Biomedical subjects

S G Shoemaker

Publications and source records attributed to S G Shoemaker.

7 recordsLinked to original sources

PubMed1994

Coordinate regulation of multiple human lymphokine genes by Oct-1 and potentially novel 45 and 43 kDa polypeptides.

The hematologic response to inflammation is critically dependent on the release of a family of glycoproteins termed CSFs. Central to this process is the coordinated T lymphocyte production of three of these proteins, granulocyte macrophage-CSF (GM-CSF), IL-3, and IL-5. In the course of our studies of the transcriptional regulation of IL-3 expression we noted that the same or similar proteins that bound to the downstream-activating region of the human IL-3 gene were also captured using DNA probes derived from sequences present in the GM-CSF and IL-5 genes. In this study, DNA-protein cross-linking techniques and reporter gene analyses were used to define the nuclear proteins that mediate transcriptional enhancement of these genes at one cis-acting site. These studies establish that Oct-1, a member of the POU family of transcription factors, binds to regions of the GM-CSF, IL-3, and IL-5 promoters and contributes to basal T cell gene transcription. In addition, although the c-jun proto-oncogene product was detected in stimulated human T cells, two additional T cell-specific proteins (45 and 43 kDa) bind to the GM-CSF, IL-3, and IL-5 promoters and are functionally and immunologically distinct from c-jun or OAP40 (jun-D). These proteins contribute to transcriptional enhancement of the IL-3 and GM-CSF genes in stimulated T cells. Together, these data suggest that the coordinate regulation of several lymphokine genes is based on responses to shared proteins. These studies further our understanding of the networks of transcriptionally active proteins responsible for the host response to inflammatory stimuli.

Base Sequence↗

PubMed1992

Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3.

IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to it receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs of these mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor.

Adult↗

PubMed1990

Muramyl dipeptide induces production of hemopoietic growth factors in vivo by a mechanism independent of tumor necrosis factor.

Monocyte products including TNF and IL-1 can stimulate hemopoietic growth factor production in vitro and in vivo. Endotoxin-resistant C3H/HeJ mice have a cellular defect in the regulation of TNF production and provide a model in which to examine the relative importance of TNF and IL-1. To assess the relative role of TNF and IL-1, we injected the synthetic bacterial cell wall derivative muramyl dipeptide (MDP) or LPS into C3H/HeJ mice. Both MDP and LPS stimulated hemopoietic growth factor production in a dose-dependent manner. Northern blot analysis of splenic and lung mRNA revealed that MDP treatment induced granulocyte-macrophage CSF and macrophage-CSF transcripts, whereas LPS treatment induced macrophage-CSF transcripts. Messenger RNA for granulocyte-CSF, IL-3, IL-4, and IL-5 was not detected in control or treated mice. MDP treatment induced IL-1 mRNA but not TNF mRNA, and TNF bioactivity was not detected in the serum of MDP-treated mice. In contrast, LPS treatment induced TNF production. These results identify the hemopoietic growth factors induced by MDP and LPS in vivo, and suggest that MDP stimulates growth factor production by a mechanism independent of TNF.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1990

Transcriptional regulation of interleukin 3 gene expression in T lymphocytes.

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.

Base Sequence↗

PubMed1989

Hematopoietic activity of granulocyte/macrophage colony-stimulating factor is dependent upon two distinct regions of the molecule: functional analysis based upon the activities of interspecies hybrid growth factors.

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 finding, 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.

Animals↗

PubMed1987

Synthesis and properties of vinyl monomer/enzyme conjugates. Conjugation of L-asparaginase with N-succinimidyl acrylate.

Monomer conjugation of an enzyme followed by copolymerization with free monomer is a useful method of enzyme immobilization. L-asparaginase was conjugated with N-succinimidyl acrylate. Analysis of the conjugated enzyme via isoelectric focusing showed that a molar ratio of 9.5 free monomers per enzyme was needed during the conjunction for each vinyl group bound. Only 3% of the enzyme activity was lost per vinyl group added, and conjugation of an average of four monomers per enzyme thermally destabilized the enzyme only at temperatures above 50 degrees C. Activity of the enzyme at physiological temperatures was relatively unaffected.

Acrylates↗

PubMedDate not supplied

The regulation of GM-CSF is dependent on a complex interplay of multiple nuclear proteins.

GM-CSF is an important mediator of hematopoiesis and its dysregulation may play a role in neoplastic and inflammatory conditions. Previous studies have demonstrated that GM-CSF production depends upon the accumulation of specific mRNA, which occurs by transcriptional and post-transcriptional mechanisms. In order to dissect the cis-acting sequences responsible for its regulation, we performed an extensive mutagenesis study spanning 54 nucleotides 5' of the GM-CSF coding region. Our analysis suggests that the previously-described functional elements of the GM-CSF promoter, kappa B and a repetitive CATTT/A motif, the former co-exists with an overlapping 9 nucleotide site which silences promoter activity, and the CATTT/A complex binds multiple polypeptides which differentially contribute to basal and inducible promoter activity. These two sites interact to provide tissue-appropriate and stimulus-specific promoter function. Using DNA-protein cross-linking and co-transfection studies, we demonstrate that the c-rel-related proteins p65 and p50 bind to the GM-CSF promoter and that p65 binding is primarily responsible for the enhancing effects at this site. In addition, we show that the GM-CSF kappa B decanucleotide is inadequate to provide full binding affinity; mutation of nucleotides flanking this site affect promoter function by altering NF-kappa B binding affinity. Together these results suggest that the transcriptional response of GM-CSF is dependent on a complex interplay of multiple DNA binding proteins.

Base Sequence↗