Modulation of Growth and Transformation of Murine MC3T3-E1 Cell Line by Murine Wild-type and Mutant p53 Genes

We studied the effects of murine wild-type and mutant p53 genes (p53-wt and p53val135) on the growth and transformation of murine osteoblastoid cell line MC3T3-El. The mutant p53val135 enhanced focus formation of MC3T3-E1 cells by the activated H-ras plus LTR-myc gene and H-ras plus adenovirus 12 E1A gene more than four fold each, while p53-wt suppressed them 0.4 and 0.3 fold, respectively. The plating efficiency of hygromycin-resistant MC3T3-E1 cells after transfection of pSV2hygro were also increased by more than three fold with the cotransfection of p53val135 and the efficiency was also decreased 0.2 fold by cotransfection of p53-wt. These indicate that p53val135 enhances and p53-wt suppresses not only oncogene focus formation but also the cellular growth of the murine MC3T3-E1 cell line. Southern blot hybridization detected the tran-sfected p53-wt sequence only in three out of ten MC3T3-E1 cell lines established from foci induced by p53-wt and oncogenes, and failed to detect the p53-wt DNA in hygromycin-resistant MC3T3-E1 cell lines transfected with pSV2hygro and p53-wt. These suggest that MC3T3-E1 cells containing p53-wt are at a dis-advantage to form transformed foci or colonies, and suggests that MC3T3-E1 provides a good in vitro system to test the biological activity of murine wild-type and mutant p53 genes

TNP-470 Suppresses the Tumorigenicity of HT1080 Fibrosarcoma Tumor Through the Inhibition of VEGF Secretion From the Tumor Cells

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer. TNP-470, a systemic analogue of fumagillin, is an angiogenesis inhibitor capable of suppressing the tumorigenicity in several animal models even though the mechanisms of action have not been completely clarified. In the current study, we investigated the effects of TNP-470 on human fibrosarcoma cells in vivo and in vitro. The administration of TNP-470 could suppress the tumorigenicity of HT1080 fibrosarcoma tumor. The conditioned medium from HT1080 fibrosarcoma cells treated with TNP-470 inhibited the proliferation and migration of human endothelial cell line, HUVEC and ECV304. The concentration of VEGF in the conditioned medium from HT1080 cells treated with TNP-470 was lower than that of the cells without TNP-470 treatment, indicating that TNP-470 downregulates the secretion of VEGF from HT1080 cells. These findings strongly suggest that the direct action of TNP-470 on sarcoma cells inhibits angiogenesis through the downregulation of VEGF secretion and this angiogenesis suppression resulted in the inhibition of tumorigenicity of HT1080 fibrosarcoma tumo

ER Stress Protein CHOP Mediates Insulin Resistance by Modulating Adipose Tissue Macrophage Polarity

Obesity represents chronic inflammatory states promoted by pro-inflammatory M1-macrophage infiltration into white adipose tissue (WAT), thereby inducing insulin resistance. Herein, we demonstrate the importance of an ER stress protein, CHOP, in determining adipose tissue macrophage (ATM) polarity and systemic insulin sensitivity. A high-fat diet (HFD) enhances ER stress with CHOP upregulation in adipocytes. CHOP deficiency prevents HFD-induced insulin resistance and glucose intolerance with ATM M2 predomination and Th2 cytokine upregulation in WAT. Whereas ER stress suppresses Th2 cytokine expression in cultured adipocytes, CHOP knockdown inhibits this downregulation. In contrast, macrophage responsiveness to Th1/Th2 cytokines is unchanged regardless of whether CHOP is expressed. Furthermore, bone marrow transplantation experiments showed recipient CHOP to be the major determinant of ATM polarity. Thus, CHOP in adipocytes plays important roles in ATM M1 polarization by altering WAT micro-environmental conditions, including Th2 cytokine downregulation. This molecular mechanism may link adipose ER stress with systemic insulin resistance

The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6β remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b−/− mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b−/− neurons were recovered by ATF6β and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b−/− and Calr+/− mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6β-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis

The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

神経細胞死を抑制する新たな分子を発見 --脳卒中やアルツハイマー病への応用に期待--. 京都大学プレスリリース. 2021-06-30.While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6β remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca²⁺-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b⁻/⁻ mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca²⁺ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b⁻/⁻ neurons were recovered by ATF6β and CRT overexpressions, or by treatment with Ca²⁺-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b⁻/⁻ and Calr⁺/⁻ mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6β-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca²⁺ homeostasis

Simulation of livestock biomass resource recycling and energy utilization model based on dry type methane fermentation system

International Conference of Biomass and Bioenergy 19-20 August 2019, West Java, IndonesiaThis study was aimed at investigating the local livestock biomass volume for dairy farms in Town A, located in eastern Hokkaido, Japan, and at presenting a model of a biogas plant that enables maximizing the use of the available livestock biomass. Using a dairy farm with 250 animals in Town A as a model for a biogas plant based on dry-type methane fermentation system (dry-type biogas plant), we set the operational conditions to an average hydraulic retention time of 20 days, digestion temperature of 55°C, and methane gas yield of 0.12 Nm3 CH4/kg VSA. We compared the biogas production of our presented model with that of a wet-type biogas plant with the same number of animals. The results showed that the dry-type biogas plant produced biogas at 859 Nm3/day, while the wet-type biogas plant produced biogas at 666 Nm3/day. These results indicate that introducing dry-type biogas plants in all dairy farms in Town A would potentially enable semi-solid livestock manure to be processed, which is not amenable to ordinary composting, in addition to the conventional processes being carried out through biogas plants, as well as lead to an increase in the amount of biogas production