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21.
Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor,
fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by
bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every
other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured
serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population
doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to
endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro. 相似文献
22.
Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR. 总被引:1,自引:0,他引:1 下载免费PDF全文
S Chandrasegaran L S Kan L O Sillerud C Skoglund A A Bothner-By 《Nucleic acids research》1985,13(6):2097-2110
13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis. 相似文献
23.
Mapping parathyroid hormone, β-globin,insulin, and LDH-A genes within the human chromosome 11 short arm by spot blotting sorted chromosomes 总被引:2,自引:0,他引:2
Roger V. Lebo Mei-Chi Cheung Barry D. Bruce Vincent M. Riccardi Fa-Ten Kao Yuet Wai Kan 《Human genetics》1985,69(4):316-320
Summary Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, -globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, -globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci. 相似文献
24.
Isolation and characterization of human placenta fibronectin 总被引:3,自引:0,他引:3
M Isemura Y Yamaguchi H Munakata J Aikawa M Kan I Yamane Z Yosizawa 《Journal of biochemistry》1984,96(1):163-169
Fibronectin was isolated from human placenta tissues and compared with human plasma fibronectin. Placenta and plasma fibronectins had similar amino acid compositions, immunological properties, and cell attachment-promoting activities, but differed in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be accounted for at least partly by the difference in carbohydrate composition. Unlike plasma fibronectin, placenta fibronectin failed to form a precipitin line with concanavalin A in a double diffusion system. The non- or low-reactivity of placenta fibronectin with this lectin was also demonstrated by affinity chromatography with concanavalin A-agarose, in which more than 90% of the radiolabeled glycopeptides derived from placenta fibronectin was not retained on the gel. The two fibronectins also differed in the reactivity with Lens culinaris agglutinin of their glycopeptide fractions. These data indicate that placenta and plasma fibronectins are different in their carbohydrate structures and, therefore, suggest the presence of a tissue- or cell-specific mechanism for processing the carbohydrates of this glycoprotein. 相似文献
25.
E A Rinnooy Kan E Platzer K Welte C Y Wang 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(6):2979-2985
We investigated the influence of monocytes on the susceptibility of the T3 antigen on human T cells to modulation induction by OKT3 antibody. In the absence of monocytes, the T3 antigen was only minimally susceptible to modulation. After the addition of 20% monocytes to the culture, however, complete modulation was readily observed. Furthermore, we found that even in the absence of OKT3 antibody, monocytes were able to down-regulate the expression of the T3 antigen, although to a lesser extent. The ability of monocytes to enhance antigenic modulation proved to be a more general phenomenon. Each individual T cell antigen, however, differed in its susceptibility to modulation by antibody, monocytes, or both, thereby establishing its own characteristic pattern. In addition, after complete modulation of the T3 antigen, the addition of monocytes to the culture thereafter had a distinct inhibitory effect on the reexpression of the T3 antigen. Monocyte enhancement of T3 modulation is significantly reduced when using the OKT3 F(ab')2 fragment, as is OKT3 mitogenesis. After pulsing the monocytes with OKT3 antibody before adding them to the culture, T3 modulation became nearly complete even in the absence of added OKT3 antibody. Monocyte-induced modulation proved not to be MHC restricted, thus allowing for comparative analysis of this effect between monocytes and other cell types. A moderate, however, incomplete modulation enhancement was observed with the human monocyte cell line U937 and with Daudi cells. This finding proved to coincide with the distinct ability of these cell lines to bind OKT3 antibody by their Fc receptors, as was the case with monocytes. In contrast, neither Fc receptor binding nor T3 modulation enhancement was observed with the cell lines Cess and G7. In addition, no effective T3 modulation was observed with glutaraldehyde-fixed monocytes. The overall results seem to indicate that effective modulation of the T3 antigen by OKT3 antibody requires the active participation of Fc receptors on monocytes. 相似文献
26.
Hiroyoshi Hoshi Mikio Kan Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1987,23(10):723-732
Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on
human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase
mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented
medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth
factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed
that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported
attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined
as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically
stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte
number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could
be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned
medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein
(1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The
results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification
and characterization of additional normal hepatocyte growth factors.
This work was supported by NIH grant DK35310.
Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes.
In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that
factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional
growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate
that the malignant transformation of these cells may involve the production of autocrine growth stimulators. 相似文献
27.
The exchange behavior of the guanine N(1) and uracil N(3) protons in the self-complementary hexanucleotide r(ApApGpCpUpU) has been studied at 5 degrees C in 80% H2O/20% D2O by proton NMR. Under these conditions, the hexanucleotide forms a stable miniduplex. The exchange rate of all Watson-Crick NH protons is unaffected by addition of trifluoroethylamine up to 0.07 M. On the other hand, addition of phosphate buffer, pH 6.9, enhances the exchange rate of the uracil N(3) protons of both terminal and internal A X U base pairs but does not influence the exchange rate of the guanine N(1) protons of the central G X C base pairs. Catalysis by increased phosphate concentrations results in an open-limited rate of the internal A X U base pairs with kex = 233 s-1, equivalent to a lifetime of 4.3 ms. The proton exchange of the central G X C is regulated by the opening rate of the central core of the miniduplex. On the other hand, the sensitivity of the exchange rate of internal as well as of terminal A X U base pairs can be explained by their reduced lifetime due to end "fraying" and a subsequent catalysis of the exchange process from the opened state. These results suggest that it may be possible to probe labilized parts of RNAs such as tRNA by gradual addition of the exchange catalyst phosphate and to monitor their exchange rates by proton NMR. 相似文献
28.
Jila H. Boal Scott F. Deamond Daniel E. Callahan Sarah A. Bruce Paul O. P. Ts'o L. L. Kan 《Cell biochemistry and biophysics》1989,14(3):245-256
The nuclear magnetic resonance (NMR) parameters, spin-lattice (T1), and spin-spin (T2) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally
been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have
both spent a short time in culture. We have previously demonstrated that although the T1 values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different
when measured at 300 MHz, the 300 MHz T2 values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics
8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible
expression of neoplasia-associated phenotypes, T1 and T2 were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz
T2 values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor
(FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted
in smaller T2 values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T2 values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells. 相似文献
29.
Expression and function of the UM4D4 antigen in human thymus 总被引:3,自引:0,他引:3
D A Fox L S Chan L Kan O Baadsgaard K D Cooper 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(7):2166-2175
UM4D4 is a newly identified T cell surface molecule, distinct from the Ag receptor and CD2, which is expressed on 25% of peripheral blood T cells, resting or activated. Monoclonal anti-UM4D4 is mitogenic for T cells and T cell clones. Since alternative activation pathways independent of Ag/MHC recognition may be important in thymic differentiation, the expression and function of UM4D4 was examined in human thymus. UM4D4 was found on the surface of 6% of thymocytes. All thymocyte subsets contained UM4D4+ cells but expression was greatest on thymocytes that were CD1- (12%), CD3+ (11%) and especially CD4-CD8- (18%). CD3+CD4- CD8- cells, most of which bear the gamma delta-receptor, were greater than or equal to 50% + for UM4D4. Moreover, anti-UM4D4 was comitogenic for thymocytes together with PMA or IL-2. Anti-UM4D4 also reacted strongly with a subset of thymic epithelial cells in both cortex and medulla. Dual color fluorescence microscopy, with anti-UM4D4 and antibodies to other thymic epithelial Ag, showed UM4D4 expression on neuroendocrine thymic epithelium but not on thymic fibrous stroma. Thus, UM4D4 is expressed on, and represents an activation pathway for, a subset of thymic T cells. In addition, this determinant, initially identified as a novel T cell activating molecule, is broadly expressed by neuroendocrine thymic epithelium. Although the function of UM4D4 on the thymic epithelial cells is not yet clear, it is possible that UM4D4 represents a pathway for the functional activation of a subset of the thymic epithelium as well as a subset of thymocytes, thus playing a dual role in T cell differentiation. 相似文献
30.
Norimichi Kan Takashi Okino Masaki Nakanishi Kohei Satoh Kazuhisa Ohgaki Takayoshi Tobe 《Cancer immunology, immunotherapy : CII》1989,28(4):260-266
Summary The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 105 tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1×107 tumor-bearer cultured lymphocytes and 4×107 immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2+ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lytl+ and Lyt2+ T-cells.A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the 51Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells. 相似文献