摘要:

AbstractA new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS),αcristallin (αC) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC50= 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of125I-bFGF bound to the Muller cell receptors did not occur at 4°C. At 37°C, however, there was a rapid decrease in receptor-bound125I-bFGF due to the downregulation of bFGF rjeceptors. Concomitantly125T-bFGF appeared inside the Muller cells. After 2 h,125I-bFGF began to be degraded and after 6 h three fragments of 16 kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these factors may be the mediators involved in the communication between glial cells and neurons.

ISSN:0897-7194    

DOI:10.3109/08977199109000260

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractA heparin-binding protein with neurotrophic activity for perinatal rat neurons, termed HBNF, was purified to homogeneity from bovine brain utilizing pH 4.5 extraction, ammonium sulfate precipitation, cation exchange and heparin-Sepharose affinity chromatographies, and reverse phase HPLC. In the presence of protease inhibitors during extraction, a protein with an apparent molecular weight of 18kDa was obtained in a yield of approximately 0.5 mg/kg brain tissue. The amino acid sequence of the first 114 residues of HBNF was determined and found to highly homologous to the cDNA-derived amino acid sequence of human HBNF, a 136-residue protein. Bovine and human HBNFs have identical molecular weights as judged by SDS gel electrophoresis and very similar amino acid compositions. This and overall sequence conservation suggest that bovine HBNF is also a 136 amino acid protein with a calculated molecular weight of approximately 15.5 kDa. The apparent discrepancy between calculated and observed molecular weights of bovine HBNF (and of human HBNF of which the complete sequence is known) is most likely a result of the highly basic nature of HBNF. If protease inhibitors were omitted during tissue extraction, two additional proteins with lower apparent molecular weights and identical N-terminal sequences were isolated, with the smallest forms being the major product. Amino acid analysis showed that the smaller forms correspond to C-terminally truncated HBNFs with calculated molecular weights of 13.6 and 12.4 kDa, lacking approximately 14 and 22 residues. Comparison of the HBNF protein sequence with sequences stored in the Protein Identification Resource/Genebank databases reveals high homology to the translation product of the MK-1 gene, which is retinoic acid-inducible in embryonic carcinoma cells and developmentally expressed during gestation in mice.

ISSN:0897-7194    

DOI:10.3109/08977199109000261

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractThe IL3-dependent cell line FDCP-2 dies within 32 h of removal of IL3. Electron microscope studies indicate that 22 h after IL3 removal the nuclei are condensed, but the morphology of mitochondria and ribosomes is preserved. This pattern is characteristic of apoptosis. IL3 removal also results in the fragmentation of DNA into nucleosome-sized pieces, suggesting that an endonuclease is activated. The protein synthesis inhibitor, cycloheximide, enhances survival on IL3 removal, suggesting that death is an active process. The nuclease inhibitor, aurintricarboxylic acid, also enhances survival, suggesting a causal role for DNA fragmentation in apoptosis.

ISSN:0897-7194    

DOI:10.3109/08977199109000262

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractWe tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype

ISSN:0897-7194    

DOI:10.3109/08977199109000263

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractIn the presence of epidermal growth factor (EGF) a human colon cell line, LIM 1215, proliferates in serum-free medium. Under these culture conditions the cells are dependent on the presence of EGF for both proliferation and survival. In order to study the action of growth factors at different stages of the LIM 1215 cell cycle, pure populations of G1S and G2/M cells were obtained by cell sorting after supravital staining of the DNA with Hoechst 33342. Conditions were established for Hoechst 33342 staining which produced satisfactory DNA histograms and greater than 80% survival of cells. The kinetics of passage for sorted S or G2/M cells into G1werenotaffected by EGF or fetal calf serum. After sorting there appeared to be a 4 h delay before the cells proceeded in the cell cycle. Sorted S cells entered G2over an 8 h period and maintained this same transition period from G2into G1. If EGF or serum was present, these cells then re-entered the cell cycle after a variable delay and in an asynchronous manner. EGF was applied to S phase and G2/M phase LIM 1215 cells for periods of 2-10 h at various times after replating in serum-free conditions. Cells in S phase only responded to EGF as they passed from G2/M into G1. Exposure to EGF in S phase resulted in little growth stimulus once the cells returned to G1For cells in G2/M phase, EGF was required immediately to give the maximum stimulus for re-entering the cell cycle. If the EGF was delayed for more than 8 h, the cells did not re-enter the cycle within the following 20 h. Exposure to EGF for less than 2 h failed to stimulate proliferation. These results indicate that EGF must be present as cells enter G; from mitosis. Once the cells have entered G1EGF is required for a 10 h period for a large number of cells to re-enter the cycle from G1

ISSN:0897-7194    

DOI:10.3109/08977199109000264

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractThe role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras.Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21rasprotein.

ISSN:0897-7194    

DOI:10.3109/08977199109000265

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor