摘要:

AbstractRecent progress has been made concerning the understanding of the molecular pathways that mediate the growth suppressive effects of inhibitory cytokines. Interferons, interleukin‐6 and transforming growth factor‐β were investigated in these studies. Cell lines that display growth sensitivity to all three cytokines and growth resistant derivates provided a suitable genetic background to determine whether common or unique post‐receptor elments mediate the effects of each cytokine. three nuclear genes, c‐myc, RB, and cyclin A were found to be common key downstream targets along the cytokine induced growth suppressive pathways. Genetic and pharmacological manipulations proved that these molecular responses fall into few complementary pathways that function in parallel to achieve the cytokine mediated GO/G1 arrest. New strategies, such as knock out anti‐sense gene cloning were developed and they currently provied powerful tools for the isolation of genes along the signaling pathways of growth arrest. © 1992 Wile

ISSN:0730-2312    

DOI:10.1002/jcb.240500102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240500103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240500104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractPancreatic adenocarcinomas induced in Syrian hamsters by treatment with N‐nitrosobis(2‐oxopropyl) amine express blood group A antigen, which is absent in normal pancreatic cells. On membrane glycoproteins purified from tumors, blood group A antigen has been found to be expressed on multiantennary Asn‐linked complex glycans. In this study, we investigated the effect of inhibitors of Asn‐glycan processing on blood group A antigen bearing glycan structures in a cell line (PC‐1) established from a primary induced pancreatic cancer. Expression of blood group A antigen on cells and in membrane preparations was blocked by treatment with 1‐deoxymannojirimycin, an inhibitor of mannosidase I, but was retained after treatment with swainsonine, an inhibitor of mannosidase II. However, swainsonine treatment altered the glycan structure associated with blood group A antigen from an endoglycosidase H resistant type to a sensitive type, indicating that the blood group A structure might shift from a complex type to a hybr‐id type glycan by this treatment. These results demonstrate that Asn‐linked glycans carry the major blood group A antigens in PC‐1 cells. © 19

ISSN:0730-2312    

DOI:10.1002/jcb.240500105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractSertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type 1 collagen‐coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane‐associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium. © 1992 Wiley‐Lis

ISSN:0730-2312    

DOI:10.1002/jcb.240500106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractGlucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the proα2(1) collagen gene required for the glucocorticoid‐mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5′ flanking region fragment from −2048 to +54 and the intronic fragment from +418 to +1524 of the mouse α2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid‐mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the ppossible role of the first intron fragment in the dexamethasone‐mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was trasnfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone‐mediated response was then localized within the 5′ flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions −2048 to −981 and −506 to −351 were required for the dexamethasone response of gene activity. However, the DNA stretch from −981 to −506 was not. Analysis of the DNA sequences of these regions revelaed a singel GRE at −1023 to −1018 and a modified doublet at −873 to −856. The doublet GRE contains and A/T strand switch of the third base pair as compared to the single GRE and is not necessary for dexamethasone regulation of gene activity. All‐trans‐retionic acid increased CAT activity of the same pR40 CAT construct transfected in the mouse fibroblasts. DNA sequencing revealed a RARE and a modified RARE in the stretch of DNA from −981 to −506. Deletion of only the latter DNA region eliminated the elevation of CAT activity elicited by all‐trans‐retinoic acid. Our results indicate that the single GRE and the RARE are required for glucocorticoid and retinoic acid regulation o

ISSN:0730-2312    

DOI:10.1002/jcb.240500107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractFour residues in the carboxy‐termianl domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site‐directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor‐ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge‐conservative replacement of glutamate 40 with asparate resulted in a decrease in receptor binding affinity to 30% relative to wild‐type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitutiono of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side‐chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274–2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor‐ligand association. The most highly conserved of the four residues studied in this report, aspartate 46, was replaced with alanine, tyrosine, and arginine, resulting in a decrease in relative receptor affinity to 23, 14, and 4 percent, respectively, and suggests the importance of an acidic group at this site of EGF. The ability to generate sufficient yields of mutant recombinant EGF protein was sensitive to the type of side‐chain substitutions generated at the sites described in this report and may indicate a role for these residues in the formation of the EGF structure apparently required for productive yields of EGF proteins in the expression system used in this study. © 19

ISSN:0730-2312    

DOI:10.1002/jcb.240500108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe report on the discovery and isolation of DNA‐ and RNA‐containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of ∼ 90 and ∼ 25kbp. The deoxyribonucleoprotein‐ribonucleoprotein complexes conatin RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large‐cell lymphoma cells by restriction digestion withMsp‐1, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two‐dimensional (isoelectric focusing/Mr) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of ∼ 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific acticvites. Using the DNP/RNP complexes a discrete DNA polymerase α product of ∼ 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase α inhibitor aphidicolin. RNA polymerase assays in the presence of excess α‐amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex‐associated enzyme activities to be cell‐cycle dependent. The DNA and RNA polymerase‐related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors. Hybridization sutides showed that the complexes contained theabloncogene which is expressed in RAW117 cells, but not the β‐casein gene which is not expressed in this ce

ISSN:0730-2312    

DOI:10.1002/jcb.240500109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractLeukoregulin(LR), a product of activated T‐cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455–1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin‐1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose‐ and time‐dependent elevation of stromelysin‐1 mRNA levels, the maximum enhnacement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/ reporter gene construct containing 1.3 kb of 5′ flanking DNA of the human stromelysin‐1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This inhancement was abolished by a single base substitution in the AP‐1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP‐1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP‐1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin‐1 gene expression by LR is mediated by AP‐1 regulatory elements which are necessary, but not sufficient, for gene respons

ISSN:0730-2312    

DOI:10.1002/jcb.240500110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe aim of this study was to address whether there is a fundamental difference in regulation of histone gene expression in cells that have become quiescent but retain the ability to proliferate, compared with those cells that have differentiated. We compared multiple levels of regulation of histone gene expression during 3T3‐L1 pre‐adipocyte differentiation. Confluent cells induced to differentiate by treatment with insulin, dexamethasone, and isobutylemethylxanthine initially exhibited an increased proliferative response compared with cells given serum alone. This initial differentiation response was associated with a twofold increase in both histone gene transcription and cellular histone mRNA levels, as well as with enhanced sequence‐specific binding of nuclear factors to the proximal cell‐cycle‐regulatory element of the H4 histone promoter. Transforming growth factor β1, an inhibitor of 3T3‐L1 differentiation, increased both the percentage of proliferating cells and the cellular levels of histone mRNA when given in addition to serum stimulation, but no enhancement of these parameters was observed upon addition of TGFβ1to the differentiation treatment. Interestingly, although TGFβ1enhanced binding of nuclear factors to the proximal cell cycle regulatory element of the histone promoter, these protein/DNA interactions were not associated with an increase in histone transcription. Our results are consistent with the down‐regulation of histone gene expression at confluency being controlled primarily at the post‐transcriptional level, in contrast to an increased involvement of transcriptional down‐regulation at the onset of differentiation. ©

ISSN:0730-2312    

DOI:10.1002/jcb.240500111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY