Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.     

MSI2通过下调circRNA_001214促进急性髓系白血病细胞HL60生长

Musashi-2（MSI2）是一种RNA结合蛋白质,对维持造血干细胞功能具有重要作用。研究表明,MSI2高表达能促进急性髓系白血病（acute myelocytic leukemia, AML）进展,但其作用机制尚不明确。本研究稳定沉默HL60细胞MSI2后,第1、2、3、4 d对照组的相对细胞生长率分别为1.931 ± 0.027、3.070 ± 0.073、4.017 ± 0.092和4.215 ± 0.246;敲减组分别为1.927 ± 0.035、2.564 ± 0.090、2.825 ± 0.097和3.223 ± 0.182,两组相比具有统计学差异,P<0.001;细胞凋亡明显增加(7.967% ± 0.698% vs 3.400% ± 0.322%., P<0.01);G₀/G₁期细胞比例明显增高（67.430% ± 4.390% vs. 50.360% ± 2.160%, P<0.01）;NUMB蛋白明显上调,LEF1明显下降。环状RNA（circular RNA, circRNA）芯片筛选和荧光定量PCR验证显示,MSI2沉默组circRNA_001214表达水平是对照组3.48倍。这一结果也在NALM6细胞得到证实。进一步用生物信息学分析,显示circRNA_001214最可能与miR-1273a、miR-1273e和miR 5095结合,进而影响参与细胞凋亡相关基因（CYCS、AKT1、BAX、TNFRSF10A、TNFRSF10D）、Wnt信号基因（WNT4、WNT2B、WNT7B、 DKK2、SFRP1、CSNKE1和LEF1）以及参与细胞代谢相关基因（RPE, PGAM4, PGAM1, TAT, CBS、RPE、SUCLG2、PGAM4、PGAM1和 IDNK）。总而言之,MSI2可能通过干扰circRNA_001214生成,减少靶miRNA对凋亡、Wnt信号及细胞代谢相关基因表达的影响,促进细胞生长。     

ATF4在内质网应激调控成骨分化中的作用

为避免内质网中未折叠蛋白质的过度累积,真核细胞能激活一系列信号通路来维持内质网稳态,这个过程称为内质网应激。在骨生长发育中,适宜的内质网应激有助于成骨细胞、破骨细胞和软骨细胞的生长,可以促进骨髓间充质干细胞向成骨细胞分化。而过度的内质网应激会抑制成骨分化,严重的甚至导致骨质疏松、成骨不全等相关骨病的发生。内质网应激时可激活未折叠蛋白质反应,其主要是通过PERK/eIF2α/ATF4信号通路,上调转录激活因子4(ATF4)的表达。ATF4位于许多成骨分化调节因子的下游,是促进成骨分化的关键因子,在内质网应激对成骨分化的调节中发挥重要作用。在成骨分化过程中,适宜的内质网应激能通过激活PERK信号通路,诱导ATF4表达增加,进而上调骨钙素、骨涎蛋白等成骨所必需基因的表达,促进成骨分化。过度的内质网应激会激活ATF4/CHOP促凋亡途径,并导致Bax、胱天蛋白酶等凋亡信号分子的大量产生,进而导致细胞凋亡,抑制成骨分化。由于ATF4在ERS和成骨分化中的重要作用,ATF4在骨质疏松、成骨不全等骨相关疾病的治疗中具有重要意义。本文通过综述ATF4在内质网应激调控成骨分化中的作用机制,为相关骨性疾病治疗提供理论依据。     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.