B7-2胚胎性癌细胞诱导分化过程中的表型转变

胚胎性癌细胞(简称EC细胞)作为一类肿瘤(畸胎瘤)的干细胞近年受到广泛的重视,从胚胎学、肿瘤学和分子生物学等许多学科领域都应用它作为实验材料,离体诱导分化研究是其中的一个方面。B 7-2 EC细胞是我们从129品系小鼠的自发睾丸畸胎瘤中分离克隆得到的一株多能EC细胞,它在同种同基因小鼠     

小鼠早期胚胎发育期间TGF-β_1免疫组织化学定位

用兔抗人血小板TGF-β_1 N末端1—29氨基酸残基人工合成多肽抗血清作探针以及免疫荧光和免疫酶染色技术,分析了1—12天小鼠早期发育期间胚胎的TGF-β_1物质分布。结果表明,着床前胚胎包括卵裂细胞,桑椹胚和胚泡的ICM及滋养外胚层等细胞均显示TGF-β_1阳性免疫荧光染色。免疫酶染色还证明,沿囊胚腔顶部单层排列的原始内胚层细胞比邻近的ICM细胞有较深的染色反应。随着胚胎着床和进一步发育,7天龄胚胎中胚层早期形成阶段,紧靠中胚层一侧的外胚层胞质中含有浓集的棕色颗粒;各胚层的部分区域也存在着染色强度上差别。8—12天龄胚胎中,体节,心壁、间质细胞和肠道以及卵黄囊的脏壁中胚层均有显著的TGF-β_1免疫酶阳性物质。这些结果表明,着床前小鼠胚胎富含TGF-β_1物质,着床后的胚外组织,例如卵黄囊也为胚胎进一步发育提供了富含TGF-β_1物质的微环境;同时也提示,小鼠早期胚胎发育期间的胚泡形成,ICM细胞分化出原始内胚层,卵柱期中胚层形成,以及以后的神经管、体节和肢芽形成阶段等一系列形态发生和器官形成过程中,TGF-β_1可能是参与重要作用的一种生长调节因子。     

Insulin-like,and insulin-antagonistic,carbohydrate derivatives. The synthesis of aryl and aralkyl d-mannopyranosides and 1-thio-d-mannopyranosides

A number of novel, aryl and aralkyl d-mannopyranosides and 1-thio-d-mannopyranosides were synthesized for evaluation of insulin-like and insulin-antagonistic properties. The substituted-phenyl α-d-mannopyranosides were prepared by the general procedure of Helferich and Schmitz-Hillebrecht, the substituted-phenyl 1-thio-α-d-mannopyranosides by a method corresponding to the Michael synthesis of aromatic glycosides, and the aralkyl 1-thio-α-d-mannopyranosides by aralkylation of 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranose (15) and subsequent O-deacetylation. Compound 15 was obtained by basic cleavage of the amidino group in 2-S-(tetra-O-acetyl-α-d-mannopyranosyl)-2-thiopseudourea hydrobromide, the product of the reaction of tetra-O-acetyl-α-d-mannosyl bromide with thiourea. Benzyl 1-thio-β-d-mannopyranoside, obtained by reaction of the sodium salt of 1-thio-β-d-mannopyranose with α-bromotoluene, and benzyl 1-thio-α-l-mannopyranoside were also synthesized, in order to assess the stereospecificity of the biological activities measured.     

Effects of orchinol and related phenanthrenes on the enzymic degradation of indole-3-acetic acid

Orchinol, hircinol, loroglossol and certain related phenanthrenes inhibited horseradish peroxidase-catalysed IAA degradation to a varied degree. Among     

ZAKβ antagonizes and ameliorates the cardiac hypertrophic and apoptotic effects induced by ZAKα

ZAK (sterile alpha motif and leucine zipper containing kinase AZK), a serine/threonine kinase with multiple biochemical functions, has been associated with various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy. In our previous reports, we found that the activation of ZAKα signaling was critical for cardiac hypertrophy. In this study, we show that the expression of ZAKα activated apoptosis through both a FAS‐dependent pathway and a mitochondria‐dependent pathway by subsequently inducing caspase‐3. ZAKβ, an isoform of ZAKα, is dramatically expressed during cardiac hypertrophy and apoptosis. The interaction between ZAKα and ZAKβ was demonstrated here using immunoprecipitation. The results show that ZAKβ has the ability to diminish the expression level of ZAKα. These findings reveal an inherent regulatory role of ZAKβ to antagonize ZAKα and to subsequently downregulate the cardiac hypertrophy and apoptosis induced by ZAKα.     

Hyperosmolarity enhanced susceptibility to renal tubular fibrosis by modulating catabolism of type I transforming growth factor‐β receptors

Hyperosmolarity plays an essential role in the pathogenesis of diabetic tubular fibrosis. However, the mechanism of the involvement of hyperosmolarity remains unclear. In this study, mannitol was used to evaluate the effects of hyperosmolarity on a renal distal tubule cell line (MDCK). We investigated transforming growth factor‐β receptors and their downstream fibrogenic signal proteins. We show that hyperosmolarity significantly enhances the susceptibility to exogenous transforming growth factor (TGF)‐β1, as mannitol (27.5 mM) significantly enhanced the TGF‐β1‐induced increase in fibronectin levels compared with control experiments (5.5 mM). Specifically, hyperosmolarity induced tyrosine phosphorylation on TGF‐β RII at 336 residues in a time (0–24 h) and dose (5.5–38.5 mM) dependent manner. In addition, hyperosmolarity increased the level of TGF‐β RI in a dose‐ and time‐course dependent manner. These observations may be closely related to decreased catabolism of TGF‐β RI. Hyperosmolarity significantly downregulated the expression of an inhibitory Smad (Smad7), decreased the level of Smurf 1, and reduced ubiquitination of TGF‐β RI. In addition, through the use of cycloheximide and the proteasome inhibitor MG132, we showed that hyperosmolarity significantly increased the half‐life and inhibited the protein level of TGF‐β RI by polyubiquitination and proteasomal degradation. Taken together, our data suggest that hyperosmolarity enhances cellular susceptibility to renal tubular fibrosis by activating the Smad7 pathway and increasing the stability of type I TGF‐β receptors by retarding proteasomal degradation of TGF‐β RI. This study clarifies the mechanism underlying hyperosmotic‐induced renal fibrosis in renal distal tubule cells. J. Cell. Biochem. 109: 663–671, 2010. © 2010 Wiley‐Liss, Inc.     

mtDNA T8993G Mutation-Induced F1F0-ATP Synthase Defect Augments Mitochondrial Dysfunction Associated with hypoxia/reoxygenation: The Protective Role of Melatonin

Background

F1F0-ATP synthase (F1F0-ATPase) plays important roles in regulating mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on hypoxia/reoxygenation (H/RO) is unknown. The aim of this study was to investigate how mtDNA T8993G mutation (NARP)-induced inhibition of F1F0-ATPase modulates the H/RO–induced mitochondrial dysfunction. In addition, the potential for melatonin, a potent antioxidant with multiple mitochondrial protective properties, to protect NARP cells exposed to H/RO was assessed.

Methods And Findings

NARP cybrids harboring 98% of mtDNA T8993G genes were established as an in vitro model for cells with F1F0-ATPase defect; their parental osteosarcoma 143B cells were studied for comparison. Treating the cells with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP significantly enhanced apoptotic death upon H/RO detected by MTT assay and the trypan blue exclusion test of cell viability. Based on fluorescence probe-coupled laser scanning imaging microscopy, NARP significantly enhanced mitochondrial reactive oxygen species (mROS) formation and mitochondrial Ca²⁺ (mCa²⁺) accumulation in response to H/RO, which augmented the depletion of cardiolipin, resulting in the retardation of mitochondrial movement. With stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia duration, or administrating secondary oxidative stress following H/RO), NARP augmented H/RO-induced mROS formation to significantly depolarize mitochondrial membrane potential (ΔΨm), and enhance mCa²⁺ accumulation and nitric oxide formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted cardiolipin, thereby promoting permanent mitochondrial permeability transition, retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly reduced NARP-augmented H/RO-induced mROS formation and therefore significantly reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa²⁺, stabilized cardiolipin, and then improved mitochondrial movement and cell survival.

Conclusion

NARP-induced inhibition of F1F0-ATPase enhances mROS formation upon H/RO, which augments the depletion of cardiolipin and retardation of mitochondrial movement. Melatonin may have the potential to rescue patients with ischemia/reperfusion insults, even those associated with NARP symptoms.     

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ‐induced responses, for example, expression of IFN‐stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress‐induced‐phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca²⁺ levels while inhibiting nuclear translocalization of STAT1 and NFAT‐1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca²⁺/calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5‐mediated inhibition of IFNβ‐induced responses, for example, IFN‐sensitive response element driven luciferase, STAT1‐dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5‐expressing cells in response to IFNβ. Results prove Ca²⁺, calreticulin, and calcineurin involvement in STAT1‐mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.