共查询到20条相似文献,搜索用时 15 毫秒
1.
B V Bronk R J Wilkins J D Regan 《Biochemical and biophysical research communications》1973,52(3):1064-1070
Human skin cells were incubated at various temperatures during and after treatment with methyl methanesulfonate and the number of single-strand breaks introduced into the cellular DNA then estimated by alkaline sucrose sedimentation. Elevation of temperature above 37° greatly enhanced damage to the DNA caused by methyl methanesulfonate. Inactivation of an essential step in the repair of DNA was indicated by the observation that rejoining of breaks in the DNA was halted above a critical temperature (about 41.5°). Enhancement of damage to DNA increased with temperature, especially above 42°. Similar results were obtained for Chinese hamster cells. A correlation of these results with cell viability is discussed. 相似文献
2.
D Holowka 《Analytical biochemistry》1981,117(2):390-397
The preparation of iodinated N-chloroacetyltyramine and its evaluation as a specific sulfhydryl reagent are described. N-Chloroacetyltyramine was synthesized by a carbodiimide-mediated condensation of chloro- or iodoacetic acid and tyramine·HCL, and the crystalline product was iodinated in a reaction with chloramine T to yield either a 3,5-[125I]diiodotyramine derivative, or a trace-iodinated product when carrier-free 125I was employed. These iodinated derivatives react specifically with sulfhydryl groups, as judged by their ability to label reduced but not unreduced ribonuclease A and immunoglobulin E. Specific activities of 1 Ci/mmole in 125I or 131I can be readily achieved with both the diiodinated and trace-iodinated (carrier-free) derivatives, and the specific activity of the former can be used directly to quantitate sulfhydryl groups in subnanomolar quantities of protein. N-Chloroacetyl 125I-labeled tyramine prepared by trace iodination with carrier-free 125I is more useful when very high specific activities (100–1000 mCi/μmol) are required. The utility of these reagents is discussed. 相似文献
3.
IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells 总被引:4,自引:0,他引:4
Yang L Dybedal I Bryder D Nilsson L Sitnicka E Sasaki Y Jacobsen SE 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(2):752-757
Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-gamma, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-gamma might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-gamma on HSC maintenance and function. Although purified cord blood CD34(+)CD38(-) cells underwent cell divisions in the presence of IFN-gamma, cycling HSCs exposed to IFN-gamma in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-gamma accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-gamma can negatively affect human HSC self-renewal. 相似文献
4.
Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. 总被引:14,自引:0,他引:14
H E Broxmeyer J Kurtzberg E Gluckman A D Auerbach G Douglas S Cooper J H Falkenburg J Bard E A Boyse 《Blood cells》1991,17(2):313-329
This paper reviews our recent laboratory and clinical studies demonstrating the efficacious use of human umbilical cord blood for HLA-matched allogeneic sibling stem/progenitor cell transplantation in cases of Fanconi's anemia. Future implications and potential problems are discussed with regards to (a) the possibility of maternal cell contamination, (b) the broadness of applicability with regards to other diseases that might be transplanted, and whether such transplants are feasible in adults, as well as in children, and (c) the immunological reactivity of cord blood cells, and whether these cells can be used to cross histocompatibility barriers more easily than that of bone marrow from adults. 相似文献
5.
Qiaoxiang Dong Danhan Wang Abhik Bandyopadhyay Hui Gao Karla M. Gorena Kim Hildreth Vivienne I. Rebel Christi A. Walter Changjiang Huang Lu-Zhe Sun 《Stem cell research》2013,10(3):396-404
Identification of murine mammary stem cells (MaSCs) has been attempted with various in vitro and in vivo assays. While, the in vivo repopulation assay remains as the most definitive assay for MaSC detection, it is expensive, time-consuming, and technically challenging. The in vitro mammosphere assay was considered unreliable because of major concerns about its clonal origin. In the current study, co-culture experiments with mammary cells from fluorescent protein transgenic mice and time-lapse video microscopy revealed that > 90% mammospheres formed from sorted basal epithelial-enriched cells were of clonal origin in terms of stem cell. These basal-cell derived mammospheres were further distinguished morphologically in a 3-dimensional extracellular matrix culture and functionally in the in vivo repopulation assay. Transplant of single mammospheres or the resultant 3-dimensional solid structures into gland-free mammary fat pads yielded a 70% success rate of multilineage mammary gland reconstitution. Thus, this in vitro sphere formation and differentiation assay is a reliable alternative to the in vivo repopulation assay for the study of MaSCs. 相似文献
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Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. 总被引:20,自引:0,他引:20
R Eiges M Schuldiner M Drukker O Yanuka J Itskovitz-Eldor N Benvenisty 《Current biology : CB》2001,11(7):514-518
Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research. 相似文献
9.
Azuma M Hirao A Takubo K Hamaguchi I Kitamura T Suda T 《Biochemical and biophysical research communications》2005,338(2):1164-1170
Homeostasis of prostate tissue is maintained by stem cells, although such cells have not been well characterized. Here, we report establishment of such a method using matrigel. Matrigel containing a single-cell suspension from adult prostatic cells was subcutaneously grafted into the flank of nude mice. Prostatic duct-like structures derived from donor tissue were observed in the gel 2 weeks after transplantation. Luminal and basal cells observed in the gel expressed several markers characteristic of prostatic and/or epithelial cells. When a mixture with both EGFP-positive and negative prostate cells was transplanted, prostatic ducts consisted of either EGFP-positive or negative cells and chimeric patterns were rarely observed, suggesting that ducts were reconstituted from a single cell. Stem cell number and function were also evaluated by competition with control cells. Overall this method revealed that cells localized in the proximal portion in prostate ducts had higher reconstitution capacity than those in the distal portion. We conclude that prostate stem/progenitor cells exist and that our method is applicable to analysis of prostate stem cells, epithelial mesenchyme interactions, and prostate cancer stem cells. 相似文献
10.
With the help of the earlier devised model of cultured cell growth kinetics it is shown that the "plateau" level on the growth curve of Chinese hamster cells decreased proportionally to a dose of the agent applied after a short-term treatment by gamma-radiation or by alkylating agent thiophosphamide. The analysis of our own and literature data enabled us to propose that the lowering of the growth curve may testify to the geropromoter character (i.e. manifesting in ageing promoting) of the investigated factor action. As the low-frequency electromagnetic field induces similar changes in the growth curve, it is related (together with both the above factors investigated) to geropromoters. 相似文献
11.
The glucose-derived alkylating agent N-bromoacetylglucosamine (GlcNBrAc) is shown to cause a time-dependent irreversible inactivation of rat muscle hexokinase type II. The kinetics of inactivation are in accord with the reversible formation of an enzyme-inhibitor complex prior to modification, indicating that the reagent is active-site-directed. A Ki of 0.57 mM obtained for this reversible complexing is in agreement with a Ki of 0.65 mM obtained for the inhibition caused by N-propionylglucosamine, an isosteric analogue of GlcNBrAc and a competitive inhibitor with respect to glucose. Glucose itself protects competitively against inactivation. A KG of 0.26 mM obtained for the formation of enzyme-glucose complex from these studies is in agreement with the kinetically-determined Km of 0.2 mM. The substrate-unrelated but chemically similar alkylating agents bromoacetic acid and N-bromoacetylgalactosamine inactivate the enzyme at 20% of the rate caused by GlcNBrAc. The inactivation rate increases rapidly over the pH range 7--9. Analysis of this pH dependence shows that a single residue of pKa 8.9 is reacting with GlcNBrAc with a kmax (pH corrected, pseudo-first-order rate constant) of 1.5 x 10(-3) S-1. These values are typical of the reaction of model thiols with alkylating agents and suggests the reacting residue is probably a cysteine. Use of radioactively labelled GlcNBrAc indicates that uptake of 1 mol of reagent per mol protein causes complete activity loss. Finally the behaviour of this enzyme with active-site-directed alkylating agents is compared with published results of similar experiments carried out with yeast hexokinase and bovine brain hexokinase type I. 相似文献
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Colony formation by surviving spermatogonial stem cells was investigated by mapping pieces of whole mounted tubuli at intervals of 6 and 10 days after doses of 0.75 and 1.50 Gy of fission neutron irradiation. Colony sizes, expressed in numbers of spermatogonia per colony, varied greatly. However, the mean colony size found in different animals was relatively constant. The mitotic indices in large and small colonies and in colonies in different epithelial stages did not differ significantly. This finding suggests that size differences in these spermatogenic colonies are not caused by differences in growth rate. Apparently, surviving stem cells start to form colonies at variable times after irradiation. The number of colonies per unit area varied with the epithelial stages. Many more colonies were found in areas that during irradiation were in stages IX-III (IX-IIIirr) than in those that were in stages IV-VII (IV-VIIirr). After a dose of 1.50 Gy, 90% of all colonies were found in areas IX-IIIirr. It is concluded that the previously found difference in repopulation after irradiation between areas VIII-IIIirr and III-VIIIirr can be explained not by differences in colony sizes and/or growth rates of the colonies in these areas but by a difference in the number of surviving stem cells in both areas. In area XII-IIIirr three times more colonies were found after a dose of 0.75 Gy than after a dose of 1.50 Gy. In area IV-VIIirr the numbers of colonies differed by a factor of six after both doses. This finding indicates that spermatogonial stem cells are more sensitive to irradiation in epithelial stages IV-VII than in stages XII-III. In control material, spermatogonia with a nuclear area of 70-110 micron2 are rare. However, especially 6 days after irradiation, single cells of these dimensions are rather common. These cells were found to lie at random over the tubular basement membrane with no preference for areas with colonies. It is concluded that the great majority of these cells were not or do not derive from surviving stem cells. These enlarged cells most likely represent lethally injured cells that will die or become giant cells (nuclear area greater than 110 micron2). 相似文献
14.
The degree of alkylation of haemoglobin was determined at different times after treatment of mice with one directly active alkylating agent, ethylene oxide, and one agent that requires metabolic activation, dimethylnitrosamine. Because of the random alkylation of red blood cells of various ages and the stability of alkylated haemoglobin, the amount of alkylated amino acids in haemoglobin decreases linearly with time, reaching the value zero after about 40 days, the life-span of erythrocytes in the mouse. This provides a basis for the use of haemoglobin as a monitor for integral doses of genotoxic environmental chemicals. 相似文献
15.
There are increasing concerns regarding intracellular accumulation of gadolinium (Gd) after multiple dynamic contrast enhanced (DCE) MRI scans. We investigated whether a low dose (LD) of Gd-based contrast agent is as effective as a high dose (HD) for quantitative analysis of DCE-MRI data, and evaluated the use of a split dose protocol to obtain new diagnostic parameters. Female C3H mice (n = 6) were injected with mammary carcinoma cells in the hind leg. MRI experiments were performed on 9.4 T scanner. DCE-MRI data were acquired with 1.5 s temporal resolution before and after a LD (0.04 mmol/kg), then again after 30 min followed by a HD (0.2 mmol/kg) bolus injection of Omniscan. The standard Tofts model was used to extract physiological parameters (Ktrans and ve) with the arterial input function derived from muscle reference tissue. In addition, an empirical mathematical model was used to characterize maximum contrast agent uptake (A), contrast agent uptake rate (α) and washout rate (β and γ). There were moderate to strong correlations (r = 0.69–0.97, p < 0001) for parameters Ktrans, ve, A, α and β from LD versus HD data. On average, tumor parameters obtained from LD data were significantly larger (p < 0.05) than those from HD data. The parameter ratios, Ktrans, ve, A and α calculated from the LD data divided by the HD data, were all significantly larger than 1.0 (p < 0.003) for tumor. T2* changes following contrast agent injection affected parameters calculated from HD data, but this was not the case for LD data. The results suggest that quantitative analysis of LD data may be at least as effective for cancer characterization as quantitative analysis of HD data. In addition, the combination of parameters from two different doses may provide useful diagnostic information. 相似文献
16.
Pluripotent stem cells (CFU-GEMM) give rise to multilineage hemopoietic colonies in culture. The cellular composition revealed that mixed colonies contain cells of different myeloid lineages and mononuclear cells with T-cell surface antigens. T-lymphocytes of primary colonies, replated secondary and tertiary colonies from a patient with Hodgkin's Lymphoma were identified by their reaction with the monoclonal antibody OKT 8. Evidence for a common progenitor of myeloid and lymphoid cells is provided by analysis of individual secondary and tertiary colonies using OKT 3, OKT 4, OKT 8, VIM-D 5, and Ig M + D antibodies for each individual colony. Primary mixed, replated secondary and tertiary colonies revealed OKT 8 positive cells. No reaction with OKT 3, OKT 4, VIM-D 5, or Ig M + D was observed. 相似文献
17.
《DNA Repair》2019
Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible.Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50–500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation. 相似文献
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19.
In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1
de Haan G Weersing E Dontje B van Os R Bystrykh LV Vellenga E Miller G 《Developmental cell》2003,4(2):241-251
The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis. 相似文献
20.
The journey of developing hematopoietic stem cells 总被引:6,自引:0,他引:6
Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites. Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs. During this process, developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. These processes have to be recapitulated to generate HSCs from embryonic stem cells. Elucidating the interactions between developing HSCs and their niches should facilitate the generation and expansion of HSCs in vitro to exploit their clinical potential. 相似文献