Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

Long chain polyunsaturated Fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells

Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE(2) and PGF(2α), matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 μM-100 μM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca(2+)](i) mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.     

Study of the factors affecting the extraction of soybean protein by reverse micelles

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l⁻¹, aqueous pH 5.5 and KCl concentration 0.8 mol l⁻¹. The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.     

创新性教育在长学制医学生人体解剖学教学中的探索与实践

人体解剖学是医学科学中一门重要的基础课程.长学制医学教育是一种新模式,创新性教育是一种新理念.为了探索长学制医学生人体解剖学的创新性教育,我系在过去五年中以国家精品课程建设为契机.以"培养创新人才"为指导思想,更新教学思想,实行PBL教学,引入"RA"的训练体系,推行青年教师培养的"六个一工程"和国内、国外互动发展的人才培养新模式,取得了初步成果.本文对此进行总结,以期抛砖引玉,引起更多同行的共鸣而促进<人体解剖学>课程的建设.     

Isolation and characterization of a novel Ganoderma lucidum gene that differentially expressed between shaking culture and liquid static culture

Suppression subtractive hybridization (SSH) was preformed to investigate the differences of gene expression between the shaking culture mode and the liquid static culture mode which favors ganoderic acids production in Ganoderma lucidum. One novel gene preferentially expressed in liquid static culture was identified and analyzed. Its full length cDNA sequence and the 5′-flanking region were then obtained by rapid amplification of cDNA ends (RACE) and self-formed adaptor PCR (SEFA-PCR), respectively. Nucleotide sequence of the gene is not homologous to any of the known Ganoderma genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 371 amino acids that has high homology with the mitogen-activated protein kinase (MAPK) of other five species-Postia (97%), Coprinopsis (91%), Neurospora (86%), Aspergillus (83%), and Saccharomyces (80%)-so that it can be defined as a G. lucidum MAPK gene (GenBank accession number: JF781125). Computer assisted analysis revealed that this new G. lucidum MAPK gene contains thirteen exons and twelve introns. The quantitative real-time RT-PCR (qRT-PCR) analysis showed that this new gene had a much higher expression level in liquid static culture than in traditional shaking culture. Results of this research established a good foundation for further study on the functions of the G. lucidum MAPK at the molecular level.     

酿酒酵母菌核糖体RNA沉降系数的初步研究

为研究酿酒酵母菌核糖体RNA(rRNA)的沉降系数,用酶解法和液氮研磨法裂解酿酒酵母菌的细胞壁,Trizol Reagent提取其总RNA,同时提取小白鼠和斑马鱼的总RNA进行比较.经紫外分光光度计检测和甲醛琼脂糖变性胶电泳后,RNA纯度好,条带清晰,无弥散或降解现象.试验发现,与酶解法相比,用液氮研磨法破碎酿酒酵母菌细胞壁提取总RNA所用的成本低,时间少,产率和纯度高,适用于少量样品RNA的提取.同时,酿酒酵母菌与斑马鱼和小白鼠总RNA电泳图谱表明,三者的"18S rRNA"在条带大小方面差异较小,而"28S rRNA"差异较大.利用分析型离心机测得的酿酒酵母菌两个较大rRNA的沉降系数分别为24.7S和18.1S.研究结果表明了真核生物rRNA种类的多样性.     

The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.