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Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75-kDa neurotrophin receptor (NTR). An O-glycosylated peptide connects the ligand-binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O-glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O-linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of disialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O-linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O-linked glycans synthesized by cultured cells had the structure (NeuNAc)(1-2-) Gal beta 1-3GalNAc. Modification of the O-linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling.

Affinity selection of a 15-mer random peptide library displayed on bacteriophage M13 has been used to identify potent ligands for the human urokinase receptor, a key mediator of tumor cell invasion. A family of receptor binding bacteriophage ligands was obtained by sequentially and alternately selecting the peptide library on COS-7 monkey kidney cells and baculovirus-infected Sf9 insect cells overexpressing the human urokinase receptor. Nineteen peptides encoded by the random DNA regions of the selected bacteriophage were synthesized and tested in a urokinase receptor binding assay, where they competed with the labeled N-terminal fragment of urokinase with IC50 values ranging from 10 nM to 10 microM. All of the isolated peptides were linear and showed two relatively short conserved subsequences: LWXXAr (Ar = Y, W, F, or H) and XFXXYLW, neither of which is found in urokinase or its receptor. Competition experiments demonstrated that the most potent peptide, clone 20, prevented binding of bacteriophage displaying the urokinase receptor binding sequence (urokinase residues 13-32). In addition, this peptide blocked other apparently unrelated receptor binding bacteriophage, suggesting overlapping receptor interaction sites for all of these sequences. These results provide a demonstration of bacteriophage display identifying peptide ligands for a receptor expressed on cells and yield leads for the development of urokinase receptor antagonists.

A series of 2-pyrimidyl-5-amidothiophenes has been synthesized and evaluated for AKT inhibition. SAR studies resulted in potent inhibitors of AKT with IC(50) values as low as single digit nanomolar as represented by compound 2aa. Compound 2aa showed cellular activity including antiproliferation and downstream target modulation. Selectivity profile is described. A co-crystal of 2aa with PKA is determined and discussed.

The human urokinase plasminogen activator (uPA) epidermal growth factor-like domain (residues 1-48) and a variant with a C-terminal epitope tag have been secreted from recombinant yeast. Purified human uPA 1-48 and uPA 1-48glu complete for binding to the human uPA receptor with Kds of 180 and 400 pM respectively, in an in vitro assay using an immobilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expression vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to infectious particles which were selectively bound and eluted from both epitope tag antibody and urokinase receptor. Sequential binding to this antibody and receptor demonstrated a substantial enrichment, where up to 10% of the infectious particles were then retained on urokinase receptor-coated plates. A PCR strategy was used to convert previously described peptide bacteriophage ligands for the urokinase receptor to phagemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to when the peptides are multivalently displayed on a bacteriophage.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

Human erythroid-potentiating activity (EPA) is a 28,000 mol wt glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line. EPA has potent protease inhibitory activity, and is also referred to as tissue inhibitor of metalloproteinases (TIMP). We observed that colony formation by K562 cells in semi-solid medium containing reduced fetal calf serum (FCS) is not directly proportional to the number of cells plated, suggesting production of autostimulatory factors by K562 cells. Using radioimmunoprecipitation and a bioassay for EPA, medium conditioned by K562 cells was found to contain high levels of biologically active EPA; Northern hybridization analysis confirmed the expression of EPA mRNA. Radiolabeled EPA was used to identify cell surface receptors on K562 cells. Together, these results suggest that EPA may act as an autocrine growth factor for K562 cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lymphotropic virus type II (HTLV-II). This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA). Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.

The argasid tick Ornithodorus moubata Murray was fed on modified blood meals, the ionic strength and/or osmotic pressure of which was varied by additions of NaCl or glucose, and by dilution with distilled water or isosmotic glucose. The following parameters were monitored: total volume increase of tick, total volume of meal ingested, net increase in haemolymph volume, volume of coxal fluid excreted and ion concentrations and osmotic pressures of the gut contents, haemolymph and coxal fluid, as well as potential differences across the gut lumen. Absorption of fluid from the gut into the haemolymph appeared to be linked to the active transport of Na+ and Cl−, although the rate of absorption was an inverse function of the prevailing osmotic gradient across the gut epithelium. The tick can maintain a reasonably constant [Na+] and [Cl−] in the haemolymph when fed meals in which the concentration of these ions varies up to 6-fold, but only if the meal remains isosmotic with blood. The rate and volume of coxal fluid production are direct functions of the rate and volume of fluid absorption by the gut epithelium. Thus, during the feeding cycle, the coxal organ appears to function chiefly in an osmo- and volume-regulatory capacity in this species of argasid tick.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.

Antigen- or lectin-stimulated T lymphocytes and human T-cell leukemia virus (HTLV)-infected cell lines secrete lymphokines that can influence the growth and function of a variety of cell types. We recently demonstrated that supernatants from the HTLV-II-infected Mo-T-cell line stimulate the proliferation of rat brain oligodendrocytes and astrocytes. We have now purified a glial growth-promoting factor (GGPF) from these supernatants. Purification from serum-free conditioned medium was accomplished by sequential concentration, ammonium sulfate precipitation, lentillectin affinity chromatography, gel filtration, and reversed-phase high-performance liquid chromatography. GGPF is assayed by its ability to stimulate DNA synthesis in oligodendrocytes, as measured by [3H]thymidine uptake. The purified GGPF has an apparent Mr of 30,000 when analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, however, GGPF appears as a single band of Mr 18,000. Both reduced and unreduced forms have biological activity, suggesting that GGPF exists in both a functional monomeric and dimeric form. Purified GGPF appears to be a biochemically and functionally distinct lymphokine.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the colony growth of myeloid progenitors in semisolid media, and enhances the function of mature effector cells, including neutrophils, monocytes, and eosinophils. Small cell carcinoma lines (SCCL) have properties of amine precursor uptake and decarboxylation (APUD) cells and express high levels of the enzyme, L-aromatic amino acid decarboxylase. We looked for possible expression of GM-CSF receptors on nonhematopoietic cells and found specific high-affinity binding of human GM-CSF to SCCL and to the SV40-transformed African green monkey kidney cell line, COS. The small cell carcinoma lines responded to GM-CSF with enhanced proliferation, and both small cells and COS cells were found to express authentic 84,000 dalton GM-CSF receptor protein. These findings indicate that nonhematopoietic cells can bind and respond to GM-CSF, suggesting additional biological activities as well as the possibility of tumor responses when GM-CSF is used therapeutically in humans. Since preliminary clinical trials using CSFs as adjunctive treatment in patients with solid tumors are underway, it will be important to consider the possible responsiveness of nonhematopoietic tumor cells to CSFs.

Adult ticks (Omithodorus moubata Murray, Acari, Argasidae) were fed on human blood at 37 °C in a beaker covered with chicken skin. 14C-labelled inulin and 3–0[14CH3]glucose were rapidly cleared from haemolymph to coxal fluid ; the coxal tubule appeared not to reabsorb either substance. Glucose was reabsorbed from tubular fluid via a phlorrhizin-sensitive mechanism. Reabsorption of amino acids varied between o and 90 % but was greatest for those amino acids which were scarce in the meal. During normal feeding conditions, there was positive correlation between haemocoelic hydrostatic pressure and rate of coxal fluid excretion. Unilateral cannulation of a coxal organ so as to eliminate back pressure of a valve at the coxal orifice led to a twofold increase in rate of coxal fluid production compared to the contralateral side. The above data confirm that coxal fluid excretion occurs by a filtration resorption mechanism.

Purified natural and biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate colony formation by myeloid progenitor cells and enhance the function of mature neutrophils. Both of these actions occur at concentrations between 1 and 100 pM, with half-maximal stimulation at 10-20 pM. We have examined specific binding of 125I-labeled GM-CSF to responsive target cells in this range of concentrations. The results show a low number (50-250) of high-affinity (15-30 pM) binding sites on GM-CSF-responsive leukemic cells (KG-1, HL-60), as well as on peripheral blood neutrophils from normal donors. This high-affinity binding component was absent from unresponsive cell lines (KG-1a, K562). These results suggest that this binding site mediates the biological activities of GM-CSF on both proliferation and function of myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine derived from activated T cells, endothelial cells, fibroblasts, and macrophages. It stimulates myeloid and erythroid progenitors to form colonies in semisolid medium in vitro, as well as enhancing multiple differentiated functions of mature neutrophils, macrophages, and eosinophils. We have examined the binding of human GM-CSF to a variety of responsive human cells and cell lines. The most mature myelomonocytic cells, specifically human neutrophils, macrophages, and eosinophils, express the highest numbers of a single class of high affinity receptors (Kd approximately 37 pM, 293-1000 sites/cell). HL-60 and KG-1 cells exhibit an increase in specific binding at high concentrations of GM-CSF; computer analysis of the data is nonetheless consistent with a single class of high affinity binding sites with a Kd approximately 43 pM and 20-450 sites/cell. Dimethyl sulfoxide induces a 3-10-fold increase in high affinity receptors expressed in HL-60 cells, coincident with terminal neutrophilic differentiation. Finally, binding of 125I-GM-CSF to fresh peripheral blood cells from six patients with chronic myelogenous leukemia was analyzed. In three of six cases, binding was similar to the nonsaturable binding observed with HL-60 and KG-1 cells. GM-CSF binding was low, or in some cases, undetectable on myeloblasts obtained from eight patients with acute myelogenous leukemia. The observed affinities of the receptor for GM-CSF are consistent with all known biological activities. Affinity labeling of both normal neutrophils and dimethyl sulfoxide-induced HL-60 cells with unglycosylated 125I-GM-CSF yielded a band of 98 kDa, implying a molecular weight of approximately 84,000 for the human GM-CSF receptor.