一株根瘤菌培养条件优化及代谢调控研究

研究了柠条根瘤菌的生理学特性 ,通过摇瓶试验确定了最佳培养基配方和最适培养条件 ,应用 10L -自动控制发酵罐在优化条件下进行了分批发酵试验 ,并以月桂酸盐为调节剂进行底物水平的代谢调控。试验结果表明 ,该菌株生长速率快 (0 .6 6 0 8g·L- 1 ·h- 1 )、发酵周期短 (2 7h)、产物PHB产量高 (6 .6 1g L) ,通过代谢调控可获得 3-羟基丁酸和 3-羟基辛酸异型共聚体 [P(HB -HO) ],是一株生产性状优良的生产菌株 ,有着良好的开发应用前景     

植物乳杆菌Lp-2的高密度发酵

高密度培养植物乳杆菌是制作其发酵剂的重要环节。首先,研究了不同的溶氧和pH对植物乳杆菌的分批发酵的影响。在分批发酵的基础上,为进一步提高发酵液中的菌体浓度,进行了补料分批发酵实验。最终通过对蔗糖反馈补料发酵试验对比改造获得了pH反馈补料发酵工艺。此发酵补料工艺可以控制蔗糖残糖量始终处于较低的水平,因此获得了最高的菌体产量。菌体干重达到13.56g/L,较分批培养提高90.05%。     

基于pH调节和有机氮源流加调控补料分批发酵过程提高ε-聚赖氨酸产量

为了解决ε-聚赖氨酸(ε-PL)补料分批发酵过程中后期ε-PL产率下降的问题,提出了在补料阶段利用调节p H值和流加有机氮源(酵母粉)两种手段来提高ε-PL产率。利用上述两种策略,实现ε-PL平均产率分别达到4.62 g/(L·d)和5.16 g/(L·d),较未调控补料分批发酵(典型补料分批发酵)分别提高了27.3%和42.15%;同时,实现ε-PL产量分别达到36.95 g/L和41.32 g/L,较未调控补料分批发酵分别提高了27.4%和42.48%。进一步细胞活性染色和关键酶活性分析发现,两种策略均能显著提高细胞活力和关键酶活性。该研究结果表明,在发酵中后期通过调节p H值和流加酵母粉两种措施能够显著增强细胞活性和产生菌代谢能力,从而达到提高ε-PL产率和产量的目的。     

L-色氨酸生产菌的选育及其发酵条件的研究

以代谢控制发酵理论为指导 ,对L -色氨酸产生菌的定向选育、摇瓶发酵条件、30L发酵罐发酵条件进行了研究。以谷氨酸棒杆菌Tx5 - 32 (Phe- +Tyr- )为出发菌株 ,经硫酸二乙酯 (DES)多次诱变处理 ,定向选育出一株L -色氨酸产生菌TQ2 2 2 3(Phe- +Tyr- +5 -MTr+5 -FTr+SGr+CINr)。以摇瓶分批发酵最优条件为基础 ,对菌株TQ2 2 2 3进行了 30L发酵罐分批发酵试验 ,该菌株发酵 6 4h ,产L -色氨酸 7.2 8g·L- 1 。     

P.chrysosporium分批发酵木素过氧化物酶动力学模型

目的:为研究黄孢原毛平革菌(P.chrysosporium AS5.776)液态分批发酵产木素过氧化物酶(LiP)发酵动力学规律,实现其发酵过程的优化,达到进一步提高酶产量.方法:对P.chrysosporium AS5.776进行5L液态分批发酵,根据发酵过程中菌体生长、基质消耗及LiP形成规律,基于Logistic方程和Luedeking-Piret方程建立了P.chrysosporium AS5.776液态分批发酵的过程动力学模型,并对模型进行了参数拟合、方差分析及验证比较.结果:模型模拟计算结果与实验值能较好地吻合,R2分别为0.9891、0.995 6和0.9925,模型显著性极高.验证实验显示所构建模型平均相对误差分别为2.9％、3.6％和12.5％.结论:该模型能较好揭示木素过氧化物酶发酵代谢过程的特征,可为工业化应用提供理论依据.     

环境因素对L-异亮氨酸发酵的影响

通过 5L自控发酵罐发酵实验 ,结合发酵过程中菌生长形态的变化 ,对L -异亮氨酸补料分批发酵进行研究 ,研究了环境因素对黄色短杆菌 (Brevibacteriumflavum)TJCN - 1的影响 ,优化出发酵最佳控制条件 ,提出分阶段发酵控制模式 ,对L -异亮氨酸生产有指导意义。     

发酵条件对毕赤酵母表达重组人干扰素ω糖基化的影响

发酵条件是影响毕赤酵母 (P .pastoris)表达外源重组糖蛋白时糖基化的重要因素。通过菌体浓度、起始pH值、甲醇诱导浓度和周期、装液量等摇瓶发酵实验 ,研究不同发酵条件对毕赤酵母表达分泌型重组人干扰素ω(rhIFNω)过程中糖基化的影响 ;同时 ,在连续培养过程中考察pH值变化对rhIFNω糖基化的影响和分批发酵过程中rhIFNω糖基化的变化。结果表明 ,控制菌体密度 250g L(WCW)、起始pH值 6 0、装液量小于 30mL、甲醇诱导浓度 15g L、甲醇诱导 3次 (每 24h诱导一次 )等发酵条件 ,有利于摇瓶发酵过程中rhIFNω的糖基化 ;控制pH值 70～75可促进rhIFNω的糖基化 ;分批发酵过程中 ,糖基化与非糖基化rhIFNω的含量有同比变化趋势 ,但糖基化rhIFNω所占比例明显低于摇瓶发酵实验的结果 ,其原因有待进一步研究。     

根瘤菌N613胞外多糖发酵条件及抗肿瘤作用研究

用摇瓶正交试验研究了根瘤菌Rhizobium sp.N613合成根瘤菌胞外多糖(rhizobium exopolysaccharide,REPS)的最佳培养基配方和最适培养条件。采用10L自动控制罐进行了分批发酵试验,获得了合成REPS的发酵动力学参数。经40h的补料分批发酵与代谢调控,REPS产量达到11.31g/L。此外,抗肿瘤实验表明,当REPS剂量为5mg/kg时,其抑瘤率可达53.40%。结果表明,REPS的发酵周期短,产量高,成本低,且具有良好的免疫活性与增稠性,有着极高的开发应用价值。     

聚-ε-赖氨酸补料发酵的初步研究

在5L自动发酵罐中,通过分批发酵和补料分批发酵,对白色链霉菌(Strepomyces albulus)生物合成聚-ε-赖氨酸(ε-PL)进行了初步研究。结果表明,在分批发酵中,利用pH控制策略,ε-PL产量从0.6 g/L提高到2.4 g/L。在分批补料发酵中,采用pH控制策略,当糖浓度在10g/L以下,补加葡萄糖和硫酸铵,则菌体大量生长,ε-PL产量提高到16.81 g/L,为分批培养的27倍。     

氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth

We have quantitated by autoradiography the binding of [¹²⁵I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G₁ phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [¹²⁵I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.     

Type 3 IP3 receptors driving oncogenesis

Type 3 Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R3s) have been identified as anti-oncogenic channels by propelling pro-apoptotic Ca²⁺ signals to mitochondria. Yet, recent studies (Rezuchova et al, Cell Death Dis, 2019; Ueasilamongkol et al, Hepathology, 2019; Guerra et al, Gut, 2019) revealed that IP₃R3 upregulation drives oncogenesis via ER-mitochondrial Ca²⁺ crosstalk, adding complexity to IP₃R3’s role in cancer.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响

长链非编码RNA (long non-coding RNA, lncRNA)是一类长度大于200nt、没有长开放阅读框架但往往具有mRNA结构特征的RNA,可以在转录及转录后水平参与基因的表达调控。近年来,有研究证实lncRNA对脂肪生成具有重要作用。Lnc-RAP3位于小鼠(Mus musculus)17号染色体,其表达量在小鼠脂肪细胞分化前后呈现显著差异,但其具体的生物学功能尚不清楚。为探讨lnc-RAP3在小鼠3T3-L1前脂肪细胞成脂分化中的作用,本文首先构建了lnc-RAP3的真核表达载体pcDNA3.1-RAP3,利用脂质体将pcDNA3.1-RAP3和人工合成的lnc-RAP3的siRNAs分别转染3T3-L1前脂肪细胞,并对转染后的细胞进行诱导分化,并通过油红O染色、qRT-PCR检测成脂分化相关基因表达等方法比较过表达和敲降lnc-RAP3对3T3-L1前脂肪细胞成脂分化的影响。结果显示,过表达lnc-RAP3后,细胞内脂滴聚集显著减少(P<0.05),在诱导分化第0 d、2 d和4 d时C/EBPα、Glut4、PPARγ、LPL和FAS的表达水平均呈显著(P<0.05)或极显著(P<0.01)下降;敲降lnc-RAP3后,细胞内脂滴聚集显著增多(P<0.05),同时在诱导分化第0 d、2 d时PPARγ、LPL、C/EBPα、FAS和Glut4的表达水平呈显著(P<0.05)或极显著(P<0.01)升高。本研究结果表明,lnc-RAP3可能通过影响成脂分化相关基因的表达来抑制3T3-L1前脂肪细胞的成脂分化。     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

Synthesis of 3-methyl-3-hydroxy-6-oxo-androstane derivatives

3alpha,17beta-Dihydroxy-3beta-methyl-5alpha-androstan-6-one (1) and 3beta,17beta-dihydroxy-3alpha-methyl-5alpha-androstan-6-one (13) were prepared by the reaction of methylmagnesium bromide with the 3-ketosteroids. Structures and configurations in position 3 were determined by NMR spectra. Substitution in the position 6 influences the ratio of the products.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.