猪传染性水疱病病毒(SVDV)在微细胞和微胞质体内的复制

本实验研究了SVDV在微细胞和微胞质体两种非完整细胞体系内的复制。用秋水仙素处理IB-RS-2细胞,在含有松胞素B的Ficoll 400不连续梯度中进行高速离心,制备了大量的微细胞。微细胞在正常的培养条件下60小时后仍有70%以上存活,用SVDV感染只需4—5小时就几乎全部脱落。并证明微细胞能繁殖出有感染力的子代病毒。用松胞素B去核技术制备的微胞质体,运用扫描电镜和显微放射自显影的方法证实了SVDV可以在微胞质体内复制病毒RNA,并引起微胞质体病变。同时纯化了用松胞素B处理悬浮的IB-RS-2细胞而制备的大量的微胞质体,SVDV感染后,可以产生一定数量的有感染力的子代病毒。虽然滴度低于完整细胞繁殖的病毒,但证明了SVDV可以利用微胞质体中少量的细胞成分和酶类完成其感染、复制和装配一系列繁殖过程,产生有感染力的子代病毒。     

松胞素B和离心术并用制备体外培养哺乳类细胞的核体与胞质体

Carter于1967年发现,松胞素B(Cytochalasin B),简称CB,能诱发体外培养哺乳类细胞发生排核作用。嗣后,     

凝集素与细胞凝集 Ⅱ细胞膜在凝集反应中的作用

凝集素对细胞的凝集作用可被秋水仙硷、长春新硷和松胞素B所抑制;已知前两种药物能破坏微管,而松胞素B可分解微丝。因此认为凝集作用是和微丝、微管相关。此外,用荧光素、酶、铁蛋白或血蓝蛋白标记的凝集素与细胞作用,发现凝集素在正常细胞表面是随机分布的;在肿瘤细胞表面则是成簇分布的。许多实验证明细胞表面受体的成簇或成帽需要有完整的微丝及微管。这就是说凝集素在微丝和微管的作用下,使表面受体成簇,造成局部受体的浓聚,终于发生细胞凝集。这些观察结     

胞松素B对黑麦花粉母细胞发育的影响

采用花药离体培养研究了胞松素B对黑麦花粉母细胞的发育及显微和亚显微结构的影响。细线期和偶线期接种的花药在含有5—40μmol/L胞松素B的White培养基上培养24小时以后,部分花药不能进一步发育,被抑制在偶线期阶段,并且偶线期花药的百分率随胞松素B浓度的增加而增加。在被抑制的偶线期的花药中,多数花药的花粉母细胞被抑制在细胞融合期或细胞融合前时期。电子显微镜观察发现,相邻花粉母细胞间的胞间连丝通道仍然处于开放状态,内部充满细胞质基质和一些细胞器,细胞核停留在胞间连丝通道口附近,通道内部及附近细胞质内的微管系统依然存在。文章讨论了胞松素B对细胞融合的作用。     

松孢素B对花粉母细胞染色质穿壁的影响

Nagai等,以及Wessels等证实在植物细胞中微丝是促进川流运动的因子。松胞素B(Cytochalasin B简称CB)有干扰微丝抑制川流运动的生物学效应。根据我们的研究,细胞质和核液的川流运动对花粉母细胞间细胞质和染色质穿壁运动起着直接的推动作用。因此,如果用不同浓度的松     

IB-RS-2细胞的微细胞的形成过程及其超微结构的研究

IB-RS-2细胞经秋水仙素(2微克/毫升)处理24—48小时,约75％的细胞形成微核,同时在细胞表面形成许多大小不一的原生质突起。微核化的细胞进一步用松胞素B(10微克/毫升)处理,在Ficoll不连续梯度(12.5％、16％、25％)中进行离心去核、形成许多微细胞。扫描电镜观察表明,微细胞是微核化细胞的原生质突起脱落而形成的。微细胞是一个不均一的群体,无论在大小与表面形态,还是在微细胞内部的成分与结构上都存在很大差异。     

核体合成自身DNA与病毒DNA的能力

近年来,用松胞素B(简称CB)诱导细胞去核的技术已被许多学者应用于细胞内大分子代谢及其调控过程的核质关系研究中(Colonne et al.,1980;Premveer et al.,1980;White et al., 1982)。我们在对CB诱导的去核产物的研究基础上(何大澄等1982),对核体内RNA的合成进行了研究,表明核体合成RNA的能力与核体贴壁和所带质膜的完整性以及胞质量多少有关(戎宪辉等1984)。此外,我们还研究了两种小RNA病毒和痘病毒的核酸在胞质体内的复制,证明它们的复制可以不依赖细胞核     

松胞素对百合花粉原生质体内肌动蛋白微丝的影响

用B_5培养基酶解分离出百合花粉原生质体。原生质体经松胞素(5μg/ml)分别处理5、10、15、30、60 min,再用荧光标记的鬼笔碱染色,共焦激光扫描镜观察,跟踪了原生质体内的肌动蛋白微丝从一个组织复杂严密的网络转变为无数颗粒体的过程。松胞素处理过的原生质体移回至不含松胞素的培养基中后继续培养15 min,肌动蛋白颗粒快速地再延伸出微丝,重组成新的网络。存在于花粉原生质体中生殖细胞的微丝网络,在经松胞素处理后同样都形成为颗粒体。之后,如果原生质体再放入不含松胞素的培养基内继续培养,生殖细胞内的颗粒体同样会再延伸出微丝,重新组成网络。从原生质体胞质以及生殖细胞内所见到的微丝和颗粒相互转化的情况,可以断定,颗粒体不但具有凝聚微丝的功能,同时也具有重组微丝的功能。     

抗菌肽天蚕素B基因在纳豆芽胞杆菌中的表达

抗菌肽是生物体内经诱导产生的一类具有生物活性的小分子多肽。天蚕素B(Cecropin B)是最早从天蚕体内分离得到的一种热稳定的可溶性多肽,在已分离的众多抗菌肽中抗性较强。纳豆芽胞杆菌具有优良的益生特性,本研究选择枯草芽胞杆菌的一种表达载体p HT43,将抗菌肽天蚕素B基因导入纳豆芽胞杆菌中,验证其在目的菌中是否能够表达和稳定传代以及进行抗菌活性分析。结果表明,天蚕素B基因在纳豆芽胞杆菌中表达,并能稳定传代,能够提高纳豆芽胞杆菌的抑菌活性,抗金黄色葡萄球菌的活性优于干酪乳杆菌和枯草芽胞杆菌。本研究为该重组菌作为饲料添加剂的应用提供了技术基础。本文首次报道天蚕素B在纳豆枯草芽胞杆菌中表达。     

植物细胞原生质流动与胞间运输的关系(简报)

紫鸭跖草雄蕊毛经DNP、NaN_3和CB处理后,细胞原生质流动完全停止。将羧基荧光索注入雄蕊毛末端细胞后,在原生质不流动的情况下叠氮化钠处理的运输速度和数量高于对照,2.4-二硝基苯酚和细胞松弛素B处理的则比对照低,表明了胞质流动与胞间运输无关。     

THE EFFECTS OF CYTOCHALASIN B ON THE MICROFILAMENTS OF BABY HAMSTER KIDNEY (BHK-21) CELLS

Attempts were made to test the motile functions of bundles of microfilaments found in baby hamster kidney (BHK-21) cells, by using cytochalasin B (CB). It was found that individual cells respond differently to the drug. These differential effects are quite obvious in both light and electron microscope preparations. Some cells contain normal bundles of microfilaments even after 24 hr in CB, and other cells form muscle-like configurations which also contain arrays of microfilaments. These varied effects suggest the existence of several types of microfilaments in BHK-21 cells, and make the interpretation of the motile role of microfilaments difficult to evaluate at the present time.     

胃肠道伤害性刺激诱导中缝背核触液神经元Fos表达

本文以ＣＢ－ＨＲＰ逆行追踪和原癌基因ｃ－ｆｏｓ表达技术相结合,观察胃肠道伤害性刺激后中缝背核触液神经元Ｆｏｓ的表达。在中缝背核发现三种标记神经元,包括ＣＢ－ＨＲＰ逆行标记神经元（３０８）、Ｆｏｓ阳性神经元（４２）和ＣＢ－ＨＲＰ／Ｆｏｓ双重标记神经元（５）。本研究提示中缝背核含有一些具有双重功能的神经元,它们既在脑－脑脊液神经体液回路中传递信息,又在胃肠道伤害性刺激的中枢传递和功能调控中起一定的作用     

MODIFICATION OF ZYMOSAN-INDUCED RELEASE OF LYSOSOMAL ENZYMES FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES BY CYTOCHALASIN B

During the process of phagocytosis, polymorphonuclear leukocytes (PMN) release lysosomal enzymes into the extracellular medium. When the antibiotic cytochalasin B (CB) is present in the incubation medium along with phagocytable particles, enhanced recovery of enzyme activities from the incubation medium has been observed. These findings have led to the interpretation that CB enhances lysosomal enzyme release. Our results contradict this interpretation. The lysosomal enzymes acid phosphatase and β-galactosidase are unstable after they are released from cells. During the first 5–15 min of phagocytosis, significant amounts of both acid phosphatase and β-galactosidase can be recovered from the extracellular medium. After this, the recovery of enzyme from the medium declines, presumably because the rate of loss of lysosomal enzyme activity exceeds the rate of release at later time periods. In the presence of CB, the appearance of lysosomal enzymes in the extracellular medium of cells exposed to zymosan is retarded for 5–10 min, after which it begins and then continues for approximately 20 min. At the end of a 30-min incubation period, therefore, in the absence of CB, extracellular levels of lysosomal enzymes (especially those which are unstable) are declining toward low levels while, in the presence of CB, extracellular enzyme levels are continuing to rise. We also measured the lysosomal enzyme remaining within cells after exposure to zymosan. CB retarded the disappearance of enzyme from cells and resulted in significantly less total cell enzyme loss. Thus, in the presence of CB, a greater proportion of the lysosomal enzyme lost from cells is recovered in the extracellular medium. In contrast to the previous conclusions that CB enhances lysosomal enzyme release, our results indicate that CB delays and decreases the zymosan-stimulated release of lysosomal enzymes from PMN. Since CB inhibits phagocytosis by PMN, our results indicate that the antibiotic modifies the mechanism of release of lysosomal enzymes, resulting in zymosan stimulation of their release independently of phagocytosis.     

金鱼精子发生中的拟染色质小体

拟染色质小体是性细胞特有的细胞器。本文报道:在金鱼精子发生过程中,精原细胞含有大量的拟染色质小体,在它上面不仅聚集着许多线粒体,而且有膜片层附着,其本身还会出现环孔和类似线粒体内嵴状结构。随着细胞的分化,拟染色质小体逐渐变少减小,直至消失。本文对拟染色质小体的形成方式与功能进行了讨论。     

去核过程中CHO细胞波形纤维的分布

本文用豚鼠抗小牛晶状体波形纤维蛋白的血清抗体,对经细胞松驰素B(CB)和秋水仙素等药物处理后再离心去核的CHO细胞及其核体、胞质体进行了间接免疫荧光染色,并对核体做了电镜观察。CB处理后离心的细胞的免疫荧光染色显示,去核过程中核的后方始终伴有强烈的荧光,核体上也有强荧光斑。在核体的电镜材料中同样观察到了中等纤维。经CB和秋水仙素合并处理后离心的细胞,去核效果比仅用CB处理有明显的增强,免疫荧光染色表明,核后的荧光并不因秋水仙素处理而消失。实验结果表明:1.微丝对维持细胞表面的完整性有重要作用,CB能破坏微丝故有利于离心去核。2.中等纤维与核之间存在密切联系,这种联系在核膜的某些区域比较集中、牢固,不易为离心力所破坏。3.微管对核固着作用有重要意义,细胞核可能通过中等纤维与微管相连而抛锚在胞质中,故秋水仙素可增强去核作用。微管对维持细胞表面强度可能也有一定作用。     

Co-culture of cord blood CD34(+) cells with human BM mesenchymal stromal cells enhances short-term engraftment of cord blood cells in NOD/SCID mice

BACKGROUND: The major challenge for cord blood transplantation (CBT) is higher rates of delayed and failed engraftment. In an attempt to broaden the application of CBT to more candidates, ex vivo expansion of hematopoietic stem/progenitor cells in CB is a major area of investigation. The purpose of this study was to employ human BM mesenchymal stromal cells (hBM-MSC) as the feeding-layer to expand CB cells ex vivo. METHODS: In this study, hBM-MSC were isolated and characterized by morphologic, mmunophenotypic and RT-PCR analysis. The hBM-MSC at passage 3 were employed as the feeding-layer to expand CB CD34(+) cells in vivo in the presence of thrombopoietin, flt3/flk2 ligand, stem cell factor and G-CSF. The repopulating capacity of the ex vivo-expanded CB cells was also evaluated in a NOD/SCID mice transplant experiment. RESULTS: After 1 or 2 weeks of in vitro expansion, hBM-MSC supported more increasing folds of CB in total nucleated cells, CD34(+) cells and colony-forming units (CFU) compared with CB without hBM-MSC. Furthermore, although NOD/SCID mice transplanted with CB cells expanded only in the presence of cytokines showed a higher percentage of human cell engraftment in BM than those with unexpanded CB CD34(+) cells, expanded CB cells co-cultured with hBM-MSC were revealed to enhance short-term engraftment further in recipient mice. DISCUSSION: Our study suggests that hBM-MSC enhance in vitro expansion of CB CD34(+) cells and short-term engraftment of expanded CB cells in NOD/SCID mice, which may be valuable in a clinical setting.     

Multicolor immunofluorescence and flow cytometry utilizing cascade blue to purify murine hematopoietic stem cells from fetal liver and bone marrow.

BACKGROUND: Here we demonstrate the utility of cascade blue (CB), to purify hematopoietic stem cells by flow cytometry. Multicolor immunofluorescence and the sensitivity (signal-to-noise) of the fluorochromes are essential for the identification and isolation of rare stem cell populations. METHODS: We isolated hematopoietic stem cells utilizing a 407 nm laser line to excite CB and propidium iodide (PI) in combination with FITC, PE, and Red670 which were excited at 488 nm. RESULTS: CB is maximally excited using a 407 nm laser line, when compared to UV or 413 nm excitation. The increase in sensitivity of CB at 407 nm can be contributed to higher absorption of CB and a reduction of autofluorescence at this excitation wavelength (Ropp et al.: Cytometry 21: 309-317, 1995). CONCLUSIONS: Despite the fact that the CB antibody conjugate has a tendency to adhere specifically to a B cell subpopulation in bone marrow, we nevertheless could purify stem cells by using CB for the detection and elimination of lineage positive cells. Isolated stem cells from mouse fetal liver (Lin-CD34(+)Sca-1(+)c-Kit(high)) and adult bone marrow (Lin-CD34(-/low)Sca-1(+)c-Kit(+)) were transplanted into lethally irradiated mice, and the sorted stem cells had the ability to efficiently repopulate all mature hematopoietic lineages in recipient mice.     

Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function.     

Cannabinoid receptor 2 is critical for the homing and retention of marginal zone B lineage cells and for efficient T-independent immune responses

The endocannabinoid system has emerged as an important regulator of immune responses, with the cannabinoid receptor 2 (CB2) and its principle ligand 2-archidonoylglycerol playing a major role. How CB2 regulates B cell functions is not clear, even though they express the highest levels of CB2 among immune cell subsets. In this study, we show that CB2-deficient mice have a significant reduction in the absolute number of marginal zone (MZ) B cells and their immediate precursor, transitional-2 MZ precursor. The loss of MZ lineage cells in CB2(-/-) mice was shown to be B cell intrinsic using bone marrow chimeras and was not due to a developmental or functional defect as determined by B cell phenotype, proliferation, and Ig production. Furthermore, CB2(-/-) B cells were similar to wild type in their apoptosis, cell turnover, and BCR and Notch-2 signaling. We then demonstrated that CB2(-/-) MZ lineage B cells were less efficient at homing to the MZ and that their subsequent retention was also regulated by CB2. CB2(-/-) mice immunized with T-independent Ags produced significantly less Ag-specific IgM. This study demonstrates that CB2 positively regulates T-independent immune responses by controlling the localization and positioning of MZ lineage cells to the MZ.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.