The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l⁻¹) and yeast extract concentrations (4, 8 and 16 g l⁻¹) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 10⁶ ml⁻¹) was obtained using a glucose concentration of 10 g l⁻¹ and a yeast extract concentration of 8 g l⁻¹ in the feeding medium. A yeast extract concentration of 16 g l⁻¹ inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.     

Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Patterns of frugivory in the columnar cactus Pilosocereus leucocephalus

In the frugivory networks of many arid and semi‐arid Mesoamerican ecosystems, columnar cacti act as keystone species that produce fruits with a high content of water and nutrients attractive to numerous vertebrates. The aim of this investigation was to assess the fruit removal patterns of two guilds of frugivores on the fruits of the woolly torch Pilosocereus leucocephalus. We assessed fruit pulp removal in two ways: by estimating the consumption of seeds given the amount of pulp removed per visit and by estimating the percentage of pulp removal over time. We put exclosures on unripe, intact fruits to keep frugivores from removing material. Once ripe, we removed the exclosures and tracked animal visitation of 69 fruits using camera traps. We obtained a total of 2,162 hr of footage (14:47 hours of them with effective pulp removal). The highest number of visitors is that of diurnal species (n = 12, all birds) versus only four nocturnal (three bats, one rodent). The most effective species in pulp removal are birds. Bats play a modest role in frugivory of this cactus. The significance of this work is twofold: (a) birds and bats consume the fruit pulp of this cactus and likely disperse its seeds, and (b) although bats rank high in pulp removal effectiveness, birds as a guild far outweigh their importance in this system, as they are not only more frequent but also remove more pulp and seeds. Both groups are known to be important in cacti seed dispersal, and our findings are essential in understanding the population dynamics of the woolly torch and in elucidating its seed dispersal ecology.     

红托竹荪多糖诱导肿瘤细胞凋亡的作用初探

探讨红托竹荪多糖对小鼠腹水瘤S180细胞凋亡的作用。分别使用不同浓度的红托竹荪多糖处理S180细胞24h,Western blot法检测Bcl-xl、Bax、caspase-9和Caspase-3等蛋白表达,使用流式细胞仪检测细胞凋亡。Western blot 法检测结果显示Bcl-xl表达量随竹荪多糖剂量增加而降低,Bax表达量则相反,每组Bax/Bcl-xl的比值增加,Caspase-9、Caspase-3表达量增加;流式细胞仪检测结果显示：0、25、50和100mg/L组细胞凋亡率分别为5.12%±0.11%、9.61%±0.61%、16.39%±0.19%和17.05%±0.13%,与对照组相比细胞凋亡率增高,差异具有统计学意义（P<0.05）,推断红托竹荪多糖具有诱导S180细胞凋亡的功能。     

~（60）Co－γ射线诱变柠檬酸产生菌黑曲霉Co9－6的研究

本试验采用￣（６０）Ｃｏ－γ射线对柠檬酸产生菌黑曲霉Ｃｏ９－６进行辐射,经两次处理后选育出Ｌ１２１７和Ｌ８０１两株优良柠檬酸产生菌。中试结果表明菌株Ｌ１２１７较Ｌ８０１更优：产酸率较菌株Ｃｏ９－６提高１７．６％、发酵周期缩短１３．４％、对糖转化率提高１３．３％。     

动物基因组编辑中提升CRISPR/Cas9介导的同源重组效率研究进展*

基因组编辑技术可以对DNA或RNA进行精准改造,极大地促进了生命科学的发展。CRISPR/Cas9系统在靶位点诱导DNA发生双链或单链损伤,细胞对损伤部位采用无供体模板的非同源末端连接(non-homologous end joining,NHEJ)或有供体模板的同源重组(homologous recombination,HR)修复。基于HR的基因组编辑策略通常被用于获得DNA的精准改造,而NHEJ在动物DNA损伤修复中起主导作用。为了提升HR效率,研究人员设计了多种方案,包括CRISPR/Cas9系统优化和DNA修复通路调控等。从DNA损伤修复途径、Cas9变体选择、sgRNA设计、供体模板设计、DNA修复途径相关蛋白功能调控、供体模板募集效率提升、细胞周期调控及编辑细胞生存效率提升等方面详细综述了相关研究成果,发现尚未开发出放之四海而皆准的HR提升策略,基于HR的基因组编辑需要针对具体案例制定个体化策略。旨在为动物基因组编辑中提升CRISPR/Cas9介导的HR效率研究提供理论参考,为动物基因功能分析、基因治疗和经济动物基因编辑育种提供帮助。     

利用CRISPR-Cas9技术失活黑曲霉中果胶酶基因及突变株性能评价*

我国果胶酶制剂使用广泛但专一性不高,高效、专一的果胶酶制剂在市场上仍然匮乏。利用基因工程技术改造果胶酶生产菌株——黑曲霉来生产单一成分的果胶酶成为解决果胶酶应用需求的一种有效方案。构建一种高效的CRISPR-Cas9基因编辑技术,可为构建高产单一性果胶酶的黑曲霉底盘菌株提供有效的基因编辑工具。首先敲除产果胶酶黑曲霉基因组上的pyrG基因构建尿嘧啶营养缺陷型菌株AnΔpyrG,并在AnΔpyrG菌株的pyrG基因位点定点整合Cas9基因表达盒和pyrG基因表达盒,构建组成型表达Cas9基因的黑曲霉菌株AnCas9,再构建含有gpdA启动子、锤头结构核酶、HDV核酶的稳定性表达sgRNA的pLM2-sgRNA质粒,建立CRISPR-Cas9基因编辑体系。利用该技术失活AnCas9菌株中的2个聚半乳糖醛酸酶基因4978020和4983861来检测构建的CRISPR-Cas9基因编辑效率并检测4978020基因功能缺失菌株的表型变化和产酶变化,结果表明果胶酶基因编辑效率大于50%,AnΔ4978020的表型和果胶酶酶活性与出发菌株均无明显变化。在黑曲霉中成功构建了高效的Cas9基因编辑技术,4978020基因功能缺失也不影响菌株表型,为构建高产单一性果胶酶黑曲霉底盘菌株奠定基础。     

FrzA基因转导干预缺血性心力衰竭小鼠心肌Wnt信号通路的研究

目的:研究重组9型腺相关病毒(recombinant adeno-associated virus serotype 9,rAAV9)携带FrzA基因转导干预缺血性心衰小鼠心肌Wnt信号通路的可行性,为基因治疗心力衰竭提供新的思路.方法:选择3月龄雄性C57BL/6J小鼠共130只,随机分为空白组(n=10),心衰组(n=40),心衰+空病毒(rAAV9-GFP)注射组(n=40),心衰+rAAV9-FrzA组(n=40),采用结扎左冠状动脉定量控制心梗面积,于术后2周行心脏超声评各组心功能变化,再经尾静脉注射已稀释好的病毒,28d后处死小鼠取心脏标本,RT-PCR检测心肌目的基因FrzA以及Dvl-1,β-catenin的表达;Western blot检测心肌Wnt信号通路关键分子Dvl-1,GSK3β,p-GSK3β,β-catenin的表达.结果:与空白组相比,成功建立心衰模型后小鼠心功能均不同程度降低(P＜0.05);FrzA组与心衰组相比,心功能明显改善(P＜0.05);经尾静脉注射可成功将rAAV9-FrzA导入小鼠体内,并且目的基因FrzA在心肌组织中高表达(P＜0.05);心衰小鼠心肌中Wnt信号通路关键分子Dvl-1,p-GSK3β,β-catenin的表达显著升高(P＜0.05),FrzA转导后小鼠心肌中Wnt信号通路中关键分子Dvl-1,p-GSK3β,β-catenin表达均降低(P＜0.05)结论:利用rAAV9-FrzA转导缺血性心衰小鼠可以有效的干预心肌Wnt信号通路,抑制其活性,为基因治疗缺血性心衰提供了新的思路.     

利用基于CRISPR-Cas9 的基因编辑技术对miR-29a在GT1-7 细胞中进 行基因敲除

目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.