血纤蛋白粘附肽与低分子量单链尿激酶融合产物活性分析

人工合成了血纤蛋白粘附肽基因,构建了粘附肽与低分子量单链尿激酶ｃＤＮＡ的融合基因,在大肠杆菌中表达了融合基因。融合基因表达产物的抗原性和天然尿激酶相同,并具有尿激酶的溶纤活性和粘附肽的抗纤维蛋白单体聚合的功能。     

Ypt32p regulates the translocation of Chs3p from an internal pool to the plasma membrane

The transport of the chitin synthase III, Chs3p, to the plasma membrane is temporally and spatially regulated. Chs3p is delivered to the plasma membrane at the beginning of the cell cycle, forming chitin rings, and at the end of the cell cycle, forming the primary septum. During the rest of the cell cycle, it is maintained in intracellular compartments, termed chitosomes that share characteristics with the late Golgi and the early endosomes. Chs5p and Chs6p are required for the cell cycle-dependent delivery of Chs3p to the cell surface, but the mechanisms underlying the temporal regulation are still unknown. The Rab proteins, Ypt31/32p, are required for exit of secretory vesicles from the late Golgi and for recycling of proteins between the late Golgi and early endosomes. Either gain of Ypt32p function, by overexpression, or loss-of-function mutations alter the localization of Chs3p-GFP. Moreover, cells overexpressing Ypt32p accumulate chitin at the cell surface independent of Chs5p. Overexpression of Ypt32p also disrupts the localization of the late Golgi protein Sec7. We propose that Ypt31/32p have a role in regulating the delivery of Chs3p to the plasma membrane and deposition of chitin at the cell surface.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

Leucine induces phosphorylation and activation of p70S6K in cortical neurons via the system L amino acid transporter

p70S6 kinase is a multipotent kinase that phosphorylates substrates in response to extracellular stimuli. This kinase activity inhibits apoptosis, regulates cell size and controls translation. In the CNS, p70S6K also participates in synaptic plasticity. In this study, we report that leucine, a branched-chain amino acid, induces phosphorylation and activation of p70S6 kinase in cortical neurons. Leucine also induces phosphorylation of S6 protein, a substrate of p70S6K. These effects of leucine are completely inhibited by rapamycin, consistent with mammalian target of rapamycin mediating p70S6 phosphorylation. Finally, we demonstrate that the action of leucine on cortical neurons is mediated by the system L amino acid transporter. Neurons express components of system L amino acid transporter LAT1, LAT2, and CD98. Leucine uptake and its effect on p70S6 kinase are both inhibited by a specific inhibitor of system L amino acid transporter. We propose that leucine plays important roles in regulating signaling by p70S6 kinase by acting as an intercellular communicator in the CNS.     

P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase complex HSP70/CHIP

The short isoform of ErbB3-binding protein 1 (Ebp1), p42, is considered to be a potent tumor suppressor in a number of human cancers, although the mechanism by which it exerts this tumor-suppressive activity is unclear. Here, we report that p42 interacts with the cSH2 domain of the p85 subunit of phosphathidyl inositol 3-kinase (PI3K), leading to inhibition of its lipid kinase activity. Importantly, we found that p42 induces protein degradation of the p85 subunit and further identified HSP70/CHIP complex as a novel E3 ligase for p85 that is responsible for p85 ubiquitination and degradation. In this process, p42 couples p85 to the HSP70/CHIP-mediated ubiquitin–proteasomal system (UPS), thereby promoting a reduction of p85 levels both in vitro and in vivo. Thus, the tumor-suppressing effects of p42 in cancer cells are driven by negative regulation of the p85 subunit of PI3K.     

灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响

目的 研究灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响,探讨灵芝多糖改善胰岛素抵抗的分子机制.方法 3T3-L1前脂肪细胞经1-甲基-3-异丁基-黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,以葡萄糖氧化酶法测定培养液中残余的葡萄糖含量.比较二甲双胍组,检测培养液中葡萄糖含量及PI-3K p85和GLUT4蛋白表达变化.结果 地塞米松联合胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,细胞对葡萄糖的摄取量减少.灵芝多糖可改善3T3-L1脂肪细胞胰岛素抵抗.胰岛素抵抗细胞的PI-3K p85和GLUT4蛋白表达明显减少;应用灵芝多糖后,相关蛋白表达增加.结论 灵芝多糖通过提高PI-3K p85和GLUT4蛋白的表达,参与胰岛素抵抗状态下3T3-L1细胞的葡萄糖代谢.     

肝细胞癌组织miR-124-3p、miR-212-5p表达与PI3K/Akt信号通路和预后的关系研究

摘要 目的：探讨肝细胞癌组织中微小核糖核酸（miR）-124-3p、miR-212-5p表达与磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）信号通路和预后的关系。方法：选择2017年9月至2019年9月徐州医科大学附属医院肝胆外科收治的93例肝细胞癌患者,采用实时荧光定量聚合酶链反应（qRT-PCR）检测肝细胞癌组织中miR-124-3p、miR-212-5p、PI3K 信使RNA（mRNA）、Akt mRNA的表达。Pearson相关性分析miR-124-3p、miR-212-5p表达与PI3K/Akt信号通路相关mRNA表达的相关性。绘制Kaplan-Meier生存曲线,Log-Rank检验不同miR-124-3p、miR-212-5p表达肝细胞癌患者3年总生存率（OS）的差异。结果：肝细胞癌组织中miR-124-3p表达低于癌旁组织（P＜0.05）,miR-212-5p、PI3K mRNA、Akt mRNA表达高于癌旁组织（P＜0.05）。肝细胞癌组织中miR-124-3p表达与PI3K mRNA、Akt mRNA表达呈负相关（P＜0.05）,miR-212-5p表达与PI3K mRNA、Akt mRNA表达呈正相关（P＜0.05）。Ⅱa期、低分化患者肝细胞癌组织中miR-124-3p表达低于Ⅰa-Ⅰb期、中高分化患者（P＜0.05）,miR-212-5p表达高于Ⅰa-Ⅰb期、中高分化患者（P＜0.05）。随访期间死亡37例, miR-124-3p低表达组3年OS为42.55%,低于miR-124-3p高表达组的78.26%（P＜0.05）,miR-212-5p高表达组3年OS为50.00%,低于miR-212-5p低表达组的71.11%（P＜0.05）。结论：肝细胞癌组织中miR-124-3p表达下调,miR-212-5p表达上调,且与肝细胞癌PI3K/Akt信号通路激活,PI3K mRNA和Akt mRNA高表达,低分化,CNLC分期Ⅱa期以及低OS有关。     

小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响

初步探讨在小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响。通过显微操作技术注射野生型、持续激活型及激酶失活型的PKB的mRNA,用免疫荧光方法检测p21蛋白的细胞定位、Western blot方法检测p21蛋白的表达。结果显示在注射不同形式的PKB mRNA后p21蛋白的表达无明显差别,但是细胞定位发生改变,PKB被激活后,p21蛋白滞留在胞浆中。因而初步认为在小鼠受精卵中,PKB/Akt通过影响p21的细胞定位而影响细胞周期的进程。     

The Immunomodulatory Activity of Peptides Related to the DNA Contacting Loop of p53 Protein

Taking into account the sequence homology existing between thymopoietin II and the DNA-binding domain of p53 protein, a series of octapeptides was synthesized, related to the wild p53 type protein as well as to its mutated forms, appearing in some human tumours. The wild type octapeptide has immunostimulative activity with regard to the humoral immune response, but is inactive in the cellular immune response. The mutated peptides of p53 differ in their immunomodulatory activity from the wild type octapeptide. The Ser5 analogue of the wild type peptide is a strong stimulant of the humoral immune response and enhances TNF-α production, while at the same time suppressing the cellular immune response. The data suggest that the mutations of p53, which favour tumour development and growth, may also change the immune activity of respective p53 fragments.     

Role of p70S6K1-mediated Phosphorylation of eIF4B and PDCD4 Proteins in the Regulation of Protein Synthesis

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5′-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.     

Akirin2 could promote the proliferation but not the differentiation of duck myoblasts via the activation of the mTOR/p70S6K signaling pathway

The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48 h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors. Flow cytometry analysis revealed that the cell cycle transition was accelerated by Akirin2 overexpression. Moreover, the overexpression of Akirin2 did not influence the myotube formation. Strikingly, when duck myoblasts were cultured in the growth medium, the overexpression of Akirin2 significantly enhanced cell viability. Although the expression of cyclin-dependent proteins did not significantly increase after transfection, the expression of the mammalian targets of rapamycin (mTOR) and p70 S6 kinase (p70S6K) increased. Furthermore, the protein expression of phospho-p70S6K (Ser 417) also increased. However, when rapamycin and pEGFP-N1-Akirin2 plasmids were added together to the growth medium, the positive impact of Akirin2 on cell viability and the mRNA expression of mTOR and p70S6K were significantly blocked. Furthermore, the expression of phospho-mTOR (Ser 2448) and phospho-p70S6K (Ser 417) were also blocked. Taken together, these results could suggest that duck Akirin2 could promote myoblast proliferation via the activation of the mTOR/p70S6K signaling pathway.     

缺血性脑卒中患者血清PTX3、Cat S、IL-17A及miR-32-3p水平及其临床意义

目的:探讨缺血性脑卒中患者血清正五聚蛋白3(Pentraxin 3,PTX3)、组织蛋白酶S(Cathepsin S,Cat S)、白细胞介素(interleukin,IL)-17A及mi R-32-3水平及其临床意义。方法:选取2015年1月至2019年2月在我院神经内科住院诊治的缺血性脑卒中患者112例作为病例组,同期选择正常健康人群80例作为对照组。检测和比较两组血清PTX3、Cat S、IL-17A及mi R-32-3p含量与全血组织mi R-32-3p的表达,评估患者的神经缺损功能并进行相关性分析。结果:病例组血清PTX3、Cat S、IL-17A含量及全血mi R-32-3p相对表达均显著高于对照组(P0.05)。病例组平均NIHSS评分为9.58±1.28分,直线相关分析显示患者的NIHSS评分与血清PTX3、Cat S、IL-17A含量和全血mi R-32-3p相对表达水平均呈显著正相关性(P0.05)。COX回归分析显示血清PTX3、Cat S、IL-17A含量和全血mi R-32-3p相对表达都为影响NIHSS评分的主要因素(P0.05)。结论:缺血性脑卒中患者血清PTX3、Cat S、IL-17A与全血组织mi R-32-3p呈高表达,可能作为评价患者神经缺损功能的参考指标。     

喉鳞状细胞癌组织miR-1207-5p、miR-186-5p表达水平与PI3K/Akt信号通路、临床病理特征和预后的关系

摘要 目的：探讨喉鳞状细胞癌（LSCC）组织miR-1207-5p、miR-186-5p表达水平与磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路、临床病理特征和预后的关系。方法：选取2017年1月至2020年1月南充市中心医院收治的120例LSCC患者,取手术切除的LSCC组织和癌旁组织。检测miR-1207-5p、miR-186-5p、PI3K mRNA、Akt mRNA表达。患者出院后随访3年,统计总生存（OS）和无复发生存（RFS）情况。分析miR-1207-5p、miR-186-5p与PI3K、Akt的相关性以及影响LSCC患者预后的因素。结果：LSCC组织miR-1207-5p、miR-186-5p表达低于癌旁组织（P＜0.05）,PI3K mRNA、Akt mRNA表达高于癌旁组织（P＜0.05）。LSCC组织miR-1207-5p、miR-186-5p表达与PI3K mRNA、Akt mRNA表达呈负相关（P＜0.05）。肿瘤直径≥1 cm、低分化、TNM分期Ⅲ期、颈部淋巴结转移LSCC组织中miR-1207-5p、miR-186-5p表达低于肿瘤直径＜1 cm、中高分化、TNM分期Ⅰ～Ⅱ期、无颈部淋巴结转移（P＜0.05）。miR-1207-5p低表达、miR-186-5p低表达LSCC患者3年总生存（OS）率和无复发生存（RFS）率低于miR-1207-5p高表达、miR-186-5p高表达LSCC患者（P＜0.05）。多因素COX回归分析显示TNM III期、颈部淋巴结转移是LSCC患者复发和死亡的危险因素（P＜0.05）,高miR-1207-5p、高miR-186-5p是保护因素（P＜0.05）。结论：LSCC组织中miR-1207-5p和miR-186-5p表达均下调,与LSCC恶性病理特征、PI3K/Akt信号通路激活以及低生存率有关。     

A comparison of the involvement of p38, ERK1/2 and PI3K in growth factor-induced chondrogenic differentiation of mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are under investigation as an alternative cell source for the engineering of cartilage tissue in three-dimensional (3D) scaffolds. However, little is known about the intracellular mechanisms involved in the chondrogenic differentiation of MSCs. This study investigated the signaling pathways evoked by TGF-β1 and IGF-1 that mediated chondrogenic differentiation in adult rat bone-marrow derived MSCs in (i) monolayer on plastic and (ii) a 3D collagen-GAG scaffold. The data demonstrated involvement of the p38 pathway, but not ERK1/2 or PI3K in TGF-β1-induced chondrogenic differentiation in monolayer. Similarly, when the MSCs were seeded onto a collagen-GAG scaffold and treated with TGF-β1, the chondrogenic differentiation was dependent upon p38. In contrast, IGF-1-induced chondrogenic differentiation in monolayer involved p38, ERK1/2, as well as PI3K. The phosphorylation of Akt occurred downstream of PI3K and phospho-Akt was found to accumulate in the nucleus of IGF-1-treated cells. When MSCs were seeded onto the collagen-GAG scaffold and exposed to IGF-1, PI3K was required for chondrogenesis. These findings highlight the respective and differential involvement of p38, ERK1/2 and PI3K in growth factor-induced chondrogenesis of MSCs and demonstrates that intracellular signaling pathways are similar when differentiation is stimulated in a 2D or 3D environment.