Enrichment for Auxotrophic Mutants of Tetrahymena Using Density Gradient Centrifugation*

SYNOPSIS. A protocol based on density differences between starved and fed cells and employing density gradient centrifugation has been devised to facilitate the isolation of auxotrophic mutants of cell lines derived from Tetrahymena thermophila strain B1868. First, a mass phenotype screening procedure was established whereby true auxotrophic mutants and slow-growing wild-type cells such as strain C* could readily be distinguished. Second, simulation experiments were performed in which wild-type cells starved first in non-nutritive buffer, then suspended in a defined medium lacking a single essential amino acid became significantly denser than the same cells when starved, then suspended in a complete defined medium. Finally, using the same protocol, a reconstruction experiment was carried out which resulted in effective separation of wild-type cells from cells of a tyrosine auxotroph. The overall procedure resulted in a 9-fold increase in the relative frequency of auxotrophic cells, while the density gradient centrifugation alone provided a 400-fold enrichment.     

嗜热四膜虫异染色质蛋白Tcd1磷酸化位点的突变分析

四膜虫异染色质蛋白Tcd1在有性生殖时期特异表达,在大核基因组重排以及修复过程中发挥作用。磷酸化蛋白质组学分析表明,Tcd1存在3个磷酸化位点：S301,S303和S535。然而,Tcd1磷酸化修饰与其功能的关系并不清楚。本研究对TCD1基因的3个磷酸化位点进行了模拟磷酸化和模拟去磷酸化定点突变,获得模拟磷酸化突变基因TCD1S301D (TCD1S1D)、TCD1S301DS303D (TCD1S2D)与TCD1S301DS303DS535D (TCD1S3D) 和模拟去磷酸化的突变基因TCD1S301A (TCD1S1A)、TCD1S301AS303A (TCD1S2A)与TCD1S301AS303AS535A (TCD1S3A)。分别构建了不同突变体的过表达载体,转化四膜虫细胞并筛选获得不同突变体细胞株。Western印迹分析表明,Tcd1S1D、Tcd1S2D、Tcd1S3D与Tcd1S1A、Tcd1S2A和Tcd1S3A在四膜虫有性生殖期表达。免疫荧光定位分析发现,Tcd1S1D点状定位于细胞质中,Tcd1S2D在有性生殖初期点状定位于细胞质中,在新大核上形成均匀的定位,Tcd1S3D无法定位于亲本大核上,只是均匀定位于新大核上。Tcd1S2A和Tcd1S3A在新大核形成异常的块状定位,并且与异染色质蛋白Pdd1不能共定位。结果表明,Tcd1不同位点的磷酸化和去磷酸化修饰的动态变化决定了其在四膜虫细胞中的定位模式。     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

嗜热四膜虫Ran结合蛋白1的表达对大小核分裂的影响

Ran是细胞内的一种具有GTP酶活性的功能蛋白,可以调节染色体稳定性、细胞核组建以及核质运输等多种细胞进程．Ran结合蛋白1（Ran-binding protein 1, Rbp1p ）是Ran的必要调控因子,促进Ran-GTP水解为Ran-GDP．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的Ran结合蛋白基因RBP1(TTHERM_00158040, http://www.ciliate.org)．实时荧光定量PCR表明,RBP1在四膜虫营养生长和有性生殖过程中都有表达,且在有性生殖过程中表达水平提高．免疫荧光定位表明,在营养 生长期Rbp1p定位于细胞质中．过表达RBP1或敲减RBP1后,细胞生长速率下降,大核的无丝分裂异常,细胞分裂末期产生了无大核的异常细胞,同时过表达RBP1导致了多小核的产生．结果表明,Rbp1p影响四膜虫细胞核的分裂进程,它的正常表达对细胞增殖过程起到重要的调节作用．     

RanGTPase激活蛋白RanGAP在嗜热四膜虫细胞中的定位及功能

RanGTPase激活蛋白（RanGTPase activating protein,RanGAP）和Ran相互作用,提高了Ran GTPase水解GTP的效率. RanGAP参与细胞内核质运输、纺锤体组装、核膜重建和异染色质的组装．生物进化过程中,不同生物的RanGAP表现出结构和功能的多样性．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的RanGTPase激活蛋白基因RanGAP（TTHERM_00766430）．实时荧光定量PCR表明,RanGAP在四膜虫营养生长、饥饿和有性生殖过程中均有表达,且在有性生殖4～6 h表达水平最高．免疫荧光定位表明,在营养生长期、饥饿期及有性生殖的早期,RanGAP定位于细胞质中| 在有性生殖后期, RanGAP定位于凋亡的大核中．过表达RanGAP的细胞增殖速率下降,大核分裂和胞质缢缩异常, 产生无大核细胞．敲减RanGAP的细胞大核形态异常,细胞增殖速率下降,无丝分裂受到抑制,进而产生无大核细胞．RanGAP的过表达或敲除分别引起四膜虫RAN1,RanBP1和RCC1基因的表达下调或上调．结果表明,RanGAP通过Ran信号通路调控了嗜热四膜虫无性生殖过程中大核的无丝分裂,并可能参与了有性生殖过程中亲本大核的凋亡．     

周期蛋白Cyc2的过量表达及突变抑制嗜热四膜虫有性生殖发育

周期蛋白在细胞增殖过程中呈现周期性表达变化,不同的周期蛋白通过结合周期蛋白激酶(cyclin-dependent kinase,CDKs)及靶向特定蛋白质参与细胞有丝分裂和减数分裂过程的精确调控。嗜热四膜虫有性生殖期特异表达的B3型周期蛋白Cyc2(cyclin 2,Cyc2)对减数分裂的启始是必需的,但Cyc2蛋白的分子调控机制并不清楚。本研究通过0.1μg/mL和0.3μg/mL镉离子诱导突变细胞株OE-CYC2-B2086和OE-CYC2-CU428中CYC2基因在金属硫蛋白1基因(metallothionein gene 1,MTT1)启动子调控下上调表达。实时荧光定量PCR检测突变株OE-CYC2-B2086和OE-CYC2-CU428中CYC2的转录水平分别上调7.8倍和9.8倍。细胞有性生殖发育进程的荧光显微观察发现CYC2的表达上调并不影响有性生殖前期减数分裂的启始,但是干扰四膜虫有性生殖后期中新大核和新小核的正确形成。同时突变株OE-CYC2-B2086和OE-CYC2-CU428交配后,在镉离子诱导下不能产生有性生殖后代,但是该突变株分别和两种不同野生型细胞或CYC2敲除的突变细胞株交配能够恢复产生3%,15%或32%的有性生殖后代,有性生殖发育异常程度与CYC2的表达水平成正相关。进一步突变Cyc2第312位磷酸化位点丝氨酸为丙氨酸,获得CYC2单位点突变细胞株CYC2-S312A-B和CYC2-S312A-C。丝氨酸单位点突变阻止了四膜虫有性生殖期小核减数第1次分裂起始。结果表明周期蛋白2的表达水平和磷酸化修饰影响了不同阶段细胞核的功能,周期蛋白2对四膜虫有性生殖发育的正常进行是必需的。     

Lysosomal Physiology in Tetrahymena. II. Effect of Culture Age and Temperature on the Extracellular Release of 3 Acid Hydrolases*

Tetrahymena cultures were grown from a single inoculum and collected on 3 successive days corresponding to the log, transitional, and early stationary phases of growth. Cells were washed and incubated for 5 hr in a dilute salt solution. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed, and extracellular activities corrected for proteolytic degradation. A marked increase in the cellular content of acid phosphatase and significant decreases in α-glucosidase and ribonuclease occurred with advancing culture age. The intracellular changes in enzyme activities during incubation were roughly similar for cells of all ages. Protein content did not change appreciably during incubation. Extracellular A₂₅₅ release, monitored as an indication of the loss of RNA breakdown products, was at a minimum during incubation of transition cells. Significant quantities of all 3 acid hydrolases were released from cells of all ages except for ribonuclease from transition cells. The release of acid phosphatase and α-glucosidase was approximately proportional to the initial cellular content of these enzymes for cells of different ages and in log cells the effect of temperature on the rates of release was described by the Arrhenius equation. Release of ribonuclease, however, was not proportional to its intracellular content nor did it vary with temperature according to the Arrhenius equation. The results suggest that acid phosphatase and α-glucosidase are released via a first-order process.     

Interdependence of Cell Cycle Events in Tetrahymena thermophila: Formation of Contractile Vacuole Pore and Fission Gap in Ciliary Meridians*

SYNOPSIS The formation of the contractile vacuole pore (CVP) in Tetrahymena thermophila. genotype mol^b/mol^b is temporally and spatially associated with the formation of the fission gap in the CVP meridian. New CVPs arise when fission gaps appear in CVP meridians, the new pores being found anterior to the gaps. When, however, CVP meridians are rotated 180°, the fission gaps develop late. In more than 1/3 of the 180°-rotated CVP meridians, the new CVPs are formed before the appearance of the fission gap. Evidently, the appearance of the gap is not a prerequisite for CVP formation. Nevertheless, mutants exist in which the absence of fission gap and CVP are correlated in some cases and in which the presence of supernumerary fission gap and CVP are correlated in other instances. It is suggested that the 2 developmental events, although not causally related to each other, may be controlled by a common morpho-genetic signal. This commits a certain site (mid-body) along a ciliary meridian to develop the fission gap as well as the CVP; however, after this step of commitment, the appearance of the fission gap is delayed in 180°-rotated CVP meridians.     

Rearrangement of the 5S ribosomal RNA gene clusters during the development and replication of the macronucleus in Tetrahymena thermophila

The organization of the 5S rRNA genes in the MACronuclear genome of Tetrahymena thermophila was examined during MAC development and replication. The 5S genes are arranged in several tandem arrays of alternating transcribed and spacer sequences in both MICronucleus and MAC. The number of EcoRI fragments bearing 5S gene clusters is similar in MIC and MAC. Most fragments occur in both the MIC and newly formed MAC genomes, a few being MIC-limited and a few MAC-limited. The same rearrangements are seen in the MACs of all four caryonides of a mating pair, and most rearrangements are seen in the newly formed MACs of different inbred strains. During replication of the MAC about half the fragments bearing 5S gene clusters disappear in different cell lines, and new fragments containing 5S genes appear. These fragments differ in size from those present in the MIC or newly formed MAC. These alterations occur in the MACs of all strains except strain B, which is more resistant to vegetative rearrangement. The losses and gains of fragments occur during clonal propagation of cell lines. The process begins by 35 fissions following conjugation, but once an alteration occurs, it is stably propagated. Clonal variation occurs with respect to which losses and gains occur, although a nonrandom distribution is seen among cell clones. We conclude that the alterations in MAC fragment size occur at two stages in the life cycle of Tetrahymena. The first stage occurs during conjugation, when the MAC develops from the MIC. The second stage becomes manifest during vegetative growth, when DNA replication occurs in the MAC and daughter molecules are distributed “amitotically” to daughter nuclei. The two-stage character to MAC alterations for the 5S genes is interpreted in terms of the two steps previously described for MAC differentiation: determination and phenotypic assortment. Possible molecular mechanisms are also discussed.     

Inhibition of P-Enolpyruvate Carboxykinase and of Glyconeogenesis in Tetrahymena by 3-Mercaptopicolinic Acid*

SYNOPSIS. Tetrahymena grown overnight in deep cultures were incubated for 1 hr with [1-¹⁴C]labeled substrates in the presence or absence of 3-mercaptopicolinic acid (3-MPA). 3-MPA inhibited appearance of label in glycogen from bicarbonate, acetate, pentanoate, octanoate, and succinate, but not from glycerol or glucose. In vitro assays of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase activity showed that both enzymes were about equally distributed between the particulate and cytosol fractions. 3-MPA inhibited phosphoenolpyruvate carboxykinase from both the cytoplasmic and particulate fractions, but had no effect on phosphoenolpyruvate carboxylase from either location. These results suggest that the in vivo effects of this drug are due to inhibition of glyconeogenesis at this site.     

An Ultrastructural Investigation of 180°-Rotated Ciliary Meridians in Tetrahymena pyriformis*

SYNOPSIS. An ultrastructural investigation has been carried out on 180°-rotated ciliary meridians (inverted meridians) in Tetrahymena pyriformis temperature-sensitive mutant (mol^b/mol^b), syngen 1, strain B. The longitudinal, transverse and postciliary microtubular bands, the kinetodesmal fiber, and the parasomal sac, are shown to be disposed at a 180° angle to their normal positions or orientations. Other abnormalities are as folows: the first 2 basal bodies of the inverted meridian fail to organize into “couplets” and the inverted meridian intrudes into the anterior pole region; an extra longitudinal microtubular band is found in one of the cell lines.     

肌肉脂肪酸转运膜蛋白及其相互关系

肌肉（骨骼肌）组织对脂肪酸的利用水平是影响机体能量稳态的关键因素.肌肉摄取的长链脂肪酸(long chain fatty acids,LCFAs)主要依赖细胞膜载体蛋白协助的跨膜转运过程.近年来,一系列与脂肪酸转运相关的膜蛋白被相继克隆鉴定,其中在肌肉中大量表达的有脂肪酸转运蛋白-1（fatty acid transport protein-1,FATP-1）、膜脂肪酸结合蛋白（plasma membrane fatty acid binding protein,FABPpm）、脂肪酸转位酶（fatty acid translocase,FAT/CD36）和小窝蛋白-1（caveolin-1）.研究上述肌肉脂肪酸转运膜蛋白的结构功能、调控机制及相互关系,可能为肥胖等脂类代谢紊乱疾病的诊治提供新的手段.     

外源5-氨基乙酰丙酸对设施番茄果实发育及糖酸品质的影响北大核心CSCD

番茄果实糖酸类物质的含量及比例直接影响其风味品质,前期研究表明,适宜浓度的外源5-氨基乙酰丙酸(ALA)能够促进果实的成熟并提高其芳香品质。该试验为探究外源ALA对番茄果实发育及其糖酸品质的影响,以番茄‘原味1号’(Solanum lycopersicum cv.Yuanwei No.1)品种为试材,于第4穗果授粉后10 d果实表面喷施0、100和200 mg·L^(-1)的ALA溶液,分析ALA对番茄果实形态、果皮色泽及果实不同部位组织中糖、酸类物质组分及含量的影响。结果表明:(1)外源ALA溶液能显著促进番茄果实横径、纵径的增加,提高果实单果重,还显著降低果实硬度,促进果实软化,提升果实口感,并提高了果实V_(C)和可溶性固形物含量。(2)果实不同部位组织(包括果肉、小柱和隔膜)糖类物质组分含量测定结果显示,外源ALA处理能够显著提高果实可溶性总糖含量(包括果糖、葡萄糖和蔗糖),并有利于糖类物质向果肉中积累。(3)在有机酸类物质中,除酒石酸含量增加外,外源ALA处理均能不同程度地降低果实各部位组织中酸类物质含量,从而显著提高番茄果实果肉部位糖酸比,提升果实糖酸品质。研究发现,在番茄果实发育过程中外源施用200 mg·L^(-1) ALA不仅能够促进果实发育及着色,提高单果重,提升果实的外观品质,还有利于果实糖酸品质的形成。     

乙型肝炎病毒的核心启动子各区段功能的研究

将一系列核心启动子区的缺失突变引入乙肝病毒（ＨＢＶ）线性转录单元,从病毒的抗原,ＲＮＡ以及子代ＤＮＡ等各个水平,分析了各缺失突变对前基因组ＲＮＡ和前核心ＲＮＡ转录的影响,对核心启动子各片段的功能进行了深入的研究。Ｃ片段缺失后检测不到ｅ抗原和前核心ＲＮＡ,却仍有核心抗原和前基因组ＲＮＡ的合成以及病毒子代ＤＮＡ的复制;而Ｂ３片段缺失后ｅ抗原和核心抗原均有显著下降,但仍能检测到两种ｍＲＮＡ的合成和病毒子     

溶血磷脂酸在皮肤损伤疾病中的作用及其分子机制

皮肤作为人体最大器官覆盖于全身,能阻挡有害物质的侵入,保护人体内环境稳态,参与人体代谢过程。皮肤损伤、炎症和纤维化等,都会导致皮肤屏障功能的减退,影响正常的生命活动。溶血磷脂酸(lysophosphatidic acid,LPA)是十分活跃的磷脂信号分子,参与多种生理和病理生理过程。LPA是维持体内平衡所必需的生物活性脂质介质,在皮肤中通过不同的信号通路发挥多功能磷脂信使作用。本文综述了皮肤中溶血磷脂酸受体(lysophosphatidic acid receptor,LPA1-6)及其细胞信号通路的作用及机制,综述了LPA在皮肤创面愈合、皮肤瘢痕、皮肤黑色素瘤、硬皮病、皮肤瘙痒、过敏性皮炎、皮肤屏障、皮肤疼痛,皮肤毛发生长中的作用及分子机制,有助于了解LPA在皮肤中的生理和病理生理作用。深入研究LPA的作用机制将有助于挖掘其在皮肤治疗中的作用,开发以LPA为靶点的药物。     

Effects of Cold Acclimation and Salicylic Acid on Changes in ACC and MACC Contents in Maize during Chilling

The effect of 0.5 mM salicylic acid (SA) pretreatment and of growing at hardening temperatures on chilling-induced changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) was investigated in young maize (Zea mays L.) plants grown in hydroponic solution at 22/20 °C. Chilling at 5 °C caused an increase in ACC content;however, this increase was less pronounced in plants cold acclimated at 13/11 °C 4 d before the chilling treatment, and in those which were pretreated with SA for 1 d before the cold stress. Changes in MACC at low temperature showed no correlation with chilling tolerance in maize.     

人参皂苷Rg1对游离脂肪酸诱导非酒精性脂肪肝细胞炎症的改善作用及机制研究

该研究探讨人参皂苷Rg1对非酒精性脂肪性肝细胞炎症反应的作用及其分子机制。用1 mmol/L游离脂肪酸处理HepG2和L02细胞24 h,再用20μg/mL或40μg/mL人参皂苷Rg1处理6 h;设置对照组、模型组、低剂量Rg1组、高剂量Rg1组。全自动生化仪检测各组细胞上清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)的含量;酶联免疫吸附法测定细胞上清IL-1β、IL-6、TNF-α。RT-qPCR及Western blot检测NF-κB通路相关基因及蛋白的改变。免疫荧光染色观察NF-κB核转移;Western blot检测各组胞质与胞核内的NF-κB P65蛋白的表达。与对照组相比,模型组培养上清炎症指标明显增加(P<0.05);Rg1能降低炎症指标的表达(P<0.05)。Rg1能减少游离脂肪酸诱导的NF-κB磷酸化及其下游IL-1β、IL-6、TNF-α的表达,减少NF-κB P65从胞质向胞核的转移(P<0.05)。Rg1可通过抑制NF-κB活化减少NASH细胞模型炎症反应,为非酒精性脂肪性肝炎的治疗提供了可能的靶点。     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.