落叶松树皮原花青素提取工艺的研究

本文考察了落叶松树皮原花青素的料液比、乙醇浓度、提取温度、超声时间对原花青素提取效果的影响。采用正交实验,对提取工艺进行了优化。结果表明各因素对原花青素的提取效果的影响程度为提取温度>提取时间>乙醇浓度>料液比。从而确定落叶松树皮中原花青素的最佳工艺条件为:提取温度为35℃,提取时间为0.5 h,乙醇浓度为50%,料液比为1∶15(g/mL)。     

蕨菜多糖超声波辅助提取及其药理活性初步研究

为优化蕨菜多糖的提取工艺,同时检测蕨菜多糖的药理学活性。实验采用超声波辅助提取法,在单因素试验的基础上,考察液料比、浸提次数、超声浸提时间、超声功率四因素对蕨菜多糖提取得率的影响。运用Design Expect 10.0软件分析,通过响应面分析法(RSM)优化提取条件,对蕨菜多糖促进小鼠脾细胞增殖能力和抑制结肠癌细胞(HTC-8)增殖能力进行分析。结果表明:蕨菜多糖的优化提取工艺为:液料比:36.2∶1;浸提次数:4次;超声浸提时间:43.1 min;超声波功率:240 W,在此条件下,蕨菜多糖提取效果最好,提取得率达8.60%。各因素对多糖提取得率的影响程度:浸提次数>液料比>超声浸提时间>超声功率。通过药理活性研究表明,本实验获得的蕨菜多糖具有脾细胞免疫增值和抑制结肠癌细胞增殖活性。     

纤维素酶提取楮实子多酚及其抗氧化性研究

以楮实子为原料,采用纤维素酶法研究楮实子多酚的提取工艺,以单因素实验(纤维素酶含量、浸提时间、液料比、浸提温度和p H)为依据,选取浸提时间、液料比、浸提温度和pH等4因素,以楮实子多酚提取率为指标,通过4因素3水平的Box-Behnken实验设计,优化了楮实子多酚提取的工艺参数,结果表明:浸提时间150 min、液料比35∶1 mL/g、浸提温度70℃、pH6.10,楮实子多酚提取率达到39.67 mg/g。并通过2种体外抗氧化实验(DPPH自由基清除力和铁氰化钾还原法),证明楮实子多酚具有较强的抗氧化活性。     

离子液体超声辅助提取桑葚中原花青素

采用离子液体超声辅助提取桑葚中原花青素,在单因素实验基础上,采用响应面优化[Bmim]Cl(1-丁基-3-甲基咪唑氯)浓度、料液比、超声功率、超声时间对桑葚中原花青素得率的影响,利用扫描电镜分析了该技术提取效果较好的原因。结果表明,桑葚中原花青素的最佳提取工艺为:[Bmim]Cl浓度为0.73 mol/L、料液比1∶21(g/mL)、超声功率188 W、超声时间11 min,此条件下原花青素得率可达3.51%,与模型预测值接近。扫描电镜观察发现,离子液体超声对桑葚粉末微观结构破坏影响较大。     

苹果渣中原花青素的提取分离条件的优化

采用单因素和正交试验优化了溶剂直接提取及微波辅助提取苹果渣中原花青素的提取条件。结果表明:溶剂直接提取苹果渣中原花青素的最佳提取条件为乙醇体积分数30%,料液比(g/mL)1∶12,浸提温度90℃,提取时间0.5h;微波辅助提取法提取苹果渣中原花青素的最佳提取条件为乙醇体积分数50%,料液比(g/mL)1∶9,微波功率为700W,提取时间3min。与溶剂直接提取法相比,微波辅助提取法能更省时、高效地提取苹果渣中的原花青素。     

响应面法优化毒三素链霉菌中利普司他汀的提取工艺

采用响应面法对毒三素链霉菌提取工艺进行优化。首先确立乙醇为最佳的溶剂。以利普司他汀提取收率为指标,以乙醇为浸提溶剂,对料液比、乙醇体积分数、浸提时间、浸提温度和浸提次数进行单因素实验。在此基础上,选取料液比、乙醇体积分数、浸提时间为自变量,采用Box-Behnken设计的方法,研究各自变量及其交互作用对利普司他汀提取收率的影响。结果表明:毒三素链霉菌中利普司他汀的最佳提取工艺条件为液料比6.32∶1 m L/g、乙醇体积分数95%、浸提时间3.61 h。在此条件下,利普司他汀提取收率的预测值为83.35%,实验验证值为84.50%,相对误差为1.36%。     

桑黄菌丝体粗多糖的提取方法研究

采用正交试验设计,以桑黄菌丝体粗多糖含量为考察指标,用苯酚—硫酸法,分别确定了热水浸提法、微波辅助提取法和超声提取法的最佳工艺。通过极差分析得出:热水浸提法的最优工艺为浸提时间3 h、浸提3次、液料质量比50∶1、浸提温度90℃,粗多糖提取率为2.10%;微波提取法的最优工艺为微波处理15 min、液料质量比50∶1、提取3次,提取率为4.18%;超声提取法的最优工艺为超声30 min、提取2次、液料质量比60∶1、温度60℃、频率60 Hz,提取率为3.02%。微波辅助法与热水浸提法相比,时间缩短,且提取率提高近1倍;与超声提取法相比,时间缩短1/2,但提取率提高40%。因此,微波辅助提取法速度更快、提取效率更高、操作更简便,优于其他2种方法。     

响应曲面法优化方格星虫多糖提取工艺

为了优化提取方格星虫多糖工艺实验条件,本研究以海南三亚海域方格星虫为主要原料,用胰蛋白酶作为酶解酶,在单因素实验基础上,采取响应面法研究超声波辅助酶法提取方格星虫多糖最佳工艺条件;探讨了p H值、液料比、超声波时间、酶底比、超声波温度、超声波功率、反应时间等7个因素的交互作用及其最佳水平。结果表明在超声波辅助酶法提取方格星虫多糖的实验过程中,单因素的最佳条件酶底比为2.5%、温度为50℃、浸提时间为2 h、料液比为1:17 g/m L、超声时间为1 h、pH值为8、超声功率为960 W,多糖的最大提取率为3.24%。该方法实验条件要求不苛刻,浸提时间短,提取率高,是一项新的实验尝试,实验结果为优化方格星虫的多糖提取理论参考。     

山楂果中原花青素提取工艺研究

优选回流提取方法,以山楂果中原花青素含量为考察指标,研究提取溶剂种类、浓度、提取温度、料液比、提取时间、提取次数等因素对原花青素提取效果的影响。通过正交实验,确定山楂果中原花青素的最佳提取工艺条件为:提取溶剂70%乙醇,提取温度80℃,料液比1∶15,提取时间2 h,提取3次。     

超声辅助提取凤丹籽总黄酮的工艺研究

本文以经脱脂处理后的凤丹籽为原料,在单因素实验的基础上,选取乙醇浓度、料液比、超声时间和超声功率为自变量,以总黄酮得率为响应值,采用Box-Behnken响应面设计分析方法优化凤丹籽总黄酮的提取工艺。通过此模型得出:超声时间对凤丹籽总黄酮提取的影响最为显著,超声时间和乙醇浓度,超声时间和料液比,超声时间和超声功率之间的交互作用显著。模型预计凤丹籽总黄酮提取的最佳工艺条件为:乙醇浓度75%,料液比1:21 g/mL,超声时间49 min,超声功率240 w,在此条件下,总黄酮得率平均值为9.015 mg/g。     

Cell separation with horizontal cell electrophoresis under near-isopycnic conditions on a “density cushion”

This report describes an improvement made to the horizontal cell electrophoresis methodology. It involves using two liquid layers differing in density to produce an interface described as a "density cushion". The electrophoretic system that employed an anti-convective porous matrix to separate red blood cells (RBC) and charged dyes effectively was found to be unsuitable for some other mammalian cells. The "density cushion" method was found to be more versatile and applicable to studies on the separation of a variety of cell types. The experiments described show the differences between the electrophoretic mobilities of a human eosinophilic leukaemia cell line (Eol-1) and RBC, both with and without the modification of the cell surface properties.     

Cell electrophoresis — a method for cell separation and research into cell surface properties

In this paper, we discuss the application of various methods of cell electrophoresis in research into cell surface properties (analytical methods), and the separation of uniform cell subpopulations from cell mixtures (preparative methods). The emphasis is on the prospects of the development of simplified and versatile methodologies, i.e. microcapillary cell electrophoresis and horizontal cell electrophoresis under near-isopycnic conditions. New perspectives are considered on the use of analytical and preparative cell electrophoresis in research on cell differentiation, neoplastic transformation, cell-cell interactions and the biology of stem cells. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication cost was covered by the organisers of this meeting.     

Polarization and Migration of Hematopoietic Stem and Progenitor Cells Rely on the RhoA/ROCK I Pathway and an Active Reorganization of the Microtubule Network

Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34⁺ progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.     

Expanisns

Biochemical dissection of the “acid-growth” process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril-matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit, meristerms and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.     

酸性磷酸酶法检测体外培养细胞数

利用小鼠成纤维细胞系(NIH3T3)、小鼠骨髓瘤细胞系(SP2/0)、人大肠癌细胞系(LO-VO)和人白血病细胞系(K562),评价酸性磷酸酶(APA)法用于检测体外各类型细胞的增殖和杀伤作用。用直线回归分析光吸收度与每孔活细胞数的关系。结果表明,APA法能准确地反映检测的活细胞数(相关系数均>0.99)。本方法不仅能很好地检测表皮生长因子对细胞的增殖作用,也能够检测顺铂对体外细胞的杀伤作用。结果表明APA法简单、灵敏,可以用于上皮和间质等贴壁和悬浮生长的细胞计数。     

An overview of the KIN1/PAR-1/MARK kinase family

Members of the KIN1/PAR-1/MARK kinase family are conserved from yeast to humans and share a similar primary structural organization. Several kinases of this family appear to be at the crossroads of various biological functions including cell polarity, cell cycle control, intracellular signalisation, microtubules stability and protein stability. Here we present an overview of known roles of KIN1/PAR-1/MARK kinases including pEg3 a newly identified member which is regulated during the cell cycle and is a potential regulator of the cell cycle progression. Some common modes of action can be deciphered for this protein kinase family.     

犬皮肤成纤维细胞的分离、培养及鉴定

目的探索和建立适用于犬皮肤成纤维细胞的体外分离、培养及鉴定的技术方法。方法采用组织贴块培养法和胰蛋白酶、胶原酶Ⅰ联合消化法对犬皮肤成纤维细胞进行体外培养、传代。并对所培养的细胞进行倒置显微镜观察和苏木素-伊红染色,观察成纤维细胞形态,并对培养细胞行波形蛋白免疫荧光染色。结果倒置相差显微镜下可见长梭形细胞生长,苏木素-伊红染色可见细胞呈漩涡状、平行排列,第5代细胞免疫荧光检测波形蛋白(vimentin)表达阳性。结论建立了高效快速分离和稳定培养成纤维细胞的方法,为诱导犬心房纤维化提供了充足的种子细胞。     

The plant cell body: a cytoskeletal tool for cellular development and morphogenesis

Summary Certain aspects of cellular behaviour in relation to growth and development of plants can be understood in terms of the cell body concept proposed by Daniel Mazia in 1993. During the interphase of the mitotic cell cycle, the plant cell body is held to consist of a nucleus and a perinuclear microtubule-organizing centre from which microtubules radiate into the cytoplasm. During mitosis and cytokinesis in meristematic cells, and also during the period of growth in post-mitotic cells immediately beyond the meristem, the plant cell body undergoes various characteristic morphological transformations, many of which are proposed as being related to changing structural connections with the actin-based component of the cytoskeleton and with specialized, plasma-membrane-associated sites at the cell periphery. In post-mitotic cells, these transformations of the plant cell body coincide with, and probably provide conditions for, the various pathways of development which such cells follow. They are also responsible, for the acquisition of new cellular polarities. Events in which the plant cell body participates include the formation of a mitotic spindle, phragmoplast, and new cell division wall, the rearrangement of a diffuse type of cell wall growth into tip growth (as occurs, e.g., during the initiation and subsequent development of root hairs), and the growth and division that occurs in reactivated vacuolate cells. If more evidence can be marshalled in support of the existence and properties of the plant cell body, then this concept could prove useful in interpreting the cytological bases of a range of developmental events in plants.Abbreviations CMT cortical microtubule - EMT endoplasmic microtubule - ER endoplasmic reticulum - MF microfilament - MT microtubule - MTOC microtubule-organizing centre - PPB preprophase band (of microtubules) - QC quiescent centre - VSC vesicle supply centre     

A Scalable Approach to Prevent Teratoma Formation of Human Embryonic Stem Cells

As the renewable source of all cell types in the body, human embryonic stem cells (hESCs) hold great promise for human cell therapy. However, one major bottleneck that hinders the clinic application of hESCs is that hESCs remaining with their differentiated derivatives pose cancer risk by forming teratomas after transplantation. NANOG is a critical pluripotency factor specifically expressed in hESCs but rarely in their differentiated derivatives. By introducing a hyperactive variant of herpes simplex virus thymidine kinase gene into the 3′-untranslated region of the endogenous NANOG gene of hESCs through homologous recombination, we developed a safe and highly scalable approach to efficiently eliminate the teratoma risk associated with hESCs without apparent negative impact on their differentiated cell types. As thymidine kinase is widely used in human gene therapy trials and is the therapeutic target of U. S. Food and Drug Administration-approved drugs, our strategy could be effectively applied to the clinic development of hESC-based human cell therapy.     

Molecular basis for T cell response induced by altered peptide ligand of type II collagen

Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.