STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.     

Triphala and its active constituent chebulinic Acid are natural inhibitors of vascular endothelial growth factor-a mediated angiogenesis

Triphala churna (THL) is a combination of three fruits that has been used for many years in India for the treatment of various diseases. There are now reports which indicate that THL can inhibit growth of malignant tumors in animals. However, the mechanisms by which THL mediates its anti-tumor actions are still being explored. Because vascular endothelial growth factor-A (VEGF) induced angiogenesis plays a critical role in the pathogenesis of cancer, we therefore investigated whether tumor inhibitory effects of THL or its active constituents are through suppression of VEGF actions. We herein report that THL and chebulinic (CI) present in THL can significantly and specifically inhibit VEGF induced angiogenesis by suppressing VEGF receptor-2 (VEGFR-2) phosphorylation. These results are of clinical significance as these inexpensive and non-toxic natural products can be used for the prevention and treatment of diseases where VEGF induced angiogenesis has an important role.     

Participation in Sports Organizations and the Prevention of Functional Disability in Older Japanese: The AGES Cohort Study

Background

We sought to examine prospectively the difference in the association between incident functional disability and exercise with or without sports organization participation.

Methods

The study was based on the Aichi Gerontological Evaluation Study (AGES) Cohort Study data. In October 2003, self-reported questionnaires were mailed to 29,374 non-disabled Japanese individuals aged 65 years or older. Of these, 13,310 individuals were introduced to the Study, and they were followed for 4 years. Analysis was carried out on 11,581 subjects who provided all necessary information for the analysis.

Results

Analysis was carried out on incident functional disability by 4 groups of different combinations of performance of exercise and participation in a sports organization Active Participant (AP), Exercise Alone (EA), Passive Participant (PP) and Sedentary (S). Compared to the AP group, the EA group had a hazard ratio (HR) of 1.29 (1.02–1.64) for incident functional disability. No significant difference was seen with the PP group, with an HR of 1.16 (0.76–1.77). When a measure of social networks was added to the covariates, the HR of the EA group dropped to 1.27 (1.00–1.61), and significant differences disappeared. In contrast, it showed hardly any change when social support was added.

Conclusion

The results suggested that, even with a regular exercise habit, incident functional disability may be better prevented when a person participates in a sports organization than when he/she does not. In addition, participation in a sports organization correlates positively with social networks, which may lead to a small decrease in incident functional disability.     

Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC₅₀ of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC₅₀ 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53^neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.     

A Dominant EV71-Specific CD4+ T Cell Epitope Is Highly Conserved among Human Enteroviruses

CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD), has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine.     

Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ～25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.     

Density-dependent processes in the life history of fishes: evidence from laboratory populations of zebrafish Danio rerio

Population regulation is fundamental to the long-term persistence of populations and their responses to harvesting, habitat modification, and exposure to toxic chemicals. In fish and other organisms with complex life histories, regulation may involve density dependence in different life-stages and vital rates. We studied density dependence in body growth and mortality through the life-cycle of laboratory populations of zebrafish Danio rerio. When feed input was held constant at population-level (leading to resource limitation), body growth was strongly density-dependent in the late juvenile and adult phases of the life-cycle. Density dependence in mortality was strong during the early juvenile phase but declined thereafter and virtually ceased prior to maturation. Provision of feed in proportion to individual requirements (easing resource limitation) removed density dependence in growth and substantially reduced density dependence in mortality, thus indicating that 'bottom-up' effects act on growth as well as mortality, but most strongly on growth. Both growth and mortality played an important role in population regulation, with density-dependent growth having the greater impact on population biomass while mortality had the greatest impact on numbers. We demonstrate a clear ontogenic pattern of change in density-dependent processes within populations of a very small (maximum length 5 mm) fish, maintained in constant homogeneous laboratory conditions. The patterns are consistent with those distilled from studies on wild fish populations, indicating the presence of broad ontogenic patterns in density-dependent processes that are invariant to maximum body size and hold in homogeneous laboratory, as well as complex natural environments.