タンパク質酸化を指標とした茶飲料の抗酸化および酸化促進作用

The ready-to-drink tea beverages from the market were examined to assess their pro-oxidant activities by incubating with bovine serum albumin in the presence of 10 mM 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), a water-soluble free radical initiator, or 0.1 mM CuC12 in sodium phosphate buffer (pH 7.4) at 37°C for 90 mM. Protein carbonyl was measured as an index of protein oxidation. In the presence of AAPH, several green tea beverages reduced the formation of protein carbonyl possibly by scavenging free radicals, whereas oolong tea and black tea enhanced the protein carbonyl formation. In the presence of Cu^2+ ions, all tea beverages examined in this study largely increased protein carbonyl content. Additionally catechins oxidized by tyrosinase increased the protein carbonyl formation. These results indicate that oxidized catechins and their derivatives, which are rich in oolong tea and black tea, may be responsible for the protein carbonyl formatio

Disulfide proteomics of rice cultured cells in response to OsRacl and probenazole-related immune signaling pathway in rice

Abstract Background Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants. Methods We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2). Results We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells. Conclusions The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice

DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs▿

Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex

Ictal Tc-Ethyl Cysteinate Dimer SPECT Findings of a Girl With Refractory Localization-Related Epilepsy Who Developed Transient Ictal Bradycardia

Ictal bradycardia, which is considered to be one of the causes of sudden unexplained death in epilepsy, is rare. A 10-year-old girl with focal cortical dysplasia in her right centroparietal region developed transient ictal bradycardia during cluster seizures. Brain magnetic resonance imaging demonstrated a high signal intensity lesion adjacent to the focal cortical dysplasia lesion. Ictal 99m Tc-ethyl cysteinate dimer single-photon emission computed tomography (SPECT) detected hyperperfusion in an area containing the high signal intensity lesion, which was located close to the insular cortex. Since the hyperperfusion zone observed on SPECT was considered to reflect seizure propagation, it is possible that the ictal bradycardia experienced in the present case was caused by the following mechanism: The repetitive seizure activity caused the high-intensity lesion seen on MRI to expand into the right insular cortex, which controls cardiac rhythm, resulting in ictal bradycardia

Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. Approximately 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) gene deletion, while ~5% carry an intragenic SMN1 mutation. Here, we investigated the stability and oligomerization ability of mutated SMN1 proteins. Plasmids containing wild- and mutant-type SMN1 cDNA were constructed and transfected into HeLa cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar abundances of transcripts from the plasmids containing SMN cDNA, but Western blotting showed different expression levels of mutated SMN1 proteins, reflecting the degree of their instability. A mutated SMN1 protein with T274YfsX32 exhibited a much lower expression level than other mutated SMN1 proteins with E134K, Y276H, or Y277C. In immunoprecipitation analysis, the mutated SMN1 protein with T274YfsX32 did not bind to endogenous SMN1 protein in HeLa cells, suggesting that this mutation completely blocks the oligomerization with full-length SMN2 protein in the patient. The patient with T274YfsX32 showed a much more severe phenotype than the other patients with different mutations. In conclusion, the stability and oligomerization ability of mutated SMN1 protein may determine the protein stability and may be associated with the clinical severity of SMA caused by intragenic SMN1 mutation