Modulation of ATR-mediated DNA damage checkpoint response by cryptochrome 1

Mammalian cryptochromes (Crys) are essential circadian clock factors implicated in diverse clock-independent physiological functions, including DNA damage responses. Here we show that Cry1 modulates the ATR-mediated DNA damage checkpoint (DDC) response by interacting with Timeless (Tim) in a time-of-day-dependent manner. The DDC capacity in response to UV irradiation showed a circadian rhythm. Interestingly, clock-deficient Cry1 and Cry2 double knockout (Cry^DKO) cells retained substantial DDC capacity compared with clock-proficient wild-type cells, although the Cry1-modulated oscillation of the DDC capacity was abolished in Cry^DKO cells. We found temporal interaction of Cry1 and Tim in the nucleus. When Cry1 was expressed in the nucleus, it was critical for circadian ATR activity. We regenerated rhythmic DDC responses by ectopically expressing Cry1 in Cry^DKO cells. In addition, we also investigated the DDC capacity in the liver of mice that were intraperitoneally injected with cisplatin at different circadian times (CT). When mice were injected at CT20, about 2-fold higher expression of phosphorylated minichromosome maintenance protein 2 (p-MCM2) was detected compared with mice injected at CT08, which consequently affected the removal rate of cisplatin-DNA adducts from genomic DNA. Taken together, our data demonstrate the intimate interaction between the circadian clock and the DDC system during genotoxic stress in clock-ticking cells.     

Cdo Interacts with APPL1 and Activates AKT in Myoblast Differentiation

Cell–cell interactions between muscle precursors are required for myogenic differentiation; however, underlying mechanisms are largely unknown. Promyogenic cell surface protein Cdo functions as a component of multiprotein complexes containing other cell adhesion molecules, Boc, Neogenin and N-cadherin, and mediates some of signals triggered by cell–cell interactions between muscle precursors. Cdo activates p38MAPK via interaction with two scaffold proteins JLP and Bnip-2 to promote myogenesis. p38MAPK and Akt signaling are required for myogenic differentiation and activation of both signaling pathways is crucial for efficient myogenic differentiation. We report here that APPL1, an interacting partner of Akt, forms complexes with Cdo and Boc in differentiating myoblasts. Both Cdo and APPL1 are required for efficient Akt activation during myoblast differentiation. The defective differentiation of Cdo-depleted cells is fully rescued by overexpression of a constitutively active form of Akt, whereas overexpression of APPL1 fails to do so. Taken together, Cdo activates Akt through association with APPL1 during myoblast differentiation, and this complex likely mediates some of the promyogenic effect of cell–cell interaction. The promyogenic function of Cdo involves a coordinated activation of p38MAPK and Akt via association with scaffold proteins, JLP and Bnip-2 for p38MAPK and APPL1 for Akt.     

Epigallocatechin gallate reduces hypoxia-induced apoptosis in human hepatoma cells

Cell detachment from extracellular matrix is closely related to induction of apoptosis. Epigallocatechin gallate (EGCG) has been shown to have antioxidant effect and to protect hypoxia-induced damage. We investigated whether EGCG reduced hypoxia-induced apoptosis and cell detachment in HepG2 cells. EGCG prevented cell death by hypoxia (0.5% O2) in a dose-dependent manner (hypoxic cell viability, 54.67%). RT-PCR and caspase3 activity assay showed that the hypoxia-induced cell death was caused by apoptosis increasing mRNA level of BAX, CASP3, and caspase3 activity. EGCG reduced increase of these mRNA and caspase3 activity. Western blot analysis and immunocytochemistry showed that EGCG increased cell adhesion proteins including E-cadherin (CDH1), tumor-associated calcium signal transducer 1 (TACSTD1), and protein tyrosine kinase 2 (PTK2) decreased by hypoxia. Hypoxia-induced apoptosis in HepG2 cells, and EGCG contributed to the HepG2 cell survival by attenuating the apoptosis.     

Recurrent hepatic alveolar echinococcosis: report of the first case in Korea with unproven infection route

Human alveolar echinococcosis (AE), a hepatic disorder that resembles liver cancer, is a highly aggressive and lethal zoonotic infection caused by the larval stage of the fox tapeworm, Echinococcus multilocularis. E. multilocularis is widely distributed in the northern hemisphere; the disease-endemic area stretches from north America through Europe to central and east Asia, including northern parts of Japan, but it has not been reported in Korea. Herein, we represent a first case of AE in Korea. A 41-year-old woman was found to have a large liver mass on routine medical examination. The excised mass showed multinodular, necrotic, and spongiform appearance with small irregular pseudocystic spaces. Microscopically, the mass was composed of chronic granulomatous inflammation with extensive coagulation necrosis and parasite-like structure, which was revealed as parasitic vesicles and laminated layer delineated by periodic acid-Schiff (PAS) stain. Clinical and histologic features were consistent with AE. After 8 years, a new liver mass and multiple metastatic pulmonary nodules were found and the recurred mass showed similar histologic features to the initial mass. She had never visited endemic areas of AE, and thus the exact infection route is unclear.     

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo^−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.     

Esco2 promotes neuronal differentiation by repressing Notch signaling

Esco2 is an acetyltransferase that is required for the establishment of sister chromatid cohesion. Roberts-SC phocomelia (RBS) syndrome caused by the mutations of Esco2 gene, is an autosomal recessive development disorder characterized by growth retardation, limb reduction and craniofacial abnormalities including cleft lip and palate. Here, we show that Esco2 protein co-immunoprecipitates with Notch but not with CBF1. Esco2 represses the transactivational activity of Notch protein in an acetyltransferase-independent manner. Chromatin immunoprecipitation experiments suggest that Esco2 might regulate the activity of NICD-CBF1 via attenuating NICD binding to CBF1 on the promoter of Hes1, the downstream target gene of Notch. Furthermore, we demonstrate that the overexpression of Esco2 promotes the neuronal differentiation of P19 embryonic carcinoma cells and C17.2 neural progenitor cells and the knockdown of Esco2 by siRNA blocks the differentiation. The inhibitory effects of Notch protein on neuronal differentiation of P19 cells was suppressed by Esco2 overexpression. Taken together, our study suggests that Esco2 may play an important role in neurogenesis by attenuating Notch signaling to promote neuronal differentiation.     

The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.     

Habitual physical activity facilitates stress-induced HSP72 induction in brain,peripheral, and immune tissues

The mechanism(s) for how physically active organisms are resistant to many damaging effects of acute stressor exposure is unknown. Cellular induction of heat-shock proteins (e.g., HSP72) is one successful strategy used by the cell to survive the damaging effects of stress. It is possible, therefore, that the stress-buffering effect of physical activity may be due to an improved HSP72 response to stress. Thus the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Adult male Fischer 344 rats were housed with either a mobile running wheel (Active) or a locked, immobile wheel [sedentary (Sed)] for 8 wk before stressor exposure. Rats were exposed to either inescapable tail-shock stress (IS; 100 1.6-mA tail shocks, 5-s duration, 60-s intertrial interval), exhaustive exercise stress (EXS; treadmill running to exhaustion), or no stress (controls). Blood, brain, and peripheral tissues were collected 2 h after stressor termination. The kinetics of HSP72 induction after IS was determined in cultured mesenteric lymph node cells. Activation of the stress response was verified by measuring serum corticosterone (RIA). Tissue and cellular HSP72 content were measured using HSP72 ELISA in cell lysates. Both Active and Sed rats had elevated levels of serum corticosterone after stress. In contrast, Active but not Sed rats exposed to IS and/or EXS had elevated HSP72 in dorsal vagal complex, frontal cortex, hippocampus, pituitary, adrenal, liver, spleen, mesenteric lymph nodes, and heart. In addition, Active rats exposed to IS demonstrated a faster induction of lymphocyte HSP72 compared with Sed rats. Thus Active rats responded to stress with both greater and faster HSP72 responses compared with Sed rats. These results indicate that previous physical activity potentiates HSP72 expression after a wide range of stressors. Facilitated induction of HSP72 may contribute to the increased stress resistance previously reported in physically active organisms.     

Presenilin-1 regulates intracellular trafficking and cell surface delivery of beta-amyloid precursor protein

Presenilins (PS1/PS2) play a critical role in proteolysis of beta-amyloid precursor protein (beta APP) to generate beta-amyloid, a peptide important in the pathogenesis of Alzheimer's disease. Nevertheless, several regulatory functions of PS1 have also been reported. Here we demonstrate, in neuroblastoma cells, that PS1 regulates the biogenesis of beta APP-containing vesicles from the trans-Golgi network and the endoplasmic reticulum. PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and/or cell surface accumulation of beta APP. In contrast, release of vesicles containing beta APP is impaired in familial Alzheimer's disease (FAD)-linked PS1 mutant cells, resulting in reduced beta APP delivery to the cell surface. Moreover, diminution of surface beta APP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest that PS1 regulation of beta APP trafficking may represent an alternative mechanism by which FAD-linked PS1 variants modulate beta APP processing.     

A novel strategy for analysis of gene homologues and segmental genome duplications

Transformation-associated recombination (TAR) cloning allows selective isolation of a desired chromosomal region or gene from complex genomes. The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae. We investigated the effect of nonhomology on the efficiency of gene capture and found that up to 15% DNA divergence did not prevent efficient gene isolation. Such tolerance to DNA divergence greatly expands the potential applications of TAR cloning for comparative genomics. In this study, we were able to use the technique to isolate nonidentical chromosomal duplications and gene homologues.