Expression of c-myc changes during differentiation of mouse erythroleukaemia cells

The transforming gene of avian myelocytomatosis virus MC29, v-myc, causes a variety of malignancies in chickens. A cellular homologue, c-myc, has been implicated in B-cell malignancies in mice and humans but is also expressed in many normal cell types and may be important in the control of normal cell proliferation. c-myc is highly conserved in vertebrates. We have been investigating the relationship between c-myc expression and the terminal differentiation of cultured mouse erythroleukaemia (MEL) cells. We find that the level of c-myc messenger RNA shows a rapid biphasic change in MEL cells induced to differentiate by dimethyl sulphoxide or hypoxanthine. The changes occur during the first few hours of the differentiation programme and require active protein synthesis. These data suggest that changes in c-myc expression may be important in the irreversible commitment of MEL cells to terminal erythroid differentiation.     

Expression of a candidate sex-determining gene during mouse testis differentiation

The development of a eutherian mammal as a male is a consequence of testis formation in the embryo, which is thought to be initiated by a gene on the Y chromosome. In the absence of this gene, ovaries are formed and female characteristics develop. Sex determination therefore hinges on the action of this testis-determining gene, known as Tdy in mice and TDF in humans. In the past, several genes proposed as candidates for Tdy/TDF have subsequently been dismissed on the grounds of inappropriate location or expression. We have recently described a candidate for Tdy, which maps to the minimum sex-determining region of the mouse Y chromosome. To examine further the involvement of this gene, Sry, in testis development, we have studied its expression in detail. Fetal expression of Sry is limited to the period in which testes begin to form. This expression is confined to gonadal tissue and does not require the presence of germ cells. Our observations strongly support a primary role for Sry in mouse sex determination.     

X-chromosome inactivation during differentiation of female teratocarcinoma stem cells in vitro

Evidence is presented that both X chromosomes are genetically active in clonal cultures of undifferentiated female mouse teratocarcinoma stem cells derived from a spontaneous ovarian tumour. As the cells differentiate in vitro one of the X chromosomes becomes inactivated.     

Androgen regulation of cell proliferation and expression of viral sequences in mouse mammary tumour cells

The role of steroids in promoting cell proliferation is well established but the molecular mechanisms are not clear. The S115 mouse mammary tumour cell line provides a model system for molecular studies in vitro in that it exhibits in tissue culture both a positive proliferative response to androgens and a change from a transformed phenotype in the presence of androgen to a normal phenotype when androgen is removed. We have considered here the possible involvement of mouse mammary tumour virus (MMTV) in these processes. We have demonstrated the presence in S115 cells of MMTV-related sequences which are transcribed into RNA only in the long-term presence of androgen. Prolonged culture in the absence of androgen, which results in loss of proliferative response to androgen, is accompanied by loss of MMTV-related RNA and increased methylation of MMTV-related sequences.     

小鼠胎肝间充质干细胞体内向骨骼肌样细胞分化的研究

目的:了解小鼠胎肝间充质干细胞在体内向骨骼肌细胞分化的潜能。方法:无菌条件下将C57BL/6 J胎鼠肝脏制成单细胞悬液,雄性胎肝悬液体外贴壁培养纯化,传3代后将贴壁细胞移植于心肌毒素(card iotoxin)造成的雌鼠骨骼肌损伤部位,2月后处死受鼠,取相应骨骼肌组织固定、制片;用免疫组织化学染色和荧光原位杂交检测雌性受体小鼠骨骼肌组织内供体小鼠胎肝间充质干细胞向骨骼肌样细胞分化情况。结果:在骨骼肌组织内发现存在Y染色体阳性的供体来源的细胞,同时呈现骨骼肌组织的部分特征,表型为desm in /F lt-1-/CD4-5/F4-/80。结论:胎肝中分离出的间充质干细胞在体内可以分化为骨骼肌样细胞。     

人间充质干细胞成骨分化早期HOX家族基因转录的表观遗传调控

人间充质干细胞(human mesenchymal stem cells,hMSCs)是一类具有多分化潜能的成体干细胞,在体内外可以被人工定向诱导分化成多种不同的细胞.有报道表明,在干细胞分化的过程中,细胞核内染色质发生重塑.HOX家族基因作为一类转录因子,在胚胎发育以及细胞分化过程中发挥着十分重要作用.通过体外定向诱导骨髓间充质干细胞向成骨细胞分化,对比分化前后细胞中HOX家族基因的表达状况,发现HOX家族基因的表达水平在hMSCs早期成骨分化过程中显著下降.进一步的研究发现,HOX家族基因的这种表达变化是由其启动子区的组蛋白H3-Lys9乙酰化和二甲基化水平发生变化而导致的.一系列实验证据表明,在间充质干细胞的成骨分化过程中,HOX家族基因表达受到抑制,而这种抑制作用是与其分化过程中发生的染色质重塑事件密切相关的.     

Presence of T-cell receptor mRNA in functionally distinct T cells and elevation during intrathymic differentiation

Understanding the differentiation of functionally distinct subsets of T lymphocytes is essential to unravel their crucial role in the immune response and awaits knowledge of the assembly and expression of genes encoding the T-cell receptor. Recently, we have cloned and sequenced complementary DNA that may specify part of the human T-cell receptor. The deduced protein sequence showed extensive similarity to the entire length of mammalian immunoglobulin light chains. In addition, sequences corresponding to this message undergo somatic rearrangements and are assembled from non-contiguous genomic sequences into a single mRNA molecule, a mechanism similar to those found in the generation of immunoglobulin messages. A related molecule from the mouse was also isolated independently by Hedrick et al. Here we show that the putative T-cell receptor mRNA is expressed at a relatively high level during intrathymic differentiation before decreasing about 10-20-fold in normal, mature peripheral blood T cells and that it can also be detected in T-cell clones with helper and cytotoxic functions, as well as in at least one clone with suppressor properties.     

Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.     

The mechanism of adipocyte differentiation and regulation of hematopoietic microenvironment

在人体生长发育与衰老过程中骨髓会逐渐由红骨髓转变成为黄骨髓,即骨髓脂肪化,其本质是骨髓中脂肪细胞增多.骨髓脂肪细胞对造血微环境具有负性调节作用,能抑制骨髓造血、抑制造血重建时造血干细胞在骨髓中的植入.深入研究骨髓脂肪化机制对促进某些疾病过程中造血恢复、提高造血干细胞移植效率具有一定意义.脂肪细胞的形成主要包括间充质干细胞向前体脂肪细胞的定向分化及其终末分化两个阶段.本文主要阐述细胞形态变化和细胞外基质通过WNT和RHO-family GTPase信号级联通路调节前体脂肪细胞的定向分化,成脂刺激因素通过PPARγ的表观遗传学途径诱导前体脂肪细胞的终末分化,以及PPARγ和C/EBP的相互作用维持脂肪细胞的基因表达.     

Epidermal growth factor receptors increase during the differentiation of embryonal carcinoma cells.

Mouse teratocarcinoma stem cells (embryonal carcinoma, or EC cells) bind very small amounts of mouse epidermal growth factor (EGF) and the latter hormone seems to have no stimulatory effect on the growth of two cloned lines of EC cells. However, when EC cells are induced to differentiate into large flat endodern-like cells (END cells), EGF receptors increase in number reaching a plateau in 6 to 8 days. At 8 to 10 days after induction, END cells multiply very slowly, but when EGF is added (3 x 10(-10) M) to the medium, cell division is stimulated and a further change in morphology occurs. This letter describes the binding characteristics and numbers of the EGF receptors on EC and END cells and shows that exogenous retinoic acid increases the numbers of EGF receptors on END cells. We were unable to find endogenous competing factors produced by EC cells. Such factors could account for the lack of detectable binding of EGF on these cells. As EC cells differentiate to END cells, so the ability of the cells to form tumours is reduced. Since this change is accompanied by an increase in the number of EGF receptors there may be a relationship between these two events.     

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.     

Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts

The derivation of embryonic stem (ES) cells by nuclear transfer holds great promise for research and therapy but involves the destruction of cloned human blastocysts. Proof of principle experiments have shown that 'customized' ES cells derived by nuclear transfer (NT-ESCs) can be used to correct immunodeficiency in mice. Importantly, the feasibility of the approach has been demonstrated recently in humans, bringing the clinical application of NT-ESCs within reach. Altered nuclear transfer (ANT) has been proposed as a variation of nuclear transfer because it would create abnormal nuclear transfer blastocysts that are inherently unable to implant into the uterus but would be capable of generating customized ES cells. To assess the experimental validity of this concept we have used nuclear transfer to derive mouse blastocysts from donor fibroblasts that carried a short hairpin RNA construct targeting Cdx2. Cloned blastocysts were morphologically abnormal, lacked functional trophoblast and failed to implant into the uterus. However, they efficiently generated pluripotent embryonic stem cells when explanted into culture.     

BOD5的动态预测方程

根据历史资料建立各采样段面(点)的BOD5-COD动态预测方程,利用计算机就能准确、快速的获得BOD5预测值.该方法适用于各种工业废水和地表水体.     

Division and differentiation of isolated CNS blast cells in microculture

The mechanism of transformation of the overtly similar cells of the neural plate into the numerous and diverse cell types of the mature vertebrate central nervous system (CNS) can better be understood by studying the clonal development of isolated CNS precursor cells. Here I describe a culture system in which blast cells (cells capable of division) isolated from embryonic day 13.5-14.5 rat forebrain can divide and differentiate into a variety of clonal types. Most clones contain only neurons or glia; 22% contain both neurons and non-neuronal cells. For the division of blast cells, live conditioning cells need to be present indicating that environmental signals influence proliferation. Heterogeneous clones develop in homogeneous culture conditions, so factors intrinsic to the blast cells are probably important in determining the number and type of clonal progeny.     

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.