Materials and methods

Materials

NM was isolated and purified from the substantia nigra of control human brains, as described previously (Gerlach et al., 1995) and was dissolved in distilled water containing 15 mM L-cysteine and 10% dimethyl sulfoxide (DMSO) (L-Cyst-DMSO solution) to be 0.5 mg/ml in the final concentration (Shamoto-Nagai et al., 2004). L-Cysteine was purchased from Sigma (St. Louis, MO, USA). 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes (Eugene, OR, USA), deferoxamine mesylate (DFX) and superoxide dismutase (SOD) from bovine erythrocytes were purchased from Sigma (St. Louis, MO, USA), and anti-polyubiquitin monoclonal antibody from NBT (Tokyo, Japan). A proteasome sensor vector, pZsProSensor-1, was purchased from BD Biosciences (Palo Alto, CA, USA). 2',7'-Dichlorofluorescein (DCF), N-acetyl cysteine (NAC), L-cysteine and catalase from bovine liver, minimum essential medium (MEM) and other reagents were from Wako (Kyoto, Japan).

Measurement of in situ 26S proteasome activity in SH-SY5Y cells expressing a proteasome sensor vector

SH-SY5Y cells were cultured as reported (Shamoto-Nagai et al., 2004). Transfectant with a proteasome sensor vector was established using a pZsProSensor-1 eukaryotic expression vector, designed to express ZsGFP fused to the degradation domain of mouse or-nithine decarboxylase, a specific substrate for 26S proteasome, by lipofection technique as reported previously (Shamoto-Nagai et al., 2004). The culture medium was changed with the medium containing L-Cyst-DMSO solution without (control) or with NM and the cells were cultured for 3 days. In addition, the cells transfected with proteasome sensor vector was incubated with various concentrations of iron in the presence or absence of 25 mM DFX or antioxidants for 20 h. The fluorescence of ZsGFP in the living cells was measured as described before (Shamoto-Nagai et al., 2004), and the fluorescence intensity of the cells was expressed as arbitrary fluorescence unit/mg protein. The protein amount was measured according to Bradford (1976).

Detection of polyubiquitinated proteins in the cells treated with NM

After treatment with NM for 1 or 3 days, the cells were gathered, washed with PBS, and lyzed in the RIPA lysis buffer (Upstate Biotechnology, Lake Placid, NY, USA) containing protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Fifty mg of protein was subjected to SDS-polyacrylamide electrophoresis using 12.5% polyacrylamide gel (Bio Craft, Tokyo, Japan), and blotted onto polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). Plolyubiquitinated proteins were visualized using anti-polyubiquitin antibody and enhanced chemofluores-cence (ECF) Western blotting kit (Amersham Biosciences, Piscataway, NJ, USA), as described previously (Shamoto-Nagai et al., 2003).

Isolation of mitochondria from SH-SY5Y cells

Mitochondria were prepared from SH-SY5Y cells according to Desagher et al. (1999). The cells were gathered, washed with PBS and suspended in the isotonic mitochondrial buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA and 10 mM HEPES, pH 7.5) supplemented with complete protease inhibitor cocktail (Roche Diagnostics, Mannhein, Germany). The mito-chondrial fraction was prepared by homogenization and two steps of centrifugation.

Measurement of ROS-RNS with H2DCFDA

The mitochondria were suspended in PBS and the production of ROS-RNS was quantified fluorometri-cally by measuring DCF produced from H2DCFDA (Crow, 1997). H2DCFDA was added to be 50 mM to the mitochondria suspension in the presence or absence of NM suspension (1-5 mg/ml) in dark at 37°C. The increase in DCF fluorescence at 504 nm with excitation at 520 nm was measured at every 30 min for 3 h in a RF-5000 spectrofluorometer (Shimadzu, Kyoto, Japan). The generation of ROS-RNS was expressed as mol DCF per min per mg protein. The effects of DFX and anti-oxidants were also examined in the same way, after 15 min pre-incubation with DFX or the antioxidants.

Statistics

Experiments were repeated at least 3 times. The data was expressed as mean ± SD and the difference was evaluated by analysis of variance (ANOVA) followed by Scheffe's F-test. A p value less than 0.05 was estimated to be statistically significant.

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