As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at ∼58% compared with a peak at ∼42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at ∼42%, relatively low compared with that of protein-coding cDNAs.

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Proteins are susceptible to attack by reactive oxygen species (ROS). Several types of ROS-induced protein damage have been identified, including amino acid modification, cross-linking, and degradation. 1,2) Glutathione (GSH) is the major intracellular non-protein thiol compound and plays a key role in the protection of cells and tissue structures from oxidative injury. GSH can exist in a reduced form (GSH), oxidized form (GSSG), or as proteinbound GSH (GSSP). GSH acts as an intracellular antioxidant that is involved in preventing protein sulfhydryl groups from oxidizing and cross-linking. In addition, the GSH redox system functions indirectly with glutaredoxin (Grx), glucose 6-phosphate dehydrogenase (G6PDH), and glutathione reductase (GR), which maintain GSH in its reduced form. An increase in oxidative stress leads to the oxidation of GSH to GSSG and, under certain conditions, the formation of glutathionyl proteins. Various glutathionyl proteins are involved in numerous physical processes (e.g., growth, differentiation, cell cycle progression, cytoskeletal functions, and metabolism), suggesting that S-glutathionylation is a general mechanism of redox regulation. [3][4][5] Red blood cells (RBCs) have been used as a model system in numerous studies to determine the significance of the GSH-dependent redox system 6,7) because they have an efficient antioxidant defense system that prevents oxidative damage.8-10) Moreover, RBCs have high levels of GSH and Grx, which are thought to utilize the reducing power of GSH to catalyze disulfide reductions in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and GR. 11)Many lines of evidence have emerged to support a fundamental role for the glutathionylation of certain proteins in normal cellular biochemical and physiological processes. It has been reported that some proteins are capable of reacting to form the glutathionyl protein. In particular, it is well known that actin 12) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 13) are oxidized to form glutathionyl proteins. Additionally, the formation of glutathionyl proteins in human RBCs has been reported in in vitro studies. These studies indicated that hemoglobin, GAPDH and actin, 14) as well as spectrin and membrane protein band 4.2, 15) are glutathionylated in response to diamide or ROS. Alternatively, GSH is likely to contribute to the protection of all thiol residues in proteins against ROS in the cell. However, the physiological susceptibility of proteins to ROS and the direct effects of GSH on oxidative stress in human RBCs remain poorly understood. Moreover, there is little information on the significance of redox regulation that is dependent on the glutathionylation of individual proteins.The objective of this study was to identify physiologically relevant glutathionyl proteins formed in RBCs by ROS exposure. Therefore, we designed experiments to disrupt GSH homeostasis.16) We used two independent treatments aimed at decreasing the level of the reduced form of GSH concretely: 1) pre-...

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