Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage.An error-free DNA replication system is required to pass accurate genetic information on to the next generation. However, various kinds of DNA damage induced by endogenous and exogenous factors impair this replication ability and cause genetic alterations, resulting in cancer predisposition (1). Cells have excellent systems for avoiding these genetic alterations by removing and repairing the damaged lesions before DNA replication for maintaining the genetic stability of the organism; these systems include base excision repair, nucleotide excision repair, mismatch repair, and recombination repair (2, 3). If a lesion on template DNA escapes these repair systems, a polymerase may stall at this point and start synthesis again downstream, resulting in a single strand gap in the DNA, which can be repaired by postreplication repair. Usually, recombination repair in postreplication repair can fix this gap without base substitution, but when this repair does not happen, DNA synthesis by a bypass formation across the lesion called translesion synthesis (TLS) 1 may take place to fill the gap. This TLS may be held in the last resort for DNA repair because mutations can be induced during this step (for reviews, see Refs. 4 -6).In budding yeast Saccharomyces cerevisiae, the scRAD30 gene, the product of which is DNA polymerase , is involved in the error-free TLS that can replicate DNA through cis-syn thymine-thymine (T-T) dimer in an error-free manner (7-10), whereas the scREV1, scREV3, and scREV7 genes are involved in the error-prone TLS that frequently induces mutations at the damaged lesions (for reviews, see . It is known that most mutations induced after UV irradiation are caused by the products of these three REV genes. scRev1 protein is a ter...

Therapeutic effects of molecular hydrogen for a wide range of disease models and human diseases have been investigated since 2007. A total of 321 original articles have been published from 2007 to June 2015. Most studies have been conducted in Japan, China, and the USA. About three-quarters of the articles show the effects in mice and rats. The number of clinical trials is increasing every year. In most diseases, the effect of hydrogen has been reported with hydrogen water or hydrogen gas, which was followed by confirmation of the effect with hydrogen-rich saline. Hydrogen water is mostly given ad libitum. Hydrogen gas of less than 4 % is given by inhalation. The effects have been reported in essentially all organs covering 31 disease categories that can be subdivided into 166 disease models, human diseases, treatment-associated pathologies, and pathophysiological conditions of plants with a predominance of oxidative stress-mediated diseases and inflammatory diseases. Specific extinctions of hydroxyl radical and peroxynitrite were initially presented, but the radical-scavenging effect of hydrogen cannot be held solely accountable for its drastic effects. We and others have shown that the effects can be mediated by modulating activities and expressions of various molecules such as Lyn, ERK, p38, JNK, ASK1, Akt, GTP-Rac1, iNOS, Nox1, NF-κB p65, IκBα, STAT3, NFATc1, c-Fos, and ghrelin. Master regulator(s) that drive these modifications, however, remain to be elucidated and are currently being extensively investigated.

Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor a (GFRa). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kB in GDNF-treated cells, respectively. Oncogene (2000) 19, 4469 ± 4475.

We developed a simple algorithm, i-Score (inhibitory-Score), to predict active siRNAs by applying a linear regression model to 2431 siRNAs. Our algorithm is exclusively comprised of nucleotide (nt) preferences at each position, and no other parameters are taken into account. Using a validation dataset comprised of 419 siRNAs, we found that the prediction accuracy of i-Score is as good as those of s-Biopredsi, ThermoComposition21 and DSIR, which employ a neural network model or more parameters in a linear regression model. Reynolds and Katoh also predict active siRNAs efficiently, but the numbers of siRNAs predicted to be active are less than one-eighth of that of i-Score. We additionally found that exclusion of thermostable siRNAs, whose whole stacking energy (ΔG) is less than −34.6 kcal/mol, improves the prediction accuracy in i-Score, s-Biopredsi, ThermoComposition21 and DSIR. We also developed a universal target vector, pSELL, with which we can assay an siRNA activity of any sequence in either the sense or antisense direction. We assayed 86 siRNAs in HEK293 cells using pSELL, and validated applicability of i-Score and the whole ΔG value in designing siRNAs.