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Biomedical subjects

J F DeLamarter

Publications and source records attributed to J F DeLamarter.

29 records · Page 2Linked to original sources
PubMed1988

The effect of stem cell proliferation regulators demonstrated with an in vitro assay.

Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.

Animals↗
PubMed1987

Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells.

Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein--lacking any carbohydrate--had by far the highest specific activity observed for the recombinant hGM-CSFs.

Animals↗
PubMed1987

Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor.

Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli. The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties. The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence. The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation. This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium. Human GM-CSF had no activity on murine bone marrow or murine leukemic cells. There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF. Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells. The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide production and lysozyme secretion. Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1985

Recombinant murine GM-CSF from E. coli has biological activity and is neutralized by a specific antiserum.

We report the production and characterization of a mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) made in Escherichia coli. The synthesis of mGM-CSF was directed by a plasmid containing a gene isolated from the EL-4 cell line. After induction of expression and accumulation of the protein in E. coli, mGM-CSF accounted for 10% of total cellular protein. This recombinant mGM-CSF was purified to 90% homogeneity by chaotrope extraction and gel filtration. Recombinant mGM-CSF, like the native molecule, stimulates the growth of granulocyte and macrophage colonies in serum-free cultures of mouse bone marrow cells. Antibodies raised against recombinant mGM-CSF not only reacted with the recombinant protein but also neutralized the biological activity of both native and recombinant mGM-CSF. These results indicate that the functional structure of the recombinant protein is similar to that of native mGM-CSF.

Amino Acid Sequence↗
PubMed1985

Vasoactive intestinal peptide: expression of the prohormone in bacterial cells.

A complementary DNA (cDNA) to the messenger RNA for the preprohormone of human vasoactive intestinal peptide (VIP) has been isolated and characterized. This cDNA extends from 65 bases 5' of the AUG translation start codon through the entire 3' untranslated region. Using this cDNA we have constructed expression plasmids which allow the synthesis of 120 out of the 150 amino acids of the prohormone in E. coli. This portion of the prohormone gene was either fused to a segment of a bacteriophage structural gene or expressed alone. When expression was induced the fusion protein constituted 15% of the total bacterial cell protein while the prohormone alone was 5%. Both proteins are recognized by antiserum raised against porcine VIP. They provide protein to study the precursor-product relationship of the hormone plus the possibility of identifying cryptic regulatory peptides contained within the prohormone.

Base Sequence↗
PubMed1981

Transcriptional control of endogenous virus genes in murine lymphocytes.

The expression of endogenous retrovirus in murine lymphocytes is under genetic control and also depends on the differentiation state of the lymphocytes. We have used a cDNA probe complementary to induced virus RNA to quantify transcription of virus sequences in lymphocytes from mitogen-stimulated lymphocytes of the AKR, 129/J and Balb/c mice. Balb/c lymphocytes show the clearest case for induction of new virus sequences in response to stimulation. All strains including 129/J show expression of virus sequences in unstimulated control lymphocytes. The data indicate that mitogen induction of endogenous retrovirus is regulated at the transcriptional level.

Animals↗