Targeting of nonexpressed genes in embryonic stem cells via homologous recombination

Gene targeting via homologous recombination-mediated disruption in murine embryonic stem (ES) cells has been described for a number of different genes expressed in these cells; it has not been reported for any nonexpressed genes. Pluripotent stem cell lines were isolated with homologously recombined insertions at three different loci: c-fos, which is expressed at a low level in ES cells, and two genes, adipsin and adipocyte P2 (aP2), which are transcribed specifically in adipose cells and are not expressed at detectable levels in ES cells. The frequencies at which homologous recombination events occurred did not correlate with levels of expression of the targeted genes, but did occur at rates comparable to those previously reported for genes that are actively expressed in ES cells. Injection of successfully targeted cells into mouse blastocysts resulted in the formation of chimeric mice. These studies demonstrate the feasibility of altering genes in ES cells that are expressed in a tissue-specific manner in the mouse, in order to study their function at later developmental stages.     

应用缺口末端标记法(TUNEL)分析犬瘟热病毒(CDV)诱导的细胞凋亡

利用犬瘟热病毒弱毒标准株ＯＰ－ＣＤＶ感染敏感细胞系非洲绿猴肾细胞,应用缺口末端标记法分析感染细胞的凋亡。结果在感染后４８ｈ的Ｖｅｒｏ细胞中检出了凋亡阳性细胞,而对照细胞内未检测到凋亡细胞,感染后２４ｈ的细胞尚未出现细胞病变,这一时期的细胞内也未检测到凋亡细胞,表明ＣＤＶ能诱导细胞凋亡,且凋亡细胞的检出时间与细胞病变的出现时间呈正向相关。     

猪脾生长抑素阳性细胞和免疫细胞的发育

用免疫组织化学技术研究猪脾生长发育过程中生长抑素 (SS)阳性细胞、IgA阳性细胞和酸性非特异性脂酶(ANAE)阳性细胞数量的变化。结果表明 ,出生时 ,猪脾SS阳性细胞数量较少 ,3日龄时数量显著增加 (P <0 0 5 ) ,2 0日龄则极显著增加 (P <0 0 1) ,并维持到 90日龄 ,12 0日龄时SS阳性细胞数量显著减少 (P <0 0 5 )。出生时 ,猪脾内就分布有IgA阳性细胞和ANAE阳性细胞 ,IgA阳性细胞数量于 2 0日龄呈显著上升趋势 ,4 5日龄达最高峰 ,于 90日龄极显著降低 (P <0 0 1) ;ANAE阳性细胞数量在 2 0日龄开始增加 ,4 5日龄时极显著增加 (P <0 0 1) ,12 0日龄时又显著降低 (P <0 0 5 )。以上结果表明 ,猪脾中SS阳性细胞的发育对免疫细胞的发育具有重要的调节作用     

HSP104 required for induced thermotolerance

A heat shock protein gene, HSP104, was isolated from Saccharomyces cerevisiae and a deletion mutation was introduced into yeast cells. Mutant cells grew at the same rate as wild-type cells and died at the same rate when exposed directly to high temperatures. However, when given a mild pre-heat treatment, the mutant cells did not acquire tolerance to heat, as did wild-type cells. Transformation with the wild-type gene rescued the defect of mutant cells. The results demonstrate that a particular heat shock protein plays a critical role in cell survival at extreme temperatures.     

褐牙鲆消化道粘液细胞的类型及分布

采用阿利新兰—过碘酸雪夫氏反应(AB-PAS)染色法和不同pH(1.0、2.5、3.1)的AB染色法,对褐牙鲆进行显微观察,发现褐牙鲆食管、胃、幽门垂和肠道均有粘液细胞分布.根据AB-PAS染色结果将褐牙鲆粘液细胞分成Ⅰ-Ⅳ4种类型:Ⅰ型呈红色,AB呈阴性反应,PAS呈阳性反应,含中性粘多糖;Ⅱ型呈蓝色,AB呈阳性反应,PAS呈阴性反应,含酸性粘多糖;Ⅲ型呈紫红色,AB和PAS均呈阳性反应,主要含有PAS呈阳性反应的中性粘多糖,同时含有少量AB呈阳性反应的酸性粘多糖;Ⅳ型呈蓝紫色,AB和PAS均呈阳性反应,主要含有AB呈阳性反应的酸性粘多糖,同时含有少量PAS呈阳性反应的中性粘多糖.AB(pH 2.5、3.1)染色显示,粘液细胞含酸性粘液(蓝色);AB(pH 1.0)染色显示,粘液细胞含弱的和强的硫酸化酸性粘液(蓝色).食管、胃、幽门垂和肠道含有大量粘液细胞,食管和中后肠以Ⅲ型和Ⅳ型细胞为主,幽门垂和前肠含有许多Ⅰ型细胞,胃只含大量的Ⅰ型细胞.通过对各部位粘液细胞的分类和比较可以看出,粘液细胞的分布和类型与其所在部位执行的功能有密切的关系.     

Lateral interactions at inner plexiform layer of vertebrate retina: antagonistic responses to change

Lateral interactions at the inner plexiform layer of the retina of the mudpuppy were studied intracellularly after they were isolated from interactions at the outer plexiform layer with a special stimulus. The isolation was confirmed by recording no surround effect at bipolar cells under conditions that elicited a strong surround effect at ganglion cells. It appears that amacrine cell, which respond to spatiotemporal change at one retinal region, inhibit the response to change in on-off ganglion cells at adjacent sites.     

Long-term potentiation of neuron-glia synapses mediated by Ca2+-permeable AMPA receptors

Interactions between neurons and glial cells in the brain may serve important functions in the development, maintenance, and plasticity of neural circuits. Fast neuron-glia synaptic transmission has been found between hippocampal neurons and NG2 cells, a distinct population of macroglia-like cells widely distributed in the brain. We report that these neuron-glia synapses undergo activity-dependent modifications analogous to long-term potentiation (LTP) at excitatory synapses, a hallmark of neuronal plasticity. However, unlike the induction of LTP at many neuron-neuron synapses, both induction and expression of LTP at neuron-NG2 synapses involve Ca2+-permeable AMPA receptors on NG2 cells.     

Increased incidence of tumor metastases in female mice

Most of the tumor cells injected into the tail vein of mice fail to survive at the site of arrest in the lungs, but the percentage of surviving cells is higher in females than in males. The surviving cells, however, grow at a similar and constant rate in both sexes.     

Cell-autonomous determination of cell-type choice in Dictyostelium development by cell-cycle phase

The developmental fate of individual cells has been examined in a system that allows Dictyostelium discoideum cells to differentiate in the absence of aggregation. The results show that the propensity of single amoebae to differentiate into either prespore or prestalk cells occurs by a cell-autonomous mechanism dependent on the cell's position in the cell cycle at the initiation of development. Cells that divide between approximately 1 1/2 hours before and approximately 40 minutes after the differentiation-inducing starvation become prestalk, whereas cells dividing at other times become prespore cells. These results suggest mechanisms by which an initial proportioning of the two cell types within the aggregate is achieved.     

Somatic cell mating and segregation in chimeric frogs

Both pentaploid and haploid cells were observed in a short-term culture of bone marrow of a diploid-triploid frog chimera. Apparently, diploid and triploid marrow cells fused to form "hybrid" pentaploid cells, which subsequently gave rise by somatic reduction to haploid daughter cells. The hybrid marrow cells and their haploid segregants are presumably at a selective disadvantage, as neither type of cell has been detected in the circulating blood of chimeric frogs.     

哲罗鱼仔稚鱼嗅囊发育及其与摄食强度的关系

为掌握哲罗鱼Hucho taimen仔稚鱼嗅囊的发育过程及其与摄食强度的关系,对哲罗鱼仔稚鱼嗅囊发育进行了组织形态学观察,研究了嗅囊发育与摄食强度之间的对应关系,描述了各发育时期的时序和形态特征。结果表明:初孵哲罗鱼仔鱼嗅囊上皮未分化,消化道未有食物充塞度;25~26日龄仔鱼仅有少数个体(11.1%)消化道有一定的食物充塞度(1级);27日龄仔鱼多数个体(占66.7%)开始少量摄食,此时嗅囊上皮仍未分化;29日龄仔鱼均摄食,消化道食物充塞度达到2级和3级的个体分别占44.4%和33.3%;30日龄仔鱼嗅囊上皮从嗅囊基部开始分化;42日龄稚鱼上皮分化更加明显,细胞分化加剧,此时嗅囊上皮分化;45日龄稚鱼均强烈摄食,消化道食物充塞度等级均达到5级,此时嗅囊上皮分化完成;55日龄稚鱼嗅囊形成第一个初级嗅板;85日龄稚鱼嗅囊分化完成,通过光镜和电镜观察,嗅上皮细胞可分为6类,即基细胞、支持细胞、纤毛非感觉细胞、纤毛感觉细胞、柱状细胞和黏液细胞。本研究结果可为哲罗鱼资源的保护和苗种培育提供理论依据。     

醋酸铅对蚕豆根尖细胞微核率的影响

为研究铅对蚕豆根尖细胞遗传物质的损伤所用,以青皮蚕豆vicia faba L.为试验材料,通过蚕豆根尖细胞微核试验分析0.5~30mg·L~(-1)的醋酸铅对蚕豆根尖细胞微核率的影响,并计算污染指数。结果表明,蚕豆根尖细胞的微核率随染毒质量浓度的增加均呈上升的趋势,经30.0mg·L~(-1)醋酸铅染毒6h的细胞微核率显著高于对照组(P0.05),污染指数大于3.5,属于重度污染;经1.0~30.0mg·L~(-1)醋酸铅溶液染毒12h的细胞微核率均极显著高于对照组(P0.01),污染指数均大于3.5,属于重度污染;相同质量浓度醋酸铅溶液染毒处理6h后的细胞微核率均明显低于染毒处理12h组,但仅在质量浓度为1mg·L~(-1)时2组之间达到显著差异水平(P0.05)。研究显示,Pb2+可以通过蚕豆根尖的吸收而毒害细胞,引起细胞DNA损伤,且损伤程度随剂量的增加和时间的延长而加重。     

5-Thio-D-glucose selectively potentiates hyperthermic killing of hypoxic tumor cells

To investigate the mechanisms by which heat affects cancer cells, we used 5-thio-D-glucose, an inhibitor of glycolysis in HeLa S-3 cells, under aerobic and hypoxic conditions at temperatures ranging from 37 degrees to 43 degrees C. Drug alone or heat alone killed a minimum number of cells under aerobic or hypoxic conditions. Exposure to drug and hyperthermia selectively increased the number of cells killed under hypoxic conditions at temperatures as low as 40.5 degrees C but had little effect on cells incubated under aerobic conditions. These results suggest that the glycolytic pathways is a primary site of hyperthermic damage leading to cell death.     

Tumor regression in cancer patients by very low doses of a T cell-engaging antibody

Previous attempts have shown the potential of T cells in immunotherapy of cancer. Here, we report on the clinical activity of a bispecific antibody construct called blinatumomab, which has the potential to engage all cytotoxic T cells in patients for lysis of cancer cells. Doses as low as 0.005 milligrams per square meter per day in non-Hodgkin's lymphoma patients led to an elimination of target cells in blood. Partial and complete tumor regressions were first observed at a dose level of 0.015 milligrams, and all seven patients treated at a dose level of 0.06 milligrams experienced a tumor regression. Blinatumomab also led to clearance of tumor cells from bone marrow and liver. T cell-engaging antibodies appear to have therapeutic potential for the treatment of malignant diseases.     

Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts

Polyadenylated RNA isolated from senescent human diploid fibroblasts (HDF) inhibited DNA synthesis in proliferation-competent cells after microinjection, whereas polyadenylated RNA from young HDF had no inhibitory effect. Polyadenylated RNA from young cells made quiescent by removal of serum growth factors had a slight inhibitory effect on DNA synthesis. The abundance level of inhibitor messenger RNA (mRNA) from senescent cells was estimated at 0.8 and that of quiescent cells at 0.005 percent. These results demonstrate the existence of one or more antiproliferative mRNA's in nonproliferating normal human cells; these RNA's code for factors that either work antagonistically to initiators of DNA synthesis or regulate the expression of the initiators in some way. The abundance level of the inhibitory mRNA in senescent cells indicates the feasibility of developing a complementary DNA probe that will be useful in studying cell cycle control mechanisms.     

Quantum yield of oxygen evolution and the emerson enhancement effect in deuterated chlorella

The maximum quantum yield of oxygen evolution in deuterated chlorella is found to be 0.075, while normal chlorella showed, in parallel experiments, a value of 0.10. Deuterated Chlorella vulgaris showed a decline in the quantum yield of oxygen evolution ("red drop") beginning at 680 millimicrons and a clear Emerson enhancement effect qualitatively similar to that obtained in normal chlorella cells. However, the ratio of quantumn yield at 680 to that at 710 millimicrons was about 1.5 times higher in normal than in deuterated chlorella cells. Action spectra of the Emerson enhancement effect in deuterated chlorella also are qualitatively similar to those of normal cells.     

表皮干细胞标记物在仿刺参正常和再生体壁中的表达研究

为探究仿刺参表皮再生的机制和再生修复过程中的表皮细胞来源,采用免疫荧光方法,观察β1整合素,α6整合素,角蛋白19 (ck19) 3种表皮干细胞标记物在仿刺参正常及创伤后再生不同时间段的体壁中的表达情况。结果表明ck19在对照组和试验组仿刺参体壁中均呈阳性,并且随着再生的进行,阳性细胞数量和分布区域均有变化。创伤第1天阳性细胞向创伤面上缘迁移,细胞数量同对照组;第3天阳性细胞数量显著下降;第5天阳性细胞数量最多;第7天阳性细胞数量有所下降;11天、13天阳性细胞的数量较第7天稍有增加;第19天阳性细胞数量同对照组,但排列很分散。β1整合素和α6整合素在正常及创伤后再生的体壁中均未见阳性信号出现。结合石蜡切片观察结果进行比较分析认为被ck19标记的细胞是参与仿刺参体壁表皮再生的干细胞和短暂扩增细胞,仿刺参表皮再生的机制属于新建再生。     

参藻混养生态系统中孔石莼、蛋白核小球藻与刺参的相互作用研究

为了探讨参藻混养生态系统中孔石莼Ulva pertusar、蛋白核小球藻Chlorella pyrenoidosa与刺参Apostichopus japonicus的相互作用,采用实验生态学方法进行了10~6、10~5 cells/mL两种浓度的蛋白核小球藻与孔石莼及刺参不换水混养的生态效应研究。结果表明:孔石莼和小球藻能有效地吸收养殖水体中的氮磷;10~6cells/mL浓度的小球藻在与孔石莼、刺参混养时对孔石莼有明显的抑制作用,而10~5 cells/mL浓度的小球藻对孔石莼有一定的抑制作用。研究表明,小球藻浓度为10~5 cells/mL的混养组在水质、刺参生长方面与孔石莼混养组相近,且能较好地抑制孔石莼过快生长。     

不同生理时期小鼠卵巢的Ghrelin定位研究

研究生长素(Ghrelin)在青春期、妊娠期、哺乳期和泌乳后期小鼠卵巢上的定位,采用免疫组化PV-9000二步法观察Ghrelin在卵巢上的分布。Ghrelin阳性细胞主要分布于4个生理时期小鼠卵巢的卵母细胞、间质细胞、髓质和黄体细胞上。但表达强度和分布范围不同,妊娠期8d黄体与哺乳期18d的卵巢髓质呈强阳性表达。妊娠8d的小鼠卵巢的生殖上皮细胞出现阳性表达。Ghrelin在小鼠卵巢青春期、妊娠期、哺乳期和泌乳后期时期均有表达,妊娠期黄体呈强表达,揭示Ghrelin可能对小鼠不同生理时期尤其是妊娠有一定的调节作用。