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Biomedical subjects

I Clark-Lewis

Publications and source records attributed to I Clark-Lewis.

At least 145 records · Page 8Linked to original sources
PubMed1989

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha.

Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.

Amino Acid Sequence↗
PubMed1988

Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity.

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.

Amino Acid Sequence↗
PubMed1988

Monoclonal antipeptide antibodies recognize IL-3 and neutralize its bioactivity in vivo.

Mixtures of synthetic peptides corresponding to segments of murine IL-3 or synthetic IL-3 were used to raise murine mAb. Several mAb able to recognize synthetic IL-3 were obtained, two of which exhibited significant cross-reactivity with native IL-3 as shown by precipitation of biosynthetically 35S-labeled IL-3 and their effectiveness as affinity reagents for the purification of IL-3 from conditioned medium. The amino acid sequence recognized by the two mAb was determined by using synthetic peptide segments of IL-3. In both cases binding of the mAb to synthetic IL-3 was inhibited best with a hexapeptide corresponding to the amino acid residues 130-135 of IL-3, although the mAb differed in other characteristics. Neither mAb neutralized IL-3 bioactivity in vitro. However, we observed that in vivo administration of one mAb abrogated the increase in splenic mast cells and their precursors that normally occurred in mice bearing a s.c. IL-3-producing tumor, WEHI-3B.

Animals↗
PubMed1988

Role of disulfide bridges in determining the biological activity of interleukin 3.

Total chemical synthesis of analog proteins was used to examine the requirement for specific disulfide bridges for the biological activity of interleukin 3 (IL-3), a growth factor that stimulates multiple lineages of hemopoietic cells. Four structural analogs of the mature, 140 amino acid murine IL-3 molecule were synthesized in which specific cysteine residues were replaced by alanines. In a quantitative IL-3 assay, based on [3H]thymidine incorporation into factor-dependent cells, the IL-3 analog with alanines substituted for all four cysteines--i.e., [Ala17,79,80,140]IL-3--had 1/500th as much activity as the molecule synthesized according to the native sequence. The two analogs [Cys17,79,Ala80,140]IL-3 and [Cys17,140,Ala79,80]IL-3 had similarly low activity, whereas the [Cys17,80,Ala79,140]IL-3 analog had 2000-fold higher activity than these three analogs, and 3-fold higher than the molecule with the native sequence. This shows that in IL-3 a single disulfide bridge, between cysteines 17 and 80, is required for biological activity that approximates physiological levels. This disulfide probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function.

Alanine↗
PubMed1987

Antipeptide antibodies of predetermined specificity recognize and neutralize the bioactivity of the pan-specific hemopoietin interleukin 3.

Ten peptides that corresponded to portions of the T cell lymphokine pan-specific hemopoietin interleukin 3 (IL 3) were synthesized, coupled to keyhole limpet hemocyanin, and used to raise antipeptide antibodies in rabbits. These antisera reacted to varying degrees with native biologically active IL 3. Antibodies directed against peptides corresponding to residues 1-29 at the NH2 terminus, 123-140 at the COOH terminus, and to residues 64-82 and 91-112 were affinity-purified on peptide columns. Immunoabsorbent columns produced from affinity-purified antibodies to the 1-29, 91-112, and 123-140 although not the 64-82 peptide were effective in depleting biologically active IL 3 from conditioned medium. However, the antibodies specific for peptides 91-112 and 123-140 had only a low affinity for native IL 3 and it was only in the case of the anti-1-29 antibodies that a significant amount of IL 3 remained bound after extensive washing and could be recovered from the column by acid elution. The affinity-purified antibodies directed to peptides 1-29, 91-112, and 123-140 significantly inhibited the biological activity of IL 3, although with different dose-response characteristics. Anti-1-29 antibodies inhibited bioactivity over a wide range of concentrations (down to 20 ng/ml) although the inhibition was never complete. In contrast, the anti-91-112 antibodies, although effective only at high concentrations, produced complete inhibition of biological activity. These experiments demonstrated that antibodies to defined peptides can be used to generate antibodies to native IL 3 and should form useful tools in analyzing the structure and function of the native molecules.

Animals↗
PubMed1987

Antipeptide antibodies define the NH2-terminal structure of the pan-specific hemopoietin interleukin 3.

Antipeptide antibodies were raised in rabbits against a synthetic peptide including the six NH2-terminal amino acids of the pan-specific hemopoietin interleukin 3 (IL 3). Affinity-purified antibody preparations specific for epitopes determined by residues 1 to 6 were immobilized and used as affinity columns. Up to 98% of IL 3 bioactivity in T cell-conditioned medium was depleted by these columns, as was 71 and 74%, respectively, of IL 3 aberrantly produced by the myeloid leukemias WEHI-274.14 and WEHI-3B. IL 3 produced in vivo in WEHI-3B tumor-bearing mice also bound to the anti-1-6 antibody column, up to 70% of the bioactivity being depleted from ascites fluid and up to 84% from the serum. These results suggest that all IL 3 secreted by T cells and the majority of the IL 3 molecules secreted by myeloid leukemias express epitopes determined by residues 1-6 and cannot have the NH2-terminal amino acid sequence initially reported for IL 3. These six NH2-terminal amino acids share similarities with the NH2-terminal amino acids of several other lymphokines, suggesting an important function for this hexapeptide.

Amino Acid Sequence↗
PubMed1986

Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3.

Interleukin-3 (IL-3), a protein of 140 amino acids, was chemically synthesized by means of an automated peptide synthesizer and was shown to have the biological activities attributed to native IL-3. Assays of synthetic analogues established that an amino terminal fragment has detectable IL-3 activity, but that the stable tertiary structure of the complete molecule was required for full activity. The results demonstrate that automated peptide synthesis can be applied to the study of the structure and function of proteins.

Amino Acid Sequence↗
PubMed1986

P cell stimulating factor release: a useful assay of T cell activation in vitro.

The coordinate production of multiple lymphokines by activated T cells allows the investigator a choice of assays for antigen recognition in vitro. In this paper, we examine the production of P cell stimulating factor (PSF, a lymphokine identical to interleukin 3) by L3T4+ Lyt-2-T cell clones. PSF production is antigen-specific, Ia-restricted and inhibited by a monoclonal antibody to L3T4, PSF, interferon-gamma (IFN-gamma) and interleukin 2 (IL 2) are all rapidly secreted after antigen or mitogen stimulation. The measurement of PSF avoids problems inherent in the IL 2 and IFN-gamma assays, namely absorption of IL 2 by the producer T cells and failure of the interferon assay to distinguish between interferons of the alpha, beta and gamma classes.

Animals↗
PubMed1985

Analysis of the binding of a hemopoietic growth factor, P-cell-stimulating factor, to a cell surface receptor using quantitative absorption of bioactivity.

Quantitative absorption of biological activity was used to study the interaction of persisting (P)-cell-stimulating factor (PSF), a T-cell-derived lymphokine, with PSF-dependent lines of hemopoietic cells (P cells). It was shown that suspension of P cells in medium containing PSF at 4 degrees C resulted in a diminution of PSF activity in the medium. Similar results were obtained with homogeneous, pure PSF or crude supernatants from cells secreting PSF. This diminution was specific and involved saturable, reversible binding of PSF to the cells rather than degradation of PSF or the release of an inhibitor. Calculations based on the measurement of PSF activity remaining after absorption and estimates of the specific activity of PSF indicated that there were approximately 1000 receptors/cell and that PSF bound with an equilibrium dissociation constant (Kd) of 5 X 10(-12) M. Increased amounts of PSF were absorbed at 37 degrees C; however, in the presence of metabolic inhibitors, the amount of PSF activity removed was equivalent to that seen at 4 degrees C.

Absorption↗
PubMed1985

Regulation of human granulocyte function by products derived from murine tumors.

Murine EL-4 thymoma was found to produce a factor that activated antibody-dependent cell-mediated cytotoxicity by human eosinophils and neutrophils. The eosinophil-activating factor had some properties similar to human eosinophil colony-stimulating factor in being heat stable, inactivated by 2-mercaptoethanol, and cochromatographing by gel filtration with a factor that promoted the growth of pure eosinophil colonies from human bone marrow. The neutrophil-activating factor from EL-4 was also heat stable but was not inactivated by 2-mercaptoethanol and was found in two peaks on chromatography, neither having human neutrophil-CSF activity but one cochromatographing with mouse granulocyte-macrophage CSF. EL-4 therefore secretes factors that activate two types of human granulocytes. Several other murine thymomas and macrophage cell lines produced a neutrophil-activating factor, the production of which could not be correlated with that of several other known lymphokines. Murine tumors produce factors that activate human granulocytes.

Animals↗
PubMed1985

Stimulation of bone marrow-derived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, persisting cell-stimulating factor.

Several lines of evidence indicated that P cell-stimulating factor (PSF), a T lymphocyte-derived lymphokine known to stimulate the growth of hemopoietic stem and progenitor cells, also acted on macrophages. PSF was absorbed from medium that had been mixed for two hours at 0 degrees C with either resident or thioglycollate-elicited peritoneal cells, suggesting the presence of receptors for PSF on cells in the population. The addition of pure PSF to populations highly enriched in either resident or elicited adherent peritoneal macrophages resulted in stimulation of macrophages with morphological changes, including increases in size, spreading, vacuolation, and the number of cytoplasmic processes, together with stimulation of proliferation and the phagocytosis of opsonized yeast. PSF also stimulated the incorporation of [3H]thymidine by bone marrow-derived adherent macrophages. Addition of pure PSF to cultures that contained only a single macrophage resulted in enhanced survival and proliferation of these isolated cells, demonstrating that the effect of PSF on macrophages was direct. These results indicate that PSF can stimulate well-differentiated functional macrophages and raise the possibility that the effects of PSF on macrophages may play a regulatory role in immune responses.

Animals↗
PubMed1985

Characterization of hemopoietic growth factors from T cells and the myelomonocytic leukemia WEHI-3B.

P-cell-stimulating factor (PSF) (also termed interleukin 3) produced by the T-cell clone A3-37.4, the T-cell hybridoma 123, the T-lymphoma EL4, spleen cells, and the myelomonocytic cell line WEHI-3B had a similar apparent mol. wt. and in each case eluted from a Waters C18 silica column at a concentration of acetonitrile of 38%. Both the PSF from the T-cell clones and from WEHI-3B stimulated the in vitro growth of cloned T-dependent mast cells and of colonies from normal bone marrow cells. The T-cell sources--but not WEHI-3B--also produced an additional, distinct hemopoietic growth factor that stimulated the growth of colonies of neutrophils and macrophages but did not support the growth of P cells. This factor was termed T-cell granulocyte-macrophage colony-stimulating factor (T-cell GM-CSF). T-cell GM-CSF eluted from a C18 silica column at an acetonitrile concentration of 41%, differing in this respect from both PSF, which eluted at 38% acetonitrile, and the GM-CSF produced by endotoxin-stimulated mouse lungs.

Animals↗
PubMed1984

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells.

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.

Amino Acid Sequence↗
PubMed1984

Effect of cloned interferon-gamma on expression of H-2 and Ia antigens on cell lines of hemopoietic, lymphoid, epithelial, fibroblastic and neuronal origin.

Interferon-gamma (IFN-gamma), tested in the form of the product of a cloned murine IFN-gamma gene, was found to increase the expression of H-2 antigens on cultured cell lines of a wide variety of cell types including factor-dependent hemopoietic cells, pre-B and B cells, macrophages, T cells, mast cells and cell lines of epithelial, fibroblastic and neuronal origin. However, IFN-gamma induced Ia antigens on only B cells, macrophages and T-dependent mast cells or persisting cells. On the basis of these results, we suggest that a major function of IFN-gamma is to potentiate immune responses by enhancing the expression of H-2 and Ia antigens on a variety of cell types.

Animals↗
PubMed1984

T-cell-lymphokines and the production and function of accessory cells.

The recent purification and cloning of T cell-derived growth factors, should lead to rapid progress in delineating their role in the generation of accessory cells and the regulation of their function. The techniques that have allowed detailed in vitro studies of the T-dependent mast cells, should be generally applicable to other bone-marrow-derived cells. Although the T-dependent mast cell (i.e. P cells) of the mouse appear to have a special propensity to persist in vitro and special techniques that promote the emergence of immortalized clones will probably be necessary to grow useful quantities of other cell-types in vitro using PSF, it should prove possible to develop factor-dependent lines of various bone-marrow-derived accessory cells such as dendritic cells and Langerhans cells. The availability of clonal populations will permit analysis of the interaction of factors such as haematopoietic growth factors, interferon, glucocorticoids and other mediators involved in inflammatory reactions such as the prostaglandins and leukotrienes - not only in the regulation of Ia-antigen expression but also in modulation of other accessory cell functions such as the secretion of IL-1 and perhaps ultimately the processing and presentation of antigens.

Animals↗
PubMed1984

P cell stimulating factor and glucocorticoids oppose the action of interferon-gamma in inducing Ia antigens on T-dependent mast cells (P cells).

The expression of Ia antigens of cultured lines of T-dependent mast cells (TDMC) or persisting (P) cells was modulated by three hormones: interferon-gamma (IFN-gamma), P cell-stimulating factor (PSF), and glucocorticoids. The induction of Ia antigens by cloned IFN-gamma was inhibited by a homogeneous preparation of PSF, the T cell-derived factor that TDMC require for their in vitro growth. This inhibitory effect of PSF was dose-dependent and could not be overcome by increasing the levels of IFN-gamma. Other cytokines such as granulocyte/macrophage colony-stimulating factor, T cell growth factor, and L cell-derived macrophage colony-stimulating factor had no inhibitory effect. Thus, two different T cell lymphokines, PSF and IFN-gamma, have opposing effects on the expression of Ia antigens on TDMC. Glucocorticoids (hydrocortisone, corticosterone, dexamethasone, prednisolone, and fludrocortisone) antagonized the induction of Ia antigens by IFN-gamma, whereas sex steroids (progesterone, estradiol, and testosterone) and a mineralocorticoid (aldosterone) did not. The inhibitory effect of glucocorticoids could not be overcome by increasing concentrations of IFN-gamma, but was significantly inhibited by progesterone (10(-6) M), indicating the likely involvement of typical glucocorticoid receptors. In contrast to their effectiveness on macrophages, prostaglandin E2 and dibutyryl cyclic AMP only slightly inhibited the induction of Ia antigens on TDMC by IFN-gamma, whereas endotoxin (1 to 60 micrograms/ml) had no effect.

Animals↗