For the self-ubiquitination reactions, we used human UbcH5a, a commercially available E2 enzyme that shares 75% sequence identity with ScUbc4p  and recombinant ScPex4p. When GST Pex10 RING was added to a reaction containing ubiquitin, E1, UbcH5a, and ATP, higher molecular weight forms of ubiquitin were observed, suggesting that ubiquitin conjugation had occurred (Fig. 1B, lane 4). Control experiments showed that the formation of higher molecular weight ubiquitin AEBSF was ATP dependent (Fig. 1B, lane 5) and did not occur when E1, UbcH5a or GST Pex10 RING was left out (Fig. 1A, lanes 1–3 and data not shown). Conjugation reactions were also analysed with anti-GST and anti-Pex10 antibodies (Fig. 1C and D). These antibodies predominantly detected a band corresponding to the mono-ubiquitinated GST Pex10 RING, indicating that self-ubiquitination had occurred. Very little higher molecular weight (poly-ubiquitinated) forms of GST Pex10 RING were observed with either antibody, suggesting that the majority of the ubiquitinated species seen with the anti-ubiquitin antibody represent unanchored poly-ubiquitin chains. Surprisingly, Pex10 RING did not exhibit E3 ligase activity when ScPex4p was included in the reaction (Fig. 1B and C).
Reductive alkylation of DhaPylSc. All methylation reaction steps to reduce DhaPylSc were carried out at 4 oC. Five hundred and twenty microliters of the native PylSc (5 mg/mL) in 50 mM Hepes, SB1518 7.5, 500 mM NaCl and 10% glycerol was placed in a glass tube and incubated with 10.4 μL of freshly prepared 1 M dimethylamine–borane complex (ABC) and 20.8 μL 1 M formaldehyde for 2 h. This was followed by a second addition of 10.4 μL 1 M ABC and 20.8 μL 1 M formaldehyde, and the reaction was incubated for a further 2 h. A final addition, consisted of only 10.4 μL 1 M ABC was added to the reaction mixture, which was then left at 4 oC overnight to ensure complete reduction of the protein. The reaction was quenched using 59.3 μL 2 M ammonium sulfate. The methylated protein was buffer-exchanged into 20 mM PBS pH 7.4, 300 mM imidazole, 500 mM NaCl, 10% glycerol, and 1 mM pyrrolysine analog 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2THF-lys), followed by concentration using a BioMax centrifugal filter unit (Millipore). Methylation of the protein had no significant effect on the activity of protein (15 min?1) in mediating 32P-pyrophosphate: ATP exchange dependent upon the addition of amino acid, as assayed after the procedure described by Blight et al. .
This study is the first to provide experimental evidence demonstrating that curcumin inhibits OVA-induced airway inflammation in a murine model of asthma through suppression of NO. These findings suggest that curcumin may be useful as an adjuvant therapy for asthmatic patients in the future.
This work was supported by Korea Health 21 R&D Project (A062406), Ministry of Health & Welfare, Republic of Korea (G.-Y. Kim).
Mammary epithelial cells; Uncoupling proten 2; Long-chain fatty acids; Insulin; Dexamethazone
In this study, we have demonstrated that UCP2 mRNA is modulated by the addition of LCFAs in mammary epithelial cells. We have also investigated the effects of certain hormones, such as Insulin and dexamethazone on the expression of UCP2 mRNA.
Materials and methods
Materials. Long-chain fatty Geldanamycin sodium salts (palmitate, stearate, oleate, and linoleate), Insulin, Dxamethazone, prolactin, Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and fraction V fatty acid-free bovine serum albumin (BSA) were purchased from Sigma–Aldrich, St. Louis, MO. Triglyceride G Test Kit was from Wako, Osaka, Japan. TRIsol and SuperScript III First-Strand Synthesis System were from Invitrogen Corp., Carlsbad, CA. DyNAmo SYBR green qPCR Kit were from Finnzymes, Espoo, Finland. Deoxyribonuclease (RT Grade) for Heat Stop was from Nippon Gene pleiotropic Co., LTD., Tokyo, Japan. Zeta-Probe Blotting Membranes were from Bio-rad Laboratories, Hercules CA. The BCA protein kit was from Pierce, Rockford, Illinois, USA. PCR DIG Probe Synthesis kit, DIG Luminescent Detection kit, and DIG Easy Hyb Granules were from Roche Diagnostics GmbH, Mannheim, Germany.