嘎拉苹果果实质地发育软化与细胞壁降解及其基因表达的关系

以嘎拉苹果为试材,分析了果实发育软化过程细胞壁组分、细胞壁酶活性及其酶基因表达的变化。结果表明,发育期,细胞壁物质(CWM)及其组分均先升后降,果胶中共价结合果胶(CSP)含量最高,纤维素含量远高于半纤维素,此期CSP和纤维素含量与硬度显著相关,细胞壁酶表现不同的活性变化和基因表达,并与细胞壁组分存在一定相关性,表明细胞壁降解参与了果实发育期的相关生理过程。采后CSP含量快速降低,水溶性果胶(WSP)含量开始增加,纤维素和半纤维素含量降低,均与硬度显著或极显著相关;细胞壁酶中,β-Gal活性和基因表达量增幅最快,α-Af和PG次之,PME活性和基因表达的增加时期相对滞后,且β-Gal和α-Af活性与硬度和各细胞壁组分的相关性强于PG和PME,PG与CSP和半纤维素表现较强相关性,而在PME上的相关性最差,说明细胞壁代谢与嘎拉果实软化密切相关,β-Gal和α-Af可能对果实软化的作用更强。     

一氧化氮对常温贮藏下芒果果实软化和细胞壁代谢的影响

为了探明一氧化氮(NO)抑制采后芒果软化的作用机理,将"台农"芒果果实在0.25 mmol/L硝普钠(SNP,NO供体)溶液浸泡处理20 min,常温(20±2) ℃贮藏20 d,定期测定果实硬度、细胞壁组分含量、细胞壁水解酶活性。结果表明,与未处理果实相比,SNP处理显著降低贮藏20 d内果实中多聚半乳糖醛酸酶(PG)活性(p<0.05),显著抑制贮藏10 d内果实纤维素酶(CX)(p<0.05)活性,极显著抑制β-半乳糖苷酶(β-Gal)和α-L-阿拉伯呋喃糖苷酶(α-L-Af)活性(p<0.01),但使贮藏15~20 d期间果实CX和β-Gal活性及贮藏第20 d的α-L-Af活性均显著增加(p<0.05)。SNP处理显著抑制贮藏5 d内果胶甲酯酶(PME)活性(p<0.05),但在贮藏10~20 d期间保持较高的PME活性(p<0.05)。此外,SNP处理极显著延缓原果胶和纤维素的降解(p<0.01),减少可溶性果胶含量的增加(p<0.05),从而降低贮藏期间果实硬度的损失。硬度与原果胶、纤维素含量均呈极显著正相关(p<0.01),而与CX活性呈显著负相关(p<0.05),与可溶性果胶含量、PG、β-Gal和α-L-Af活性均呈极显著负相关(p<0.01)。可溶性果胶含量与纤维素含量呈极显著负相关(p<0.01),而与α-L-Af活性均呈显著正相关(p<0.05),与PG和β-Gal活性均呈极显著正相关(p<0.01)。因此,采后SNP处理可以通过调节果实细胞壁降解酶活性,减少细胞壁组分的降解,从而延缓芒果采后软化,延长贮藏期。     

不同成熟度橄榄果实冷藏期间细胞壁代谢对采后冷害的响应特性

研究冷藏期间橄榄果实细胞壁代谢的变化,探讨不同成熟度橄榄果实冷害发生与细胞壁组分含量、细胞 壁降解酶活性的关系。以白露、寒露、立冬、大雪节气时采摘的‘檀香’橄榄果实为材料,在温度（2±1）℃、 相对湿度85%～90%冷库内贮藏,定期测定橄榄果实冷害指数、果肉细胞壁组分含量和细胞壁降解酶活力的变化。 结果表明,不同成熟度橄榄果实冷藏期间冷害发生与其细胞壁组分降解密切相关,冷害指数与离子结合型果胶 （ionic-soluble pectin,ISP）、共价结合型果胶（covalent-soluble pectin,CSP）、半纤维素和纤维素含量呈负相 关;且果胶甲酯酶（pectin methylesterase,PME）、多聚半乳糖醛酸酶（polygalacturonase,PG）、β-半乳糖苷酶 （β-galactosidase,β-Gal）和纤维素酶（cellulase,CEL）等细胞壁降解酶的活力变化不平衡或活力提高是导致冷藏 橄榄果实细胞壁结构解体、细胞壁代谢异常、冷害发生的主要原因。同时,与成熟度Ⅰ、Ⅲ和Ⅶ的橄榄果实相比, 成熟度Ⅴ保持较低的果实冷害指数及冷藏中后期果肉PME、PG、β-Gal和CEL活力,延缓冷藏中后期果肉水溶性果 胶、ISP、CSP、半纤维素和纤维素含量降低。因此认为,成熟度Ⅴ的橄榄果实可较好维持细胞壁结构的完整性, 有效减轻冷害发生。     

茄子果实采后软化过程中细胞壁组分及其降解酶活性的变化

研究了细胞壁组分及其降解酶活性的变化与茄子果实采后软化的关系。结果表明,采后茄子果肉硬度随贮藏时间的延长而不断下降。贮藏期间果肉水溶性果胶(WSP)含量在贮藏前12天不断增加,之后快速下降,而共价结合型果胶(CSP)、半纤维素和纤维素等细胞壁组分含量持续减少。果肉果胶甲酯酶(PME)、多聚半乳糖醛酸酶(PG)和纤维素酶(CX)活性均呈先升高后下降趋势,分别在贮藏至第6、9、12天达到最大值;β-半乳糖苷酶(β-Gal)活性始终保持较高水平,且在整个贮藏期间活性变化不明显。相关性分析结果表明,CSP、半纤维素和纤维素的降解与采后茄子果实软化密切相关,PG和CX在茄子果实采后软化过程中起着重要的作用。     

微环境气调对蓝莓贮藏期软化的调控作用

为探讨微环境气调对蓝莓贮藏期果实软化的影响,采用自发气调(mMAP)、1-甲基环丙烯(1-MCP)、微环境气调(mMAP+1-MCP)处理蓝莓,以未经处理的蓝莓为对照。将处理过的蓝莓置冰温库(-0.5±0.3) ℃贮藏,分别于贮藏0,20,40 d和60 d时测定果实硬度、细胞壁多糖含量、细胞壁降解酶活性和关键降解酶基因表达量。结果表明:与对照组相比,3个处理组均能显著降低果实的软果率(P < 0.05),其中mMAP+1-MCP处理效果最佳。贮藏60 d时果实硬度显著高于其它处理组(P < 0.05),维持较高的纤维素和原果胶含量,半纤维素在贮藏前期高于其它处理,而可溶性果胶含量在贮藏中前期保持较低水平。分析细胞壁降解酶,mMAP+1-MCP处理组贮藏40 d时纤维素酶(Cx)、多聚半乳糖醛酸酶(PG)和果胶甲酯酶(PME)活性最低,贮藏60 d时β-半乳糖苷酶(β-Gal)和α-L-阿拉伯呋喃糖苷酶(α-Af)活性显著低于其它处理组(P < 0.05)。正交偏最小二乘判别分析(OPLS-DA)表明,mMAP+1-MCP与其它处理组差异性指标为Cx和β-Gal活性。对这两种细胞壁降解酶基因的表达分析结果:mMAP+1-MCP处理可有效延缓蓝莓贮藏过程中Cx和β-Gal基因表达量峰值的出现时间。结论:mMAP+1-MCP形成的微环境气调环境通过延缓Cx和β-Gal基因表达量出峰时间,抑制贮藏后期Cx和β-Gal活性,保持果实的纤维素和原果胶含量,进而减缓果实的软化。     

高能电子束辐照对猕猴桃细胞壁降解相关酶活性和基因表达的影响

以‘海沃德’猕猴桃为试材,经剂量0（对照）、300、400和500 Gy高能电子束辐照后,于0~1 ℃、RH 90%~95%冷库中贮藏90 d,研究电子束辐照对果实硬度、细胞壁组分、软化相关酶活性及其基因表达量的影响。结果表明:高能电子束辐照显著维持了果实的硬度,有效抑制了细胞壁骨架物质原果胶和纤维素的分解,延迟了果实后熟软化。同时,辐照抑制了多聚半乳糖醛酸酶（polygalacturonase,PG）、果胶甲酯酶（pectin methylesterase,PME）、β-半乳糖苷酶（β-D-galaetosidase,β-Gal）和纤维素酶（cellulase,Cx）的活性,降低了PG、PME、β-Gal和Cx编码基因的表达。综合认为,以400 Gy高能电子束辐照对抑制细胞壁降解相关酶活性及基因表达,保持细胞结构的完整性,维持贮藏期间果实硬度效果最好。研究结果为高能电子束用于猕猴桃采后保鲜提供理论依据。     

60Co-γ辐照对冷藏蓝莓果实软化相关指标的影响

为探讨辐照对蓝莓果实硬度的影响机制,本研究围绕不同辐照剂量对冷藏蓝莓腐烂率、失重率、果实硬度、细胞壁物质、细胞壁多糖含量以及细胞壁水解酶活性的影响展开系统的研究。结果发现较低剂量的辐照处理（0.5 kGy）对蓝莓冷藏保鲜的效果不显著,较高剂量的辐照剂量（3.0 kGy）加速了冷藏后期蓝莓果实的软化进程,2.5 kGy辐照处理能够降低冷藏期间蓝莓果实中PE（果胶甲酯酶）、PG（多聚半乳糖醛酸酶）、Cx（纤维素酶）三种水解酶的活性,抑制WSP（水溶性果胶）含量升高和ESP（离子型果胶）、SCSP（共价型果胶）、4KSF（连接松散半纤维素）、24KSF（连接紧密半纤维素）含量的下降,从而降低果胶不溶性向可溶性的转变和保证半纤维素的连接作用,最终有效延缓冷藏蓝莓果实软化进程。     

低温贮藏期间‘长富2’果实细胞壁酶及基因表达的变化

以‘长富2’苹果为实验材料,测定冷藏(1~4℃)期间果实多聚半乳糖醛酸酶(poly galacturonase,PG)、β-半乳糖苷酶(β-Gal)活性及基因表达变化,同时分析果实硬度、可溶性固形物(soluble solid content,SSC)、可滴定酸和还原糖含量。结果表明,果实的硬度和可滴定酸持续下降且变化显著;SSC和还原糖含量在贮藏前期有少量增加后缓慢下降;PG活性及Md PG1、Md PG2表达量均增加,PG活性和Md PG2表达量均在120 d达到峰值,Md PG1表达量在60 d达到最大值。β-Gal活性持续增加,与果实硬度呈显著负相关;Md GAL1和Md GAL2表达量在60 d之前增加,60 d以后迅速下降。总体上,PG和β-Gal基因表达先于酶活增加,对两种酶活性变化有重要作用。     

外源草酸对采后‘蜂糖李’果实软化和细胞壁降解的影响

为探讨外源草酸处理对采后李果实软化进程的影响,以‘蜂糖李’果实为试材,采用5 mmol/L草酸(oxalic acid, OA)溶液浸泡处理10 min,以蒸馏水浸泡处理10 min为对照(CK),自然晾干后置于室温(25±1)℃条件下贮藏20 d。分析果实硬度、纤维素、原果胶和可溶性果胶含量的变化以及多聚半乳糖醛酸酶(polygalacturonase, PG)、果胶甲酯酶(pectin methylesterase, PME)、纤维素酶(cellulase, Cx)、β-半乳糖苷酶(β-galactosidase, β-Gal)、α-L-阿拉伯呋喃糖苷酶(α-L-arabinofuranosidase, α-L-Af)和木葡聚糖内糖基转移酶(xyloglucan endotransglucosylase, XET)活性。结果表明,外源OA处理能使‘蜂糖李’果实保持较高的硬度,延缓原果胶和纤维素含量的下降以及可溶性果胶含量的增加,抑制果实PG、PME、Cx、β-Gal、α-L-Af和XET活性上升。果实硬度与原果胶、纤维素、可溶性果胶含量、Cx、β-Gal、XET和α-L-Af活性均...     

采前氯吡脲处理对‘秦美’猕猴桃贮藏期间果实硬度及细胞壁降解的影响

以‘秦美’猕猴桃果实为试材,于盛花期后28 d分别采用0（对照,清水）、5、10、20 mg/L 4 个质量浓度的氯吡脲（1-(2-chloropyridin-4-yl)-3-phenylurea,CPPU）溶液进行蘸果处理,蘸果时间3～5 s,研究采前CPPU处理对‘秦美’猕猴桃贮藏期间果实硬度及细胞壁降解酶活力的影响。结果表明：CPPU处理加速了果实硬度、原果胶和纤维素质量分数的下降,提高了可溶性果胶质量分数及多聚半乳糖醛酸酶（polygalacturonase,PG）、果胶甲酯酶（pectin methylesterase,PME）、纤维素酶（cellulase,Cx）和β-半乳糖苷酶（β-D-galaetosidase,β-Gal）细胞壁降解活力。各处理组果实硬度与可溶性果胶质量分数和PG、Cx活力呈极显著负相关（P＜0.01）,与原果胶、纤维素质量分数呈极显著正相关（P＜0.01）;20 mg/L CPPU处理组果实的β-Gal活力与硬度呈显著负相关（P＜0.05）。CPPU处理提高了果实细胞壁降解酶的活力,促进了细胞壁的降解,加速了贮藏期间果实的软化,降低了果实的耐贮藏性。为维持猕猴桃采后果实硬度,延长贮藏期,生产中不宜使用CPPU处理,或使用的质量浓度不宜超过5 mg/L。     

芹菜素对甲状腺癌BCPAP细胞生长及细胞周期的影响

研究芹菜素对人乳头状甲状腺癌BCPAP细胞生长的抑制作用及对细胞周期的影响。采用MTT法检测不同浓度芹菜素在24h对BCPAP细胞的抑制作用,以及12.5,25.0,50.0μmol/L芹菜素分别在24,48,72h对BCPAP细胞的抑制作用;通过明场细胞形态学照片分析,对比不同浓度芹菜素对BCPAP细胞形态的影响,评价其对细胞生长的抑制作用。利用流式细胞仪检测BCPAP细胞周期和凋亡。结果表明,不同浓度(12.5~100.0μmol/L)芹菜素对BCPAP细胞的毒性有明显剂量和时间依赖性,其24h的IC50值为40.65μmol/L。芹菜素对BCPAP细胞的形态学变化有显著影响,高浓度芹菜素强烈抑制BCPAP细胞数目的增长。芹菜素可使BCPAP细胞周期的构成发生明显的变化并诱导细胞凋亡。芹菜素对BCPAP细胞有较明显的细胞毒性和生长抑制作用,其抑制机制可能是使BCPAP细胞生长停滞在G2/M期并诱导细胞凋亡,使得细胞生存率下降,从而抑制细胞活性和数目增长。     

Fundamentals of cell proliferation: control of the cell cycle

Cell proliferation in higher eukaryotes is controlled by the extracellular environment and the state of differentiation. Many cells exist in a nondividing growth state termed quiescence. Some quiescent cells cannot proliferate and are said to be terminally differentiated. Others can be stimulated to divide in response to environmental signals or when cell replacement is needed. Finally, some cells undergo continual proliferation and differentiation. Growth regulatory factors generally act at specific stages of the cell cycle, most commonly during the first gap phase of the cell cycle. Once cells initiate DNA synthesis, they are generally committed to complete DNA replication. After DNA synthesis, additional signals determine whether cells in the last gap phase proceed through mitosis. In recent years, genes that appear to be critical for progression through the first two gap phases have been identified. Many are proto-oncogenes and therefore can neoplastically transform certain cells when mutated or inappropriately expressed. Growth factors that stimulate proliferation induce the expression of several proto-oncogenes; growth inhibitory factors often suppress proto-oncogene expression. As cells differentiate, the response to extracellular factors changes. In many cases, this may be due to intracellular controls that alter the response of certain proto-oncogenes to external signals.     

Composition of cell walls isolated from cell types of grain sorghum stems

Cell types were isolated from sorghum stems at two stages of development, anthesis and grain maturity, to study cell wall characteristics. Cell walls were isolated from epidermis (EPID), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) and analysed for total carbohydrate, acid insoluble lignin, total uronosyls, neutral sugars and hydroxycinnamic acids. In addition, walls from SCL, VBZ, IVB and PITH were subjected to chemical fractionation to separate wall carbohydrate into polysaccharide groups. Although wall characteristics were similar at both plant maturities, there were differences in lignin concentration, hydroxycinnamic acids, and carbohydrate composition among the cell wall types. Lignin was lowest in the PITH walls (169 g kg⁻¹) and highest in SCL and EPID (c 211 g kg⁻¹). Cellulose was most abundant in VBZ and SCL walls with greater secondary wall formation. Pectic materials were most abundant in PITH walls. Xylans were similar among wall types except for EPID that contained higher amounts of xylose. Releasable hydroxycinnamates were not as consistent among the cell wall types. Total ferulates, including ester linked and releasable ether linked, tended to increase from PITH to SCL (8 to 15 g kg⁻¹ CW) with an increase in the proportion etherified within the wall matrices (PITH 51%; SCL 66%). Total p‐coumarates showed opposite trends with PITH walls having significantly more (35 g kg⁻¹ CW) than VBZ or SCL (19 and 13 g kg⁻¹ CW). EPID walls contained the least pCA (6.5 g kg⁻¹ CW). Except for the hydroxycinnamates, compositional trends for the different wall types would reflect changes from primary walls to increased amounts of secondary wall. Neutral sugar analysis of indigestible residues indicated similar carbohydrate compositions among the cell wall types, with xylose being less degradable than all other wall sugars. © 1999 Society of Chemical Industry     

单细胞蛋白的利用

概括了单细胞的定义、种类、来源、生产工艺及其在饲料和食品工业中的应用;阐明了单细胞蛋白开发和生产的前景。     

单细胞蛋白的利用

概括了单细胞的定义、种类、来源、生产工艺及其在饲料和食品工业中的应用；阐明了单细胞蛋白开发和生产的前景。     

Practical cell labeling with magnetite cationic liposomes for cell manipulation

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10³–10⁵ cells for personalized cell processing, we determined that 10 μg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method.     

Single cell reporter assay using cell surface displayed Vargula luciferase

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.     

Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

The effects of bovine cumulus cell-conditioned medium on cloned bovine embryonic development and subsequent chromosome complement were examined using an air-dry procedure. Conditioned media were prepared using CR1aa supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). Nuclear-transferred embryos were reconstructed with nuclei from cumulus cells. Similar cleavage, morula, and blastocyst development was observed in conditioned media groups compared with the co-culture group. No differences (P > 0.05) were observed in the composition of blastocyst chromosomes after co-culture in different media, either with or without starvation of donor cells. The overall diploid blastocyst rate ranged from 75% to 84%. Chromosomal complement of blastocysts, however, was very different between conditioned medium and co-culture treatments. Overall incidence of chromosomal anomalies was 40% in conditioned medium, which was significantly higher (P < 0.001) than the co-culture group (20%). Moreover, a higher incidence (P < 0.05) of chromosomally abnormal blastocysts (41.5%) was observed after culture with FBS-containing conditioned medium than those cultured in BSA-containing conditioned medium (31.4%). No diploid improvement was observed after exchange of the culture system from conditioned medium to co-culture, or from co-culture to conditioned medium after the first 72 h of culture. The results of this study also indicated that the overall cell number was much lower (P < 0.01) in blastocysts with chromosomal abnormalities than those with a normal diploid state. We have concluded that medium conditioned with bovine cumulus cells increases the incidence of chromosomal anomalies in nuclear reconstructed embryos.