Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

miR-145通过靶向吞噬和细胞活力蛋白1抑制乳腺癌细胞侵袭

吞噬和细胞活力蛋白1（engulfment and cell motility protein 1,ELMO1）可以促进多种癌细胞的侵袭和转移,但ELMO1的表达是否受miRNA的调控鲜有研究。本研究旨在探讨miR-145与ELMO1表达的相关性,以及miR-145通过结合ELMO1的mRNA对乳腺癌侵袭的影响。通过TargetScan (http://www.targetscan.org/)靶基因预测软件预测与ELMO1的3′UTR结合的miR-145。荧光素酶结果证实两者互补结合。Transwell侵袭结果显示,miR-145组和siELMO1+miR-145组MDA-231乳腺癌细胞穿膜数较对照组分别降低40%（P＜0.05）和79%（P＜0.05）。siELMO1+miR-145组和siELMO1组细胞穿膜数则无显著差异（P>0.05）。结果提示,miR-145通过与ELMO1的mRNA结合抑制细胞侵袭。qRT-PCR显示,低侵袭的MCF-7乳腺癌细胞miR-145的表达量较高侵袭的MDA-435细胞高80%（P＜0.05）,较MDA-231乳腺癌细胞高75%（P＜0.05）,即miR-145与癌细胞侵袭能力呈负相关。Western印迹结果表明,miR-145组ELMO1表达量低于阴性对照组,miR-145 抑制组ELMO1表达量高于抑制剂NC组（P＜0.05）,证明miR-145抑制ELMO1的表达。qRT-PCR显示,过表达miR-145后ELMO1 mRNA含量与对照组无显著差异（P>0.05）。结果提示,miR-145对ELMO1的调控作用通过抑制其翻译实现。F-肌动蛋白聚合实验表明,miR-145组和阴性对照组于20 s和60 s时F-肌动蛋白聚合结果存在明显区别（P＜0.05）。Western 印迹结果表明,miR-145组活化的Rac1表达量较阴性对照组降低60%（P<0.05）,抑制剂NC组活化的Rac1较miR-145 抑制组降低55%（P<0.05）;miR-145组磷酸化的整合素β1较对照组于15 min时降低42%（P<0.05）,于30 min时降低31%（P<0.05）。由此得出的miR-145过表达显著促进乳腺癌细胞F-肌动蛋白聚合、Rac1活化和整合素β1磷酸化结论。综上所述,miR-145通过靶向ELMO1的 mRNA抑制ELMO1翻译,从而抑制乳腺癌的侵袭。     

Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line

Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/CRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/CRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

Hectogram-scale synthesis of [Aib8, Arg34]-GLP-1 (7–37) by liquid-phase fragment condensation

Based on small-scale synthesis (0.3 g), a 100-g scale-up synthesis of crude [Aib⁸, Arg³⁴]-glucagon-like peptide-1 (GLP-1) (7–37) was completed. The crude [Aib⁸, Arg³⁴]-GLP-1 (7–37) was purified using a dynamic axial compression column 200 (DAC-200). Approximately 61 g of [Aib⁸, Arg³⁴]-GLP-1 (7–37) with a purity of >99% was obtained through one-step reverse-phase chromatography. The purification yield was approximately 92%. The yield from the total reaction was approximately 60%. In summary, we developed an economical and environmentally friendly route to the synthesis and purification of crude [Aib⁸, Arg³⁴]-GLP-1 (7–37), laying a foundation for subsequent industrial production.     

circPVT1通过miR-24-3p/let-7a-5p/FSCN1轴促进鼻咽癌细胞侵袭迁移

目的 鼻咽癌是一种来源于鼻咽上皮的恶性肿瘤,其临床特征之一是易发生淋巴转移,但是目前鼻咽癌转移的分子机制尚未阐明。circPVT1是由PVT1基因2号外显子反向拼接形成的环状RNA (circRNA),在多种肿瘤中表达上调,本文探讨了circPVT1在鼻咽癌侵袭迁移中的作用和分子机制。方法 通过RT-qPCR检测circPVT1及其下游miRNA和FSCN1在鼻咽癌细胞的表达情况,Transwell和划痕愈合实验检测circPVT1对鼻咽癌细胞侵袭迁移的影响,RNA pull-down实验检测circPVT1结合的miRNA,双荧光素酶报告实验检测miR-24-3p和let-7a-5p靶向抑制FSCN1 mRNA表达。结果 在鼻咽癌细胞中过表达circPVT1可以促进鼻咽癌细胞侵袭迁移,而敲低circPVT1则可以抑制鼻咽癌细胞的侵袭迁移。进一步研究发现,circPVT1可以通过竞争性吸附miR-24-3p和let-7a-5p,上调FSCN1的表达,从而促进鼻咽癌细胞的侵袭迁移。结论 circPVT1通过miR-24-3p/let-7a-5p/FSCN1轴促进鼻咽癌细胞侵袭迁移,证实c...     

MiR-150-5p靶向TP53对结直肠癌细胞侵袭和增殖的影响

摘要 目的：探索miR-150-5p靶向调控TP53基因对结直肠癌（colorectal cancer, CRC）在临床和生物学的相关性,研究miR-150-5p调控TP53基因在结直肠癌细胞增殖和侵袭病变中的作用。方法：收集临床手术切除并病理证实的结直肠癌患者的手术及血浆样本（结直肠癌和癌旁组织）60例,另选取癌旁正常粘膜10例,腺瘤30例。根据瘤体直径分为肿瘤＞5 cm（n=30）和≤5 cm（n=30）。qRT-PCR法测定样本中miR-150-5p表达,荧光素酶活性测定以确定TP53是否为miR-150-5p的靶基因。使用SW480细胞株,进行Transwell小室检测细胞侵袭能力,CCK-8检测细胞增值能力,对miR-150-5p进行生物信息学分析。结果：TP53是miR-150-5p的下游基因。癌组织及血浆中miR-150-5p表达量低于癌旁组织,直径＞5 cm瘤体中的miR-150-5p表达量显著低于直径≤5 cm瘤体,Ⅰ-Ⅱ期结直肠癌组织中的miR-150-5p表达显著高于Ⅲ-Ⅳ期（P＜0.05）。上调miR-150-5p后,细胞中TP53表达下降,下调miR-150-5p后,TP53表达升高;CCK-8增殖试验显示细胞中miR-150-5p过表达组抑制细胞增殖（P＜0.05）。结论：MiR-150-5p在结直肠癌组织和细胞中显著低表达,miR-150-5p通过靶向调节TP53抑制人CRC细胞的侵袭和增殖,有望成为结直肠癌治疗的新靶点。     

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。