cDNA cloning of rat major AP endonuclease (APEX nuclease) and analyses of its mRNA expression in rat tissues.

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic (AP) endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. It is also a redox factor (Ref-1), stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun. In the present paper, a cDNA for the enzyme was isolated from a rat brain cDNA library using mouse Apex cDNA as a probe and sequenced. The rat Apex cDNA was 1221 nucleotides (nt) long, with a 951-nt coding region. The amino acid sequence of rat APEX nuclease has 98.4% identity with mouse APEX nuclease. Using the rat Apex cDNA as a probe for Northern blot analysis, the size of rat Apex mRNA was shown to be approximately 1.5 kb. Its expression was compared in 9 rat organs on postnatal days 7 and 28. Although Apex mRNA was expressed ubiquitously, the levels varied significantly, suggesting organ- or tissue-specific expression of the Apex gene. The highest level was observed in the testis, relatively high levels in the thymus, spleen, kidney and brain, and the lowest level in the liver. The level of expression at postnatal day 28, with the exception of the testis, was almost the same as or lower in respective organs than that at postnatal day 7. Postnatal developmental changes of Apex mRNA expression in the testis and thymus were further studied. The expression in testis was markedly increased on postnatal days 21 and 28. The expression in thymus increased once at postnatal day 14, and then decreased. The developmental changes of Apex mRNA expression in testis and thymus suggest that APEX nuclease is involved in processes such as recombinational events.</p

Detection and analyses by gel electrophoresis of cisplatin-mediated DNA damage.

DNA damage induced by cis-diamminedichloroplatinum (II) (cisplatin: cis-DDP), an anticancer drug, was studied in vitro by monitoring the drug-induced conformational change of pUC18 plasmid DNA, the sensitivity to some restriction enzymes of the damaged DNA and the sequence-dependent termination of DNA synthesis caused by cisplatin. Closed circular, superhelical pUC18 DNA was treated at 37 degrees C for 16 h with various concentrations of cisplatin. Cisplatin-dose-dependent conformational change due to unwinding of the treated DNA was detected by agarose gel electrophoresis. To analyze the base-specificity of the cisplatin damage, the measurement for sensitivity of cisplatin-treated DNA to various types of restriction enzyme and sequence gel analysis of the treated DNA were conducted. The results suggested that cisplatin attacked preferentially the sequence of GG &#62; AG &#62; GNG in the order. In the present assay condition, the cisplatin/DNA nucleotide ratios required for the DNA damage detection were roughly 0.025 for the conformational analysis, 0.001 or more for the restriction enzyme analysis, and less than 0.001 for the sequence gel analysis. By using the present method, it was demonstrated that the cisplatin-mediated DNA damage was inhibited by NaCl, KCl, CaCl2 or MgCl2 at their nearly physiological concentrations, and by reducing agents such as thiourea and 2-mercaptoethanol in the reaction mixture.</p

Peplomycin-induced DNA repair synthesis in permeable mouse ascites sarcoma cells.

DNA repair synthesis induced in permeable mouse ascites sarcoma cells by peplomycin, an antitumor antibiotic, was studied. Mouse ascites sarcoma (SR-C3H/He) cells were permeabilized with a low concentration of Triton X-100 in an isotonic condition. Permeable cells were treated with an appropriate concentration of peplomycin to introduce single-strand breaks in permeable cell DNA. DNA repair synthesis in peplomycin-treated permeable cells was measured by incubating the cells with four deoxynucleoside triphosphates in an appropriate buffer system. The DNA repair synthesis was enhanced by ATP and NaCl at near physiological concentrations. More than 90% of DNA synthesis in the present system depended on the peplomycin-treatment. The repair nature of the DNA synthesis was confirmed by a BrdUMP density shift technique. The repair patches were largely completed and ligated in the presence of ATP. Analyses using selective inhibitors for DNA polymerases showed that both DNA polymerase Beta and aphidicolin-sensitive DNA polymerases (DNA polymerase alpha and/or delta) were involved in the repair DNA synthesis.&#60;/P&#62;</p

cDNA cloning, sequence analysis and expression of a mouse 44-kDa nuclear protein copurified with DNA repair factors for acid-depurinated DNA

We purified a 44-kDa nuclear protein from salt-extract of permeable mouse ascites sarcoma cells in an effort to isolate factors involved in the repair of acid-depurinated DNA. It was copurified with a major AP endonuclease (APEX nuclease) by sequential column chromatography then further purified by sodium dodecyl sulphate-poly-acrylamide gel electrophoresis as a possible DNA repair support factor. Its partial amino acid sequences were determined, and a cDNA clone for the protein was isolated from a mouse T-cell cDNA library using long degenerate oligonucleotide probes deduced from the amino acid sequence. The complete nucleotide sequence of the cDNA (1.7 kilobases) was determined. Northern hybridization using this cDNA detected two transcripts: 1.8kb being the major one and 2.6 kb being the minor one. The complete amino acid sequence for the protein predicted from the nucleotide sequence of the cDNA indicates that the 44-kDa protein consists of 394 amino acids with a calculated molecular weight of 43,698. In tests performed thus far, the recombinant 44-kDa protein expressed in Escherichia coli has not expressed any repair-support activity. It remains to be analyzed whether the protein attains this activity after appropriate posttranslational modifications. Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protein p38 -2G4, recently reported as a cell cycle-specifically modulated nuclear protein of 38kDa. The p38-2G4 may be a truncated form of the present 44-kDa protein.</p

Genomic structure of the rat major AP endonuclease gene (Apex) with an adjacent putative O-sialoglycoprotease gene (Prsmg1/Gcpl1) and a processed Apex pseudogene (Apexp1).

Genomic sequencing and chromosomal assignment of the gene encoding rat APEX nuclease, a multifunctional DNA repair enzyme, were performed. An active Apex gene and a processed pseudogene were isolated from a rat genomic library. The active Apex gene consists of 5 exons and 4 introns spanning 2.1 kb. The putative promoter region of the Apex gene lacks the typical TATA box, but contains CAAT boxes and a CpG island having putative binding sites for several transcription factors, such as Sp1, AP-2, GATA-1 and ATF. A putative O-sialoglycoprotease (a homologue of Pasteurella haemolytica glycoprotease, gcp; abbreviated as Prsmg1/Gcpl1) gene consisting of 11 exons and 10 introns spanning 7.3 kb lies immediately adjacent to the Apex gene in a 5'-to-5' orientation. The Apex gene locus was mapped to rat chromosome 15p12 using in situ hybridization. The processed pseudogene (designated as rat Apexp1) has a nucleotide sequence 87.1% identical to that of the rat Apex cDNA, although several stop codons interrupting the coding sequences and multiple nucleotide deletions were observed. The Apexp1 is located in an inactive LINE sequence. Calculation of nucleotide substitution rates suggests that the immediate, active progenitor of Apexp1 arose 23 million years ago and that the non-functionalization occurred 15 million years ago.</p

Neurocognitive Evaluation of Japanese Childhood Cancer Survivors

Long-term cognitive effects following acute lymphoblastic leukemia treatment have been reported for Caucasians; however, these data remain unclear for other ethnicities and the treatment of other cancers. Here, we assessed cognitive function in Japanese childhood cancer survivors. This study enrolled 53 Japanese survivors of childhood cancer （mean age, 9.5 years; 36 boys and 17 girls）. We evaluated performance-based cognitive function using the Wechsler Intelligence Scale for Children-Fourth Edition （WISC-Ⅳ）. Deviation intelligence quotients （IQ） for verbal comprehension （VC）, perceptual reasoning （PR）, processing speed （PS）, and working memory （WM） were compared with the standardized mean and standard deviation （SD; 100 and 15, respectively）. We classified patients into three groups depending on the cumulative methotrexate （MTX） dose （none, 1–19g/m2, and＞20g/m2）. Full-scale IQ was within normal range at 104.8 （SD, 12.9）, although there were significant differences among the four WISC–Ⅳ index scores （P＜0.001）. The PS score （97.1±15.5） was significantly lower than the VC （107.2±15.8） and PR （105.3±14.2） scores （both P＜0.005）. WM performance decreased in an MTX dose-dependent manner （107.8, 102.6, and 96.5 for none, 1–19g/m2, and higher than 20g/m2, respectively; P=0.05）. Japanese childhood cancer survivors, including those in the non-MTX group, demonstrated significant PS impairment. High-dose MTX treatment might be associated with neurocognitive deficiencies, particularly in WM. Although current treatments are associated with high cure rates, future research and interventions are required to improve cognitive function in these patients

Deletion of both p62 and Nrf2 spontaneously results in the development of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwide. However, details of pathogenetic mechanisms remain unknown. Deletion of both p62/Sqstm1 and Nrf2 genes spontaneously led to the development of NASH in mice fed a normal chow and was associated with liver tumorigenesis. The pathogenetic mechanism (s) underlying the NASH development was investigated in p62:Nrf2 double-knockout (DKO) mice. DKO mice showed massive hepatomegaly and steatohepatitis with fat accumulation and had hyperphagia-induced obesity coupled with insulin resistance and adipokine imbalance. They also showed dysbiosis associated with an increased proportion of gram-negative bacteria species and an increased lipopolysaccharide (LPS) level in feces. Intestinal permeability was elevated in association with both epithelial damage and decreased expression levels of tight junction protein zona occludens-1, and thereby LPS levels were increased in serum. For Kupffer cells, the foreign body phagocytic capacity was decreased in magnetic resonance imaging, and the proportion of M1 cells was increased in DKO mice. In vitro experiments showed that the inflammatory response was accelerated in the p62:Nrf2 double-deficient Kupffer cells when challenged with a low dose of LPS. Diet restriction improved the hepatic conditions of NASH in association with improved dysbiosis and decreased LPS levels. The results suggest that in DKO mice, activation of innate immunity by excessive LPS flux from the intestines, occurring both within and outside the liver, is central to the development of hepatic damage in the form of NASH

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model “Mitomouse” (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial

Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription

Background: The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms. Methodology/Principal Findings: C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2) upregulated glycosaminoglycan (GAG) production and expression of type II collagen a1 (Col2a1) mRNA, and these effects were evident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expression of Col2a1 mRNA. Dual luciferase reporter assays revealed that WT-Nkx3.2 upregulated Col2a1 enhancer activity in a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assays revealed that Nkx3.2 bound to the Col2a1 enhancer element. Conclusions/Significance: Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independen