSO4, 25 mM Na-HEPES (pH 7.4), 12 mM glucose, 1 mM CaCl2, and the following protease inhibitors: 1 mM benzamidine, 0.1 µM leupeptin, 0.7 µM antipain, 0.7 µM pepstatin, and 0.7 µM chymostatin.
Incubation.
After preincubation, the medium was changed, and incubation was carried out for 30 min at 30°C with 100 µl of the basic medium containing 80 µCi of [32P] ortho-phosphate with or without addition of the different drugs. When indicated, ebselen (5, 15, 30, 50, 100 µM), diphenyl diselenide (PhSe)2 (1, 15, 50 µM), or methylmercury (1, 5 µM) was added to the incubation medium. In some experiments, 5 µM ebselen plus 1 µM methyl mercury and 15 µM diphenyl diselenide (PhSe)2 plus 1 µM methyl mercury were added to the incubation medium. The labeling reaction was allowed to proceed for 30 min at 30 °C and stopped with 1 ml of cold stop buffer (150 mM NaF, 5 mM, EDTA, 5 mM EGTA, TrisHCl 50 mM, pH 6.5) and the protease inhibitors described above. Slices were then washed twice by decantation with stop buffer to remove excess radioactivity.
Preparation of the high salt-Triton insoluble cytoskeletal fraction from slices of the cerebral cortex.
After treatment, preparations of total IF were obtained from the cerebral cortex of 17-day-old rats as described by Funchal et al. (2003). Briefly, after the labeling reaction, slices were homogenized in 400 µl of ice-cold high-salt buffer containing 5 mM KH2PO4, (pH 7.1), 600 mM KCl, 10 mM MgCl2, 2 mM EGTA, 1 mM EDTA, 1% Triton X-100 and the protease inhibitors described above. The homogenate was centrifuged at 15800 x g for 10 min at 4°C, in an Eppendorf centrifuge, the supernatant was discarded, and the pellet was homogenized with the same volume of the high-salt medium. The resuspended homogenate was centrifuged as described, and the supernatant was discarded. The Triton-insoluble intermediate filament-enriched pellet, containing neurofilament subunits, vimentin and GFAP, was dissolved in 1% SDS, and protein concentration was determined by the method of Lowry et al., 1951
.
Total protein homogenate.
Tissue slices were homogenized in 100 µl of a lysis solution containing 2 mM EDTA, 50 mM TrisHCl, pH 6.8, 4%( w/v) SDS. For electrophoresis analysis, samples were dissolved in 25% (v/v) of a solution containing 40% glycerol, 5% mercaptoethanol, 50 mM TrisHCl, pH 6.8 and boiled for 3 min.
Immunoblotting analysis.
The IF-enriched cytoskeletal fractions or the total protein homogenates were analyzed by 10% SDSPAGE (10, 25, 50, and 75 µg/lane) and transferred (Trans-blot SD semi-dry transfer cell, BioRad) to nitrocellulose membranes for 1 h at 15 V in transfer buffer (48 mM Trizma, 39 mM glycine, 20% methanol, and 0.25% SDS). The blot was then washed for 10 min in Tris-buffered saline (TBS; 0.5 M NaCl, 20 mM Trizma, pH 7.5), followed by 2-h incubation in blocking solution (TBS plus 5% defatted dried milk). After incubation, the blot was washed twice for 5 min with TBS plus 0.05% Tween-20 (T-TBS), and then incubated overnight at 4°C in blocking solution containing one of the following monoclonal antibodies: anti NF-150 (clone NN-18) diluted 1:100, anti NF-68 (clone NR-4) diluted 1:300, anti vimentin (clone vim 13.2) diluted 1:400, and anti glial fibrillary acidic protein (GFAP) (clone G-A-5) diluted 1:400. The blot was then washed twice for 5 min with T-TBS and incubated for 2 h in TBS containing peroxidase-conjugated rabbit anti-mouse IgG diluted 1:4000. The blot was washed twice again for 5 min with T-TBS and twice for 5 min with TBS. The blot was then developed using a chemiluminescence ECL kit.
Polyacrylamide gel electrophoresis (SDSPAGE).
The cytoskeletal fraction was prepared as described above. Equal protein concentrations were loaded onto 10% polyacrylamide gels and analyzed by SDSPAGE according to the discontinuous system of Laemmli (1970). After drying, the gels were exposed to X-ray films (Kodak X-Omat XK1) at 70°C with intensifying screens, and finally the autoradiograph was obtained. Cytoskeletal proteins were quantified by scanning the films with a Hewlett-Packard Scanjet 6100C scanner and determining optical densities with an Optiquant version 02.00 software (Packard Instrument Company). Density values were obtained for the studied proteins.
Statistical analysis.
Data were analyzed statistically by one-way analysis of variance (ANOVA) followed by the Tukey test when the F-test was significant. All analyses were performed using the SPSS software program on an IBM-PC compatible computer.
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RESULTS |
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DISCUSSION |
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The developing nervous system has been described to be especially sensitive to methylmercury exposure (Aschner et al., 2000; Castoldi et al., 2001
; Clarkson et al., 2003b
; Grandjean et al., 1997
; WHO, 1990
). The action of methylmercury on cytoskeletal proteins has also been described. In this context, methylmercury has been reported to cause a marked disruption of the cytoskeleton in different cultured cell lines (Miura et al., 1999
; Trombetta and Kromidas, 1992
; Vignani et al., 1992
). In addition, Yagame et al. (1994)
showed that methylmercury caused differential effects on the phosphorylation level of many proteins, including tubulin subunits. However little is known about the effects of methylmercury on the phosphorylating system associated with IF proteins. Nonetheless, the results of the present investigation indicate that methylmercury inhibits to the same extent the in vitro phosphorylation of each IF subunit studied. Immunoblot analysis with anti NF-M, anti NF-L, anti vimentin, and anti GFAP demonstrated that the in vitro treatment with 1 µM methylmercury was not able to alter the immunocontent of these proteins either in tissue homogenate or in the Triton-insoluble cytoskeleton. These results strongly suggest that the effects induced by methylmercury on the in vitro 32P incorporation are due to the action of this compound on the phosphorylating system associated with the cytoskeletal proteins, rather than on protein expression or on the different amounts of IF proteins recovered on the cytoskeletal fraction. These findings probably reflect a physiological role of phosphorylation on the regulation of the cytoskeleton (de Almeida et al., 2003
; de Mattos-Dutra et al., 1997
; Funchal et al., 2002
) leading to a response of the cell to the insult.
Although we cannot at present precisely explain the effects of methylmercury on the IF-associated phosphorylating system, they could reflect an imbalance on regulatory mechanisms preceding a reorganization of the cytoskeletal proteins. In this context, hipophosphorylated cytoskeletal proteins have been related with neurotoxicity in other systems, such as beta,beta'-iminodipropionitrile (IDPN) exposure, ultimately giving rise to brain damage (Tashiro et al., 1994).
Although the antioxidant properties of selenium could be related to a protection against certain neurotoxic effects, such as quinolinic acid-induced neurotoxicity (Rossato et al., 2002a; Santamaria et al., 2003
), epidemiological studies have shown that chronic exposure to selenium compounds is associated with several adverse health effects in humans (Vinceti et al., 2001
). In this context, Nogueira et al. (2003a)
recently described diselenide-induced seizures in mice. However, our results showed that diselenide, at the concentrations used, was not able to alter the activity of the phosphorylating system associated with the IF proteins. In addition, we have shown a neuroprotective effect of diselenide (15 µM), preventing the action of methylmercury on the IF-associated phosphorylation system. Also, ebselen, a selenium compound, has been widely described in the literature as a neuroprotective in stroke and ischemia (Saito et al., 1998
; Yamaguchi et al., 1998
). Moreover, in our experimental conditions, 5 µM ebselen showed a neuroprotective effect on the parameter studied. Corroborating with these findings, it is important to emphasize that ebselen has also been described to be nontoxic to cultured neurons in vitro (Porciuncula et al., 2001
). In line with our results, Farina et al. (2003)
have recently described the protective effect of ebselen against the neurotoxicity induced by in vivo exposure to methylmercury. The mechanisms underlying the neuroprotection afforded by the selenium compounds is still not completely understood, but protection was tentatively ascribed to its antioxidant properties. It is well established that ebselen inhibits both nonenzymatic and enzymatic lipid peroxidation in cells and present anti-inflammatory activity in various animal models (Bosch-Morell et al., 1999
, 2002
; Mugesh et al., 2001
; Nogueira et al., 2003b
; Porciuncula et al., 2003
; Rossato et al., 2002a
,b
; Shimohashi et al., 2000
).
Although the mechanisms underlying the effects induced by methylmercury and ebselen in our experimental approach are not clear, we could speculate that they are ascribed to the ability of methylmercury to oxidize SH groups, while diphenyl diselenide and ebselen would be transformed in reducing intermediates phenyl selenol and selenol, respectively (Zeni et al., 2000, 2003
, 2004
). These mechanisms would explain the stimulatory effects of ebselen in contrast with the inhibitory effect of methylmercury. The high thiol reactivity of methylmercury has been suggested to be the basis of its harmful biological effects (Aschner et al., 2000
; Sanfeliu et al., 2003
). In this context, Rajanna et al. (1995)
described that kinases are extremely sensitive to sulfhydryl blocking agents, including methylmercury. In fact, in vitro and in vivo exposure to methylmercury have been reported to decrease protein kinase C (PKC) activity (Haykal-Coates et al., 1998
; Rajanna et al., 1995
), which phosphorylates the major mammalian neurofilament subunit (NF-L) (Gonda et al., 1990
), vimentin (Geisler et al., 1989
), and GFAP (Inagaki et al., 1990
).
There is considerable evidence in the literature that phosphorylation of cytoskeletal proteins modulates the reciprocal interactions of their filamentous components, such as microfilaments, microtubules, and intermediate filaments (Hashimoto et al., 1998; Inada et al., 1998
; Inagaki et al., 1996
; Ku and Omary, 1997
). In this scenario, it has been demonstrated that the cAMP-dependent phosphorylation of the neurofilament proteins NF-L and NF-M inhibits their coassembly into filaments in vitro (Streifel et al., 1996
). It is also known that the phosphorylation level of IF protein regulates calpain-mediated hydrolysis (Litersky and Johnson, 1995
). Besides this, alterations in protein phosphorylation have been shown in Alzheimer's disease (Iqbal et al., 2002
) and following exposure to various neurotoxicants, such as tri-o-cresyl phosphate, carbon disulfide, and glycidamide (Jensen et al., 1992
; Reagan et al., 1995
; Wilmarth et al., 1993
).These observations indicate that aberrant cytoskeletal phosphorylation/dephosphorylation may have serious consequences for cellular function and structure, and it may be one of the mechanisms causing neuronal damage in neurodegenerative pathologies as well as following exposure to neurotoxicants.
It is difficult to extrapolate our results to the human conditions and to correlate the alterations of the phosphorylating system in cerebral cortex slices with the neurotoxicity provoked by methylmercury. However, considering the great body of evidence in the literature showing that alterations of cytoskeletal proteins may lead to disorganization of cellular structure, it is tempting to speculate that this may be at least one of the factors associated with the neurotoxicity induced by methylmercury. Regarding the ability of selenium compounds to protect against the methylmercury toxicity toward the phosphorylating system associated with the cytoskeletal proteins, the present findings show a promising route to be exploited.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Aschner, M., Yao, C. P., Allen, J. W., and Tan, K. H. (2000). Methylmercury alters glutamate transport in astrocytes. Neurochem. Int. 37, 199206.[CrossRef][ISI][Medline]
Bosch-Morell, F., Roma, J., Marin, N., Romero, B., Rodriguez-Galietero, A., Johnsen-Soriano, S., Diaz-Llopis, M., and Romero, F. J. (2002). Role of oxygen and nitrogen species in experimental uveitis: Anti-inflammatory activity of the synthetic antioxidant ebselen. Free Radic. Biol. Med. 33(5), 669675.[CrossRef][ISI][Medline]
Bosch-Morell, F., Roma, J., Puertas, F. J., Marin, N., Diaz-Llopis, M., and Romero, F. J. (1999). Efficacy of the antioxidant ebselen in experimental uveitis. Free Rad. Biol. Med. 27(34), 388391.[CrossRef][ISI][Medline]
Branco, T., Meirelles, R., da Rocha, B. B., de Mattos-Dutra, A., Wajner, M., and Pessoa-Pureur, R. (2000). Alpha-ketoisocaproate increases the in vitro 32P incorporation into intermediate filaments in cerebral cortex of rats. Neuroreport 11, 35453550.[ISI][Medline]
Castoldi, A. F., Coccini, T., Ceccatelli, S., and Manzo, L. (2001). Neurotoxicity and molecular effects of methylmercury. Brain Res. Bulletin. 55, 197203.[CrossRef][ISI][Medline]
Castoldi, A. F., Coccini, T., and Manzo, L. (2003). Neurotoxic and molecular effects of methylmercury in humans. Rev. Environ. Health. 18, 1931.[Medline]
Clarkson, T. W. (1997). The toxicology of mercury. Crit. Rev. Clin. Lab. Sci. 34, 369403.[ISI][Medline]
Clarkson, T. W., Magos, L., and Myers, G. J. (2003a). The toxicology of mercury-current exposures and clinical manifestations. N. Engl. J. Med. 349, 17311737.
Clarkson, T. W., Magos, L., and Myers, G. J. (2003b). Human exposure to mercury: The three modern dilemmas. J. Trace Elem. Exp. Med. 16, 321343.[CrossRef][ISI]
Clarkson, T. W., and Strain, J. J. (2003). Nutritional factors may modify the toxic action of methyl mercury in fish-eating populations. J. Nutr. 133, 1539S1543S.
Davalos, A. (1999). New treatments in cerebrovascular diseases. Neurologia 14, 7783.[Medline]
Dawson, D. A., Masayasu, H., Graham, D. I., and Macrae, I. M. (1995). The neuroprotective efficacy of ebselen (a glutathione-peroxidase mimic) on brain-damage induced by transient focal cerebral-ischemia in the rat. Neurosci Lett. 185, 6569.[CrossRef][ISI][Medline]
de Almeida, L. M. V., Funchal, C., Pelaez, P. L., Pessutto, F. D. B., Loureiro, S. O., Vivian, L., Wajner, M., and Pessoa-Pureur, R. (2003). Effect of propionic and methylmalonic acids on the in vitro phosphorylation of intermediate filaments from cerebral cortex of rats during development. Metab. Brain Dis. 18, 207219.[CrossRef][ISI][Medline]
de Mattos-Dutra, A., de Freitas, M. S., Schroder, N., Lisboa, C. S. F., Pessoa-Pureur, R., and Wajner, M. (1997). In vitro phosphorylation of cytoskeletal proteins in the rat cerebral cortex is decreased by propionic acid. Exp. Neurol. 147, 238247.[CrossRef][ISI][Medline]
Dunkley, P. R. (1992). Autophosphorylation of neuronal calcium/calmodulin stimulated protein kinase II. Mol. Neurobiol. 5, 179202.[ISI]
Engman, L. (1989). Expedient synthesis of Ebselen and related compounds. J. Org. Chem. 54, 29642966.[CrossRef][ISI]
Farina, M., Dahm, K. C. S., Schwalm, F. D., Brusque, A. M., Frizzo, M. E. S., Zeni, G., Souza, D. O., and Rocha, J. B. T. (2003). Methylmercury increases glutamate release from brain synaptosomes and glutamate uptake by cortical slices from suckling rat pups: Modulatory effect of ebselen. Toxicol. Sci. 73, 135140.
Flohé, L., Gunzler, W. A., and Schock, H. H. (1973). Glutathione peroxidasea selenoenzyme. Febs Lett. 32, 132134.[CrossRef][ISI][Medline]
Funchal, C., de Almeida, L. M., Oliveira Loureiro, S., Vivian, L., de Lima Pelaez, P., Dall Bello Pessutto, F., Rosa, A. M., Wajner, M., and Pessoa-Pureur, R. (2003). In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of rats. Brain Res. Prot. 11, 111118.[CrossRef][ISI][Medline]
Funchal, C., de Lima Pelaez, P., Oliveira Loureiro, S., Vivian, L., Dall Bello Pessutto, F., de Almeida, L. M. V., Wofchuk, S. T., Wajner, M., and Pessoa-Pureur, R. (2002). -Ketoisocaproic acid regulates phosphorylation of intermediate filaments in postnatal rat cortical slices through ionotropic glutamatergic receptors. Dev. Brain Res. 139, 267276.[ISI][Medline]
Geisler, N., Hatzfeldt, M., and Weber, K. (1989). Phosphorylation in vitro of vimentin by protein kinases A and C is restricted to the head domain. Identification of the phosphoserine sites and their influence on filament formation. Eur. J. Biochem. 183, 441447.[Abstract]
Ghisleni, G., Porciuncula, L. O., Cimarostia, H., Rocha, J. B. T., Salbego, C. G., and Souza, D. O. (2003). Diphenyl diselenide protects rat hippocampal slices submitted to oxygen-glucose deprivation and diminishes inducible nitric oxide synthase immunocontent. Brain Res. 986, 196199.[CrossRef][ISI][Medline]
Gonda, Y., Nishizawa, K., Ando, S., Kitamura, S., Minoura, Y., Nishi, Y., and Inagaki, M. (1990). Involvement of protein kinase C in the regulation of assembly-disassembly of neurofilaments in vitro. Biochem. Biophys. Res. Commun. 167, 13161325.[CrossRef][ISI][Medline]
Grandjean, P., Weihe, P., White, R. F., Debes, F., Araki, S., Yokoyama, K., Murata, K., Sorensen, N., Dahl, R., and Jorgensen, P. J. (1997). Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol. Teratol. 19, 417428.[CrossRef][ISI][Medline]
Harada, M. (1995). Minamata disease: Methylmercury poisoning in Japan caused by environmental pollution. Crit. Rev. Toxicol. 25, 124.[ISI][Medline]
Hashimoto, R., Nakamura, Y., Goto, H., Wada, Y., Sakoda, S., Kaibuchi, K., Inagaki, M., and Takeda, M. (1998). Domain- and site- specific phosphorylation of bovine NF-L by Rho-associated kinase. Biochem. Biophys. Res. Commun. 245, 407411.[CrossRef][ISI][Medline]
Haykal-Coates, N., Shafer, T. J., Mundy, W. R., and Barone, S. (1998). Effects of gestational methylmercury exposure on immunoreactivity of specific isoforms of PKC and enzyme activity during post-natal development of the rat brain. Dev. Brain Res. 109, 3349.[ISI][Medline]
Hunter, A. M., and Brown, D. L. (2000). Effects of microtubule-associated protein (MAP) expression on methylmercury-induced microtubule disassembly. Toxicol. Appl. Pharmacol. 166, 203213.[CrossRef][ISI][Medline]
Inada, H., Goto, H., Tanabe, K., Nishi, Y., Kaibuchi, K., and Inagaki, M. (1998). Rho-associated kinase phosphorylates desmin, the myogenic intermediate filament protein, at unique amino-terminal sites. Biochem. Biophys. Res. Commun. 253, 2125.[CrossRef][ISI][Medline]
Inagaki, I., Gonda, Y., Nishizawa, K., Kitamura, S., Sato, C., Ando, S., Tanabe, K., Kikuchi, K., Tsuiki, S., and Nishi, Y. (1990). Phosphorylation sites linked to glial filament disassembly in vitro locate in a non-alpha helical head domain. J. Biol. Chem. 265, 47224729.
Inagaki, N., Tsujimura, K., Tanaka, J., Sekimata, M., Kamei, Y., and Inagaki, M. (1996). Visualization of protein kinase activities in single cells by antibodies against phosphorylated vimentin and GFAP. Neurochem. Res. 21, 795800.[ISI][Medline]
Iqbal, K., Alonso, A. C., El-Akkad, E., Gong, C. X., Haque, N., Khatoon, S., Pei, J. J., Tsujio, I., Wang, J. Z., and Grundke-Iqbal, I. (2002). Significance and mechanism of Alzheimer neurofibrillary degeneration and therapeutic targets to inhibit this lesion. J. Mol. Neurosc. 19, 9599.[ISI][Medline]
Imai, H., Graham, D. I., Masayasu, H., and Macrae, I. M. (2003). Antioxidant ebselen reduces oxidative damage in focal cerebral ischemia. Free Rad. Biol. Med. 34, 5663.[CrossRef][ISI][Medline]
Jensen, K. F., Lapadula, D. M., Knoth-Anderson, J., Haycal-Coates, N., and Abou-Donia, M. B. (1992). Anomalous phosphorylated neurofilament aggregations in central and peripheral axons of hens treated with tri-o-cresyl phosphate (TOCP). J. Neurosci. Res. 33, 455460.[CrossRef][ISI][Medline]
Kinoshita, Y., Ohnishi, A., Kohshi, K., and Yokota, A. (1999). Apparent diffusion coefficient on rat brain and nerves intoxicated with methylmercury. Environ. Res. 80, 348354.[CrossRef][ISI][Medline]
Ku, N. O., and Omary, M. B. (1997). Phosphorylation of human keratin 8 in vivo at conserved head domain serine 23 and at epidermal growth factor-stimulated tail domain serine 431. J. Biol. Chem. 272, 75567564.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680685.
Litersky, J. M., and Johnson, G. V. W. (1995). Phosphorylation of tau in situ: Inhibition of calcium-dependent proteolysis. J. Neurochem. 65, 903911.[ISI][Medline]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265267.
Meotti, F. C., Borges, V. C., Zeni, G., Rocha, J. B. T., and Nogueira, C. W. (2003). Potential renal and hepatic toxicity of diphenyl diselenide, diphenyl ditelluride and ebselen for rats and mice. Toxicol. Lett. 143, 916.[CrossRef][ISI][Medline]
Miura, K., Koide, N., Himeno, S., Nakagawa, I., and Imura, N. (1999). The involvement of microtubular disruption in methylmercury-induced apoptosis in neuronal and nonneuronal cell lines. Toxicol. Appl. Pharmacol. 16, 279288.
Moretto, M. B., Rossato, J. I., Nogueira, C. W., Zeni, G., and Rocha, J. B. T. (2003). Voltage-dependent ebselen and diorganochalcogenides inhibition of Ca-45(2+) influx into brain synaptosomes. J. Biochem. Mol. Toxicol. 17, 154160.[CrossRef][ISI][Medline]
Mugesh, G., du Mont, W. W., Sies, H. (2001). Chemistry of biologically important synthetic organoselenium compounds. Chem. Rev. 101, 21252179.[CrossRef][ISI][Medline]
Mugesh, G., and Singh, H. B. (2000). Synthetic organoselenium compounds as antioxidants: Glutathione peroxidase activity. Chem. Soc. Rev. 29, 347357.[CrossRef][ISI]
Muller, A., Cadenas, E., Graf, P., and Sies, H. (1984). A novel biologically active seleno-organic compound. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen). Biochem Pharmacol. 15, 32353239.[CrossRef]
Myers, G. J., Davidson, P. W., Cox, C., Shamlaye, C., Cernichiari, E., and Clarkson, T. W. (2000). Twenty-seven years studying the human neurotoxicity of methylmercury exposure. Environ. Res. 83, 275285.[CrossRef][ISI][Medline]
Nestler, E. J., and Greengard, P. (1999). Serine and threonine phosphorylation. In Basic NeurochemistryMolecular, Cellular and Medical Aspects (G. Siegel, B. W. Agranoff, R. W. Alberts, S. K. Fisher, and, M. D. Ulher, Eds.), 6th ed., p. 471495. Lippincott-Raven Publishers, New York.
Nogueira, C. W. Meotti, F. C., Curte, E., Pilissão, C., Zeni, G., and Rocha, J. B. T. (2003a.) Investigations into the potential neurotoxicity induced by diselenides in mice and rats. Toxicology 183, 2937.[CrossRef][ISI][Medline]
Nogueira, C. W., Quinhones, E. B., Jung, E. A., Zeni, G., and Rocha, J. B. T. (2003b). Anti-inflamatory and antinociceptive of diphenyl diselenide. Inflamm. Res. 52, 5663.[CrossRef][ISI][Medline]
Parnham, M., and Sies, H. (2000). Ebselen: Prospective therapy for cerebral ischaemia. Exp. Opin. Investig. Drugs. 9, 607619.[ISI]
Paulmier, C. (1986). Selenium Reagents and Intermediates in Organic Synthesis. Pergamon Press, New York.
Ponce, R. A., Kavanagh, T. J., Karle Mottet, N., Whittaker, S. G., and Faustman, E. M. (1994). Effects of Methyl Mercury on the cell cycle of primary rat CNS cells in vitro. Toxicol. Appl. Pharmacol. 127, 8390.[CrossRef][ISI][Medline]
Porciuncula, L. O., Rocha, J. B. T., Boeck, C. R., Vendite, D., and Souza, D. O. (2001). Ebselen prevents excitotoxicity provoked by glutamate in rat cerebellar granule neurons. Neurosci. Lett. 299, 217220.[CrossRef][ISI][Medline]
Porciuncula, L. O., Rocha, J. B. T., Cimarosti, H., Vinade, L., Ghisleni, G., Salbego, C. G., and Souza, D. O. (2003). Neuroprotective effect of ebselen on rat hippocampal slices submitted to oxygen-glucose deprivation: Correlation with immunocontent of inducible nitric oxide synthase. Neurosci. Lett. 346, 101104.[CrossRef][ISI][Medline]
Rajanna, B., Chetty, C. S., Rajanna, S., Hall, E., Fail, S., and Yallapragada, P. R. (1995). Modulation of protein kinase C by heavy metals. Toxicol. Lett. 81, 197203.[CrossRef][ISI][Medline]
Reagan, K. E., Wilmarth, K. R., Friedman, M. A., and Abou-Donia, M. B. (1995). In vitro calcium and calmodulin-dependent kinase-mediated phosphorylation of rat brain and spinal cord neurofilament proteins is increased by glycidamide administration. Brain Res. 671, 1220.[CrossRef][ISI][Medline]
Rodnight, R., and Wofchuk, S. T., (1992). Roles for protein phosphorylation in synaptic transmission. Ess. Biochem. 27, 91100.
Rossato, J. I., Ketzer, L. A., Centuriao, F. B., Silva, S. J. N., Ludtke, D. S., Zeni, G., Braga, A. L., Rubin, M. A., and Rocha, J. B. T. (2002b). Antioxidant properties of new chalcogenides against lipid peroxidation in rat brain. Neurochem. Res. 27, 297303.[CrossRef][ISI][Medline]
Rossato, J. I., Zeni, G., Mello, C. F., Rubin, M. A., and Rocha, J. B. T. (2002a). Ebselen blocks the quinolinic acid-induced production of thiobarbituric acid reactive species but does not prevent the behavioral alterations produced by intra-striatal quinolinic-acid administration in the rat. Neurosci. Lett. 318, 137140.[CrossRef][ISI][Medline]
Sager, P. R. (1988). Selectivity of methyl mercury effects on cytoskeleton and mitotic progression in cultured cells. Toxicol. Appl. Pharmacol. 94, 473486.[CrossRef][ISI][Medline]
Saito, I., Asano, T., Sano, K., Takakura, K., Abe, H., Yoshimoto, T., Kikuchi, H., Ohta, T., and Ishibashi, S. (1998). Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery 42, 269278.[CrossRef][ISI][Medline]
Sanfeliu, C., Sebastia, J., Cristofol, R., and Rodriguez-Farre, E. (2003). Neurotoxicity of organomercurial compounds. Neurotox. Res. 5, 283305.[ISI][Medline]
Santamaria, A., Salvatierra-Sanchez, R., Vazquez-Roman, B., Santiago-Lopez, D., Villeda-Hernandez, J., Galvan-Arzate, S., Jimenez-Capdeville, M. E., and Ali, S. F. (2003). Protective effects of the antioxidant selenium on quinolinic acid-induced neurotoxicity in rats: In vitro and in vivo studies. J. Neurochem. 86, 479489.[CrossRef][ISI][Medline]
Shimohashi, N., Nakamuta, M., Uchimura, K., Sugimoto, R., Iwamoto, H., Enjoji, M., and Nawata, H. (2000). Selenoorganic compound, ebselen, inhibits nitric oxide and tumor necrosis factor-alpha production by the modulation of Jun-N-terminal kinase and the NF-Kappa B signaling pathway in rat Kupffer cells. J. Cell. Biochem. 78, 595606.[CrossRef][ISI][Medline]
Sies, H. (1993). Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free Rad. Biol. Med. 14, 313323.[CrossRef][ISI][Medline]
Sirois, J. E., and Atchison, W. D. (2000). Methylmercury affects multiple subtypes of calcium channels in rat cerebellar granule cells. Toxicol. Appl. Pharmacol. 167, 111.[CrossRef][ISI][Medline]
Streifel, D. T., Avalos, R. T., and Cohlberg, J. A. (1996). cAMP dependent phosphorylation of neurofilament proteins NF-L and NF-M inhibits their coassembly into filaments in vitro. Biophys. Res. Commun. 222, 646651.[CrossRef][ISI][Medline]
Tashiro, T., Imai, R., and Komiya, Y. (1994). Early effects of beta,beta'-iminodipropionitrile on tubulin solubility and neurofilament phosphorylation in the axon. J. Neurochem. 63, 291300.[ISI][Medline]
Takasago, T., Peters, E. E., Graham, D. I., Masayasu, H., and Macrae, I. M. (1997). Neuroprotective efficacy of ebselen, an anti-oxidant with antiinflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. Brit. J. Pharmacol. 122, 12511256.[CrossRef][ISI][Medline]
Trombetta, L. D., and Kromidas, L. (1992). A scanning electron-microscopic study of the effects of methylmercury on the neuronal cytoskeleton. Toxicol. Lett. 60, 329341.[CrossRef][ISI][Medline]
Vignani, R., Milanesi, C., and Disimplicio, P. (1992). Disruption of cytoskeleton by methylmercury in cultured CHO cells. Toxicol. In Vitro 6, 6170.[CrossRef][ISI]
Vinceti, M., Wei, E. T., Malagoli, C., Bergomi, M., and Vivoli, G. (2001). Adverse health effects of selenium in humans. Rev. Environ. Health 16, 233251.[Medline]
Wallas, S. T., and Greengard, P. (1991). Protein phosphorylation and neuronal function. Pharmacol. Rev. 43, 299349.[Abstract]
Wendel, A., Fausel, M., Safayhi, H., Tiegs, G., and Otter, R. (1984). A novel biologically active seleno-organic compoundII. Activity of PZ 51 in relation to glutathione peroxidase. Biochem. Pharmacol. 15, 32413245.[CrossRef]
WHO1990. Environmental Health Criteria 101: Methylmercury (Review No. ISBN 92 4 157101 2) World Health Organization.
Wilmarth, K. R., Viana, M. E., and Abou-Donia, M. B. (1993). Carbon disulfide inhalation increases Ca+2/calmodulin-dependent kinase phosphorylation of cytoskeletal proteins in the rat central nervous system. Brain Res. 628, 293300.[CrossRef][ISI][Medline]
Yagame, H., Horigome, T., Ichimura, T., Uchiyama, J., and Omata, S. (1994). Differential-Effects of methylmercury on the phosphorylation of protein species in the brain of acutely intoxicated rats. Toxicology 92, 101113.[CrossRef][ISI][Medline]
Yamaguchi, T., Sano, K., Takakura, K., Saito, I., Shinohara, Y., Asano, T., and Yasuhara, H. (1998). Ebselen in acute ischemic stroke: A placebo-controlled, double-blind clinical trial. Stroke 29, 1217.
Zeni, G. R., Barros, O. S., Moro, A. V., Braga, A. L., and Peppe, C. (2003). Hydrotelluration of aminoalkynes. Chem. Commun. 11, 12581259.[CrossRef]
Zeni, G. R., Formiga, H., and Comasseto, J. V. (2000). Improved procedure for the hydrotelluration of alkynes. Tetrahedron Lett. 41, 13111313.[CrossRef][ISI]
Zeni, G. R., Stracke, M. P., Nogueira, C. W., Braga, A. L., Menezes, P. H., and Stefani, H. A. (2004). Hydroselenation of alkynes by lithium butylselenolate: An approach in the syntheis of vinylic selenides. Org. Lett. 6, 11351138.[CrossRef][ISI][Medline]
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