摘要:

AbstractGenomic DNA extending over 10 kb 5′of the transforming growth factor-β2 (TGF-β2) coding region was isolated from a human lung fibroblast lambda phage library. A 5.6 kb Hind III fragment containing the 5′-untranslated region and flanking sequences was sub-cloned and sequenced. SI nuclease protection analysis identified a transcriptional initiation site 1357 nucleotides 5′of the methionine initiation codon (ATG). A“TATA box”consensus sequence was identified 30 bp from this transcriptional start site; however, consensus“CAT box”sequences were not observed. Approximately 50 nucleotides of homopurine-pyrimidine [d(GA.CT)50] sequence were identified in the 5′-untranslated region, as well as two short open reading frames of 5 and 45 amino acids. Several AP-1, AP-2, CRE and SPl-like DNA consensus sequence elements were also identified surrounding the transcription initiation site. 5′-deletion mutants of the promoter region were fused to the chloramphenicol acetyl transferase (CAT) gene and promoter activity of the isolated genomic DNA was demonstrated in several cell lines. DNA constructs containing nucleotides between -508 to +63 demonstrated high levels of promoter activity. However, sequences between–778 and–508 nucleotides modulated this promoter activity in a manner which was dependent upon the cell line utilized, suggesting that regulation of TGF-β2 gene transcription may be dependent upon the cellular background. The TGF-β2 promoter is markedly different from the promoters that have been recently characterized for TGF-β1 and TGF-β3.

ISSN:0897-7194    

DOI:10.3109/08977199109043910

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractImmuno-localization of BUdr was used to identify DNA synthesis in vitro in chicken embryonic bone cells stained positively or negatively for alkaline phosphatase activity. The results were similar to, but more sensitive than, our standard bioassay which assesses3H-thymidine incorporation into DNA by liquid scintillation counting, and more rapid than autoradiographic localization of3H-thymidine. SGF/IGF-II and bFGF stimulate cellular proliferation equally in ALP(+) and ALP(-) cells. In contrast, IGF-I and TGF-/J stimulate proliferation more in the ALP(-) than ALP(+) cells. The greatest increase in DNA replication of ALP(-) cells occurred following incubation with SGF/IGF-II or TGF-/3, and in the ALP(+) cells with SGF/IGF-II or bFGF. TGF-/3 stimulated cellular proliferation at the lowest dose (1 ng/ml). The differential effect of the growth factors on each population of cells indicates that all these bone-matrix derived growth factors may play different roles in the local regulation of skeletal metabolism.

ISSN:0897-7194    

DOI:10.3109/08977199109043911

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractThe single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not one but multiple proteins of 24, 23, 22 and 18 kD. Although bioactivities of the 18 kD protein are currently used to define bFGF gene function, it is not yet known if the three larger proteins have these same bioactivities or whether they will serve to define new bFGF gene functions. In this report we present a comparative study describing the de novo synthesis, transport, processing and intracellular location of individual bFGF isoforms. Data from cDNA mutagenesis and COS cell expression experiments show that individual isoforms are differentially localized to either the cell surface or to the nucleus. The 24, 23 and 22 kD proteins (CUG-mediated initiation) exclusively localize in the nucleus while the 18 kD protein (AUG-mediated initiation) is preferentially exported onto the cell surface, but is not released into the surrounding culture medium. Specific CUG or AUG translation initiation codons are necessary and sufficient for the synthesis of each isoform examined and thereby, indirectly, mediate differential localization. Since bFGF does not contain the characteristic signals predicted for cell surface or nuclear targeting, our continuing studies will either unmask its functionallv equivalent domain(s) or will identify the requisite participation of yet unknown cellular components.

ISSN:0897-7194    

DOI:10.3109/08977199109043912

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractIn order to understand the regulation of basic fibroblast growth factor (bFGF) gene expression, we have cloned and characterized the human bFGF gene and its regulatory elements. Using restriction endonuclease digestion, we have mapped the entire gene and sequenced all intron/exon boundaries to confirm authenticity and to determine organization. The data show that intron 1 is at least 16 kb long while intron 2 is 16 kb long. The human bFGF gene, including its three exons, is therefore at least 36 kb long. There are five GC boxes which may represent SP-1 binding sites and one potential AP-1 binding site within the core promoter region. Primer extension analysis indicates the presence of one bFGF-RNA transcription start site. We used a standard bacterial CAT gene expression system to identify the DNA sequence containing the functional bFGF gene promoter. Deletion analysis suggests the presence of two negative regulatory elements; one in the non-transcribed 5′-promoter region and the other within transcribed (but non-translated) sequences 3′of the promoter core.

ISSN:0897-7194    

DOI:10.3109/08977199109043913

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractMonoclonal antibodies, each recognizing interleukin 2 receptor (IL-2R)α, orβ, were used to see the regulatory mechanisms of the expressions on leukemic cells from a patient with T4 chronic lymphocytic leukemia (T4-CLL). Cells from this patient expressed only IL-2Rβ, and the expression was enhanced by medium cultivation. IL-1 enhanced the expression of not only IL-2Rβbut also IL-2Rαon the cells. Binding studies using125l-IL-2 snowed the presence of an intermediate receptor (734 sites/cell, Kd = 1.2nM) and a few high-affinity receptor (172 sites/cell, Kd = 132pM) on cells cultured with IL-1. IL-2 and IL-1 synergisti-cally promoted the proliferation of the cells, suggesting that the induced IL-2R was functional. In addition, anti-IL-1 antibodies inhibited IL-2Rβexpression by cultured cells, suggesting that it was dependent on IL-1 produced by the leukemic cells. These findings suggested that IL-1 might enhance the expression of IL-2Rβin a subset of human T cells, and implied a role of IL-1 in the proliferation of the leukemic T cells.

ISSN:0897-7194    

DOI:10.3109/08977199109043914

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractThe antioxidant enzyme superoxide dismutase (SOD) was previously shown to inhibit both the proliferation of murine erythroid DA-1 cells growing in the presence of Interleukin-3 (IL-3) and the DNA synthesis of marrow erythroid progenitor cells (BFU-E) in vitro. We show here that the inhibition of marrow cell DNA synthesis by SOD is specific for BFU-E and erythroid precursors (CFU-E), with other myeloid progenitors (CFU-GM) and stem cells (CFU-S) being unaffected, and IL-3 blocks the inhibitory effects of SOD on BFU-E in a dose-dependent manner. Extending earlier observations on the effects of SOD on cell proliferation, it was found that SOD was capable of inhibiting DA-1 cell proliferation supported by either IL-3 or erythropoietin (epo), but had no effect on IL-3 dependent FDCP-1 cells, nor on epo-dependent HCD-57 cells. Of several murine erythroleukemia cell lines tested, only those transformed with Friend SFFVa virus were inhibited by SOD, while those transformed with Friend SFFVp or MuLV virus were not affected. These results show that the effects of SOD are not antagonistic to particular growth factors but rather the inhibition is specific for eryrthroid cells, and cells of the proper stage can be inhibited even if they have been transformed to factor independence.

ISSN:0897-7194    

DOI:10.3109/08977199109043915

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractMany receptors for cellular growth factors are known to be protein tyrosine kinases which become activated upon ligand binding at their extracellular domain. We describe here a method to detect the activation state of Epidermal Growth Factor receptor (EGFr) with a monoclonal antibody (mAb74). This antibody was found to preferentially recognize the ligand-activated EGFr as detected by immunoprecipitation, Western blotting and immuno-cytochemical techniques. mAb74 did not recognize other tyrosine-phosphorylated proteins and was not inhibited by phosphotyrosine, suggesting that it is recognizing an epitope specific for the ligand-activated EGF receptor. The reactivity of mAb74 towards EGFr was closely correlated with the EGF-dependent tyrosine phosphorylation of endogenous substrates. This antibody allows one to detect the activated EGF receptor in vitro or in vivo even in a complex mixture of other tyrosine kinases and substrates.

ISSN:0897-7194    

DOI:10.3109/08977199109043916

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractHsp90 is a heat-shock protein constitutively expressed in most cells. Besides regulation by thermal stress, the expression of hsp90 is also positively regulated by developmental and mitogenic stimuli. The effect of serum and insulin on protein and hsp90ar-mRNA levels has been studied in the chicken hepatoma cell line DU249. The culture of cells in serum-free medium resulted in a decrease of hsp90α-mRNA level. A transient increase was observed at 6–9 h after serum restimulation. The expression of hsp90 gene was also increased by insulin alone in a dose-dependent manner and was maximum between 6 and 9 h treatment. The insulin induced increase of hsp90α-mRNA was suppressed by cycloheximide (10μg/ml) but not by an inhibitor of DNA synthesis, demonstrating that this induction requires protein neosynthesis. In serum starved cells, other growth factors (IGFi, EGF and bFGF) showed a positive effect on hsp90α-mRNA level which took place before DNA synthesis with the same time-course as that of insulin. With PDGF, the induction of hsp90α-mRNA occurred earlier. The time interval between the maximum of hsp90α-mRNA induction and that of DNA synthesis was the same for all growth factors studied. From these results, we conclude that growth factors acting via tyrosine kinase receptors up-regulate hsp90α-mRNA level in a DNA synthesis independent manner, possibly in late G1.

ISSN:0897-7194    

DOI:10.3109/08977199109043917

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor