摘要:

AbstractIt is possible to convert milk glands of transgenic animals into bioreactors producing heterologous proteins such as scarce human pharmaceuticals. To predictably and successfully engineer the milk gland, we will need a thorough understanding of its physiology. Expression studies in transgenic animals have located mammary specific and hormone inducible transcription elements in the promoter/upstream regions of milk protein genes, and transfection studies in cell lines or primary cells have identified constitutive and hormone inducible elements. Most importantly, it appears that in addition to individual promoter based transcription elements structural features of milk protein chromosomal loci may contribute to the tight developmental and hormonal regulation.I will discuss milk protein gene regulation with emphasis on regulatory differences between genes and species, and the possibility that transcription elements function only properly within genetically defined chromatin domains. Novel strategies to build mammary expression vectors and to test their functionality without pursuing the standard transgenic route will be presented. Finally, I will discuss homologous recombination with the goal to target milk protein genes. Only through the domestication of milk protein genes will we be able to use their full potential in the mammary bioreactor.

ISSN:0730-2312    

DOI:10.1002/jcb.240490402

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

年代:1992

数据来源: WILEY

摘要:

AbstractMilk is a complex bio‐colloid which presents some unique problems for the protein isolation chemist, but the majority of the processing criteria for purifying recombinant proteins are the same as with any complex biological mixture. The casein micelles and fat globules behave as separate phases; they prevent filtration of the milk and interfere with the usual separation methods. The usual first step is to centrifuge the milk to remove the fat and precipitate the casein micelles with low pH or precipitating agents. Some recombinant proteins may associate to some degree with the micelles which may necessitate solubilizing them with chelating agents. If the majority of the product protein associates with either the fat or micelles, this can be used to advantage. Once the casein micelles have been removed or disrupted, the clarified milk can be processed by the usual separation methods. There also are proteases in milk which can degrade recombinant proteins. The greatest advantage of producing recombinant proteins in milk is the high concentration which can be obtained. The high levels of product protein can alleviate many problems associated with the application of classical purification strategies to transgenic milk protein

ISSN:0730-2312    

DOI:10.1002/jcb.240490403

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

年代:1992

数据来源: WILEY

摘要:

AbstractBy use of indirect immunofluorescence it was shown that the phosphatidylinositol transfer protein (PI‐TP) in 3T3 mouse fibroblast cells is associated with the Golgi system. This was concluded from double‐labeling experiments with TRITC‐labeled Ricin which binds to sugar residues that are specifically processed in the Golgi system. Independent evidence for this association was provided by the fact that dissociation of the Golgi system by brefeldin A was reflected in an extensive redistribution of PI‐TP labeling. In addition, PI‐TP is localized in the cytoplasm and in the nucleus. In exponentially growing cells an enhanced labeling of PI‐TP was observed in the cytosol and in the Golgi system in comparison with quiescent cells. By Western blot analysis and by transfer activity assays, it was confirmed that the concentration of PI‐TP was increased in exponentially growing cells. These results strongly suggest that PI‐TP fulfills a role in the functioning of th

ISSN:0730-2312    

DOI:10.1002/jcb.240490404

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

年代:1992

数据来源: WILEY

摘要:

AbstractNuclear factors from HeLa cells were isolated by elution of DNA‐cellulose bound proteins with a double stranded synthetic oligonucleotide corresponding to the region from −34 to −79 of the human transferrin receptor (TR) gene promoter. The eluted proteins were further purified and separated from the oligonucleotide by ion exchange chromatography. Proteins within the resulting fraction bound with specificity to the TR promoter. Retardation gel analysis and competition with specific double‐stranded oligonucleotides show that multiple factors present in this fraction compete for binding within the same region of the TR promoter. Footprinting experiments demonstrate that these factors contact a GC‐rich element that is within the region that is required for enhanced expression of the gene in proliferating cells. One of the factors protects an extended DNA sequence but, still contacts the GC‐r

ISSN:0730-2312    

DOI:10.1002/jcb.240490405

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe overall coordination of cell structure and function that results in gene expression requires a spatial and temporal precision that would be unobtainable in the absence of structural order within the cell. Cells contain extensive and elaborate three‐dimensional skeletal networks that form integral structural components of the plasma membrane, cytoplasm, and nucleus. These skeletal networks form a dynamic tissue matrix and are composed of the nuclear matrix, cytoskeleton, and extracellular matrix. The tissue matrix is an interactive network which undergoes dynamic changes as cells move and change shape.Pathologists have long recognized cancer in pathologic specimens based on the altered morphology of tumor cells compared to their normal counterparts. The structural order of cells appears to be altered in transformed cells. This structural order is reflected in the altered morphology and motility observed in transformed cells compared to their normal counterparts, however, it is unclear whether the structural changes observed in cancer cells have any functional significance. We report here on the nature of the physical connections between the nucleus and cell periphery in nontransformed cells and demonstrate that the nucleus is dynamically coupled to the cell periphery via actin microfilaments. We also demonstrate that the dynamic coupling of the nucleus to the cell periphery via actin microfilaments is altered in Kirstenk‐ras transformed rat kidney epithelial cells. This loss of structure‐function relationship may be an important factor in the process of cell transform

ISSN:0730-2312    

DOI:10.1002/jcb.240490406

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

年代:1992

数据来源: WILEY

摘要:

AbstractMurine embryonal carcinoma (EC) F9 cells do not produce interferon (IFN) at the protein or RNA level in response to inducing agents, while retinoic acid differentiated F9 cells do produce IFN. A probe was constructed spanning positions −104 to −39 of the human β‐IFN upstream regulatory region to examine this developmental control at the level of a transcriptional regulatory mechanism. Gel mobility shift analyses were used to examine this molecular mechanism to determine whether the differential expression of positive or negative trans‐acting factors may act to control β‐IFN expression in undifferentiated EC cells. These analyses showed that while nuclear extracts from poly‐l,C induced L929 cells, in the IFN producing cell line, showed two shifted bands, nuclear extracts from both induced and uninduced F9 cells showed only one shifted band using the −104/ −39 probe. While this single shifted band co‐migrated with the faster migrating species of L929 cell extracts, competition analysis revealed differences between the two complexes. An oligonucleotide representing the positive regulatory domain PRDII competed efficiently for the probe when induced F9 cell extracts were examined, but failed to compete when induced L929 cell extracts were examined. In contrast, an oligonucleotide representing the positive regulatory domain PRDI competed very well when induced L929 cell extracts were examined but had only a minimal effect when induced F9 cell extracts were examined. These data suggest the involvement of developmentally regulated transcriptional factors(s) which have yet

ISSN:0730-2312    

DOI:10.1002/jcb.240490407

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

年代:1992

数据来源: WILEY

摘要:

AbstractOkadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26‐fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphory‐lated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno‐staining with anti‐vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two‐dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent‐solubility of the protein. In untreated cells, the detergent‐soluble and ‐insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure‐function regulation of cytoske

ISSN:0730-2312    

DOI:10.1002/jcb.240490408

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

年代:1992

数据来源: WILEY

摘要:

AbstractNuclear extract of human erythroleukemic cell line K562contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 μM hemin. When globin synthesis was inhibited by cycloheximide (100 μg/ml) or Actinomycin D (1 μg/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to Gγ and Aγ globin promoters but not to β‐globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene exp

ISSN:0730-2312    

DOI:10.1002/jcb.240490409

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe effect of a tumor promoter, 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventiricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): “Biology of Isolated Adult Cardiac Myocytes.” In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284–287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50–250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA‐treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of α‐actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular

ISSN:0730-2312    

DOI:10.1002/jcb.240490410

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

年代:1992

数据来源: WILEY

摘要:

AbstractAlpha‐1 antitrypsin messenger RNA (A1AT mRNA) was determined in alveolar macrophages and in peripheral blood monocytes of healthy individuals using a sensitive RNase protection assay. Determinations were made of bacterial lipopolysaccharide (LPS) stimulated and unstimulated cells. We found that the amount of A1AT mRNA increased 7.3 and 14 times after 4 h of incubation with LPS for monocytes and macrophages, respectively (relative to total RNA). The increase was 12.3 and 14.8 times, respectively, when expressed as increase per cell. In both cell types there was wide interindividual variation in LPS response: 2–36 and 5–12 times for monocytes and macrophages, respectively.The possible significance of A1AT production of monocytes and macrophages may be the local control of granulocytic proteases such as elastase and cathep

ISSN:0730-2312    

DOI:10.1002/jcb.240490411

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

年代:1992

数据来源: WILEY