Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.
Interleukin 3 (IL-3 or multi-colony-stimulating factor) plays an important role in the hematopoietic response to inflammatory stimuli through its action on both immature and mature blood cells. Like other lymphokines, IL-3 is produced in response to activation of the T-cell receptor and protein kinase C pathways. By using nuclear run-on assays of quiescent and stimulated T-cell lines, we demonstrate that IL-3 gene expression is controlled, at least in part, at the level of transcription. Functional reporter gene analysis was used to delineate two regions of the IL-3 5' flanking sequence responsible for transcriptional stimulation. DNA binding proteins that potentially mediate these responses were then recognized by mobility-shift and DNase footprinting assays. One region responsible for transcriptional enhancement was localized to the sequence GATGAATAAT, the cognate site of a transcription factor, here termed NF-IL3-A. A second region of functional activity and protein binding was localized to a single transcription factor AP-1 site. In addition three functionally inhibitory regions were identified. These results, along with the further characterization of NF-IL3-A, will contribute to the understanding of IL-3 gene regulation in stimulated T cells.Among the growth factors involved in hematopoiesis, interleukin 3 (IL-3 or multi-colony-stimulating factor) affects the widest variety of cell types. In conjunction with other growth factors, IL-3 acts on marrow-derived progenitor cells to stimulate the development of erythrocytes, granulocytes, macrophages, and megakaryocytes (1, 2). In addition, it influences the growth and activation of lymphocytes (3), promotes the self-renewal of hematopoietic progenitor cells (4), and acts upon mature leukocytes to enhance their functional activity (5,6). In this way, IL-3 is thought to be involved in the host response to infectious, inflammatory, and immunologic stimuli.Constitutive expression of IL-3 has not been observed in any normal cell yet examined. By using the polymerase chain reaction, mRNA specific for human IL-3 has been identified in a number of cell sources (7), but IL-3 mRNA has only been found in T lymphocytes or T-cell lines treated with stimulators of the T-cell receptor or of the protein kinase C pathway (8). The molecular basis for this highly inducible and selective tissue specificity is uncertain.To further understand the regulation of IL-3 production, we have cloned the gene for human IL-3 and have performed a detailed analysis of the 682-base-pair (bp) region upstream of the start of IL-3 transcription. Our results suggest that IL-3 gene expression is controlled, at least in part, at the level of transcription and that binding of trans-acting factors at two distinct sites mediate much of this response. Nuclear Run-On Analysis. Cell lines were grown to 1 X 106 cells per ml and then stimulated with concanavalin A (Con A; MATERIALS AND METHODSBoehringer Mannheim) at 10 gg/ml and phorbol 12-myrisitate 13-acetate (PMA; Sigma) (100 ng/ml) for 15 min to ...
Granulocyte/macrophage colony-stimulating factor (GM-CSF) is an acidic glycoprotein that stimulates hematopoiesis in vitro and in vivo. Despite a high degree of sequence homology, the GM-CSFs from human and murine sources fail to crossreact in their respective colony-forming assays. On the basis of this rinding, a series of hybrid molecules containing various proportions of human-and murine-specific amino acid sequences were generated by recombinant DNA techniques and assayed for species-specific activity against human and murine marrow target cells. Two regions of GM-CSF, residues 38-48 and residues 95-111, were found to be critical for hematopoietic function. These regions are structurally characterized by an amphiphilic helix and by a disulfide-bonded loop, respectively, and are homologous in position in the human and murine growth factors. In addition, competition assays suggested that, together, these regions bind to the GM-CSF receptor.
Over the past 18 years we have been deeply involved with the synthesis and applications of stimuliresponsive polymer systems, especially polymerbiomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymerprotein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pHsensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.
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