PubMed Health⌕ Search

Biomedical subjects

U Gullberg

Publications and source records attributed to U Gullberg.

At least 55 records · Page 3Linked to original sources
PubMed1994

The heterogeneity of azurophil granules in neutrophil promyelocytes: immunogold localization of myeloperoxidase, cathepsin G, elastase, proteinase 3, and bactericidal/permeability increasing protein.

Azurophil granules of myeloid cells form in promyelocytes. They store cytotoxic and digestive agents which when released are involved in the defense against infection. In order to characterize the intragranular distribution of these agents, ultrastructural methods using immunogold were used on promyelocytes. Azurophil granules were divided into nucleated, large spherical (large azurophil) and small electron-dense (small azurophil) granules. Myeloperoxidase showed a peripheral distribution of large azurophils and a uniform distribution of small and nucleated azurophils, consistent with previous findings. Likewise, the major neutral proteases of azurophils, cathepsin G, granulocyte elastase, and proteinase 3, displayed a similar distribution, with a peripheral localization in large azurophils and a uniform distribution in small and nucleated azurophils, except for proteinase 3, which was associated with the crystalloid structure in nucleated azurophils. In contrast, the bactericidal/permeability increasing protein, which is bacteristatic and bactericidal for Gram-negative bacteria, was localized to the membrane area in all types of azurophil granules, consistent with a suggested association of this protein with the granule membrane. The observed differences in intragranular distribution of the proteins investigated may reflect variations in binding to matrix structures and granule membranes.

Antimicrobial Cationic Peptides↗
PubMed1994

Processing and intracellular transport of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937-modulation by brefeldin A, ammonium chloride, and monensin.

The effects of brefeldin A, monensin, and the weak base NH4Cl on the biosynthesis and processing of cathepsin G and neutrophil elastase of myeloid cells were investigated. Monoblast-like U-937 cells were biosynthetically labeled with [35S]methionine, followed by subcellular fractionation, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Brefeldin A inhibited proteolytic processing, intracellular transport, and secretion. The effects were reversible inasmuch as removal of brefeldin A resulted in a normal pattern of processing and transfer to high-density organelles, corresponding to lysosomes, and restitution of constitutive secretion of precursor forms. Both cathepsin G and neutrophil elastase acquired resistance to endoglycosidase-H, suggesting that conversion to complex oligosaccharide side chains also occurs in the presence of brefeldin A. Monensin and NH4Cl inhibited final proteolytic processing, indicating either that acidification is necessary for directing cathepsin G and neutrophil elastase to lysosomal-like organelles or that the protease(s) responsible for processing requires an acid pH. We conclude that pH-dependent proteolytic processing of cathepsin G and neutrophil elastase occurs in post-Golgi structures and that transfer to lysosomes or an immediately prelysosomal compartment is mandatory for complete processing.

Ammonium Chloride↗
PubMed1994

Mechanisms involved in the processing of the p55 and the p75 tumor necrosis factor (TNF) receptors to soluble receptor forms.

The two tumor necrosis factor (TNF) receptors (TNF-R55 and TNF-R75) can release soluble TNF-binding proteins (TNF-R55-BP and TNF-R75-BP) by proteolytic cleavage. The proteolytic processing of the TNF receptors was investigated in monoblastic THP-1 and promyelocytic HL-60-10 leukemic cell lines. The release of soluble forms of both receptors was rapidly stimulated by staurosporine-sensitive protein kinase C activation by phorbol myristate acetate (PMA) and more slowly stimulated by TNF. No receptor release was seen below a temperature of 16 degrees C. NH4Cl (10 mmol/liter) and monensin (1 mumol/liter), known to increase intracellular pH, inhibited to some extent PMA- and TNF-induced release of both TNF-R55-BP and TNF-R75-BP. The inhibitory effect of monensin might be explained by a diminished translocation of newly synthesized receptor to the plasma membrane. The weak inhibitory effect of NH4Cl on PMA-induced release of soluble receptor forms could be due to effects on a pH-sensitive compartment. PMA-induced down-regulation of receptors was not dependent on acidity as it occurred also in the presence of monensin and NH4Cl when the release of TNF-BPs is partially blocked. Dibutyryl cAMP inhibited the PMA-induced release of TNF-R55-BP but not of TNF-R75-BP in both cell lines investigated. In addition, dibutyryl cAMP alone stimulated the release of both receptors but only in THP-1 cells. Our data show that the generation of soluble forms of both TNF receptors can be regulated by both PKC and PKA.

Alkaloids↗
PubMed1993

Phorbol ester-induced degranulation in adherent human eosinophil granulocytes is dependent on CD11/CD18 leukocyte integrins.

Secretion of unique eosinophil granule constituents may play a role in allergic and parasitic reactions. Therefore we have investigated possible mechanisms for regulation of secretion in eosinophils. A hemolytic plaque assay and an enzyme-linked immunospot (ELISPOT) assay were developed for detection of secreted eosinophil cationic protein (ECP) from single adherent eosinophils. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) induced release of ECP in a dose-dependent fashion but 4-alpha-PMA, an analogue that does not activate protein kinase C, did not cause degranulation. Staurosporine and K252a, inhibitors of protein kinase C, decreased PMA-induced ECP secretion. Low concentrations of cytochalasin B enhanced PMA-induced secretion but high concentrations had an inhibitory effect. The calcium ionophores A23187 and ionomycin were weaker secretagogues than PMA. Tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, interleukin-5, N-formylmethionyl-leucyl-phenylalanine, and lipopolysaccharide caused little or no degranulation in adherent eosinophils. Preincubation of eosinophils with antibodies to CD18, the common beta chain of leukocyte adhesion proteins, resulted in inhibition of PMA-induced ECP release from adherent cells. 1,2-Bis(O-aminophenyl)-ethane-ethane-N,N,N',N'-tetraacetic acid (BAPTA), an agent that acts intracellularly by chelation of calcium, also inhibited PMA-mediated ECP release. In conclusion, PMA induces release of ECP from single adherent eosinophils and the effect appears to be mediated via protein kinase C and, in contrast to that in neutrophils, to be dependent on CD11/CD18 leukocyte integrins.

Antibodies↗
PubMed1993

Independent transcriptional and posttranscriptional regulation of the two tumor necrosis factor receptors in promyelocytic HL-60 cells.

Two separate tumor necrosis factor (TNF) receptors of approximately 55 kDa (TNF-R55) and 75 kDa (TNF-R75) have been identified. The role of protein kinase A activation by dibutyryl cAMP (dbcAMP) and of protein kinase C activation with phorbol myristate acetate (PMA) for transcriptional and posttranscriptional regulation of the two receptors was investigated in promyelocytic HL-60 cells. Incubation with dbcAMP or the adenylate cyclase agonist forskolin caused an increase in the level of TNF-R75 mRNA while TNF-R55 mRNA was unaffected. The half-life of transcripts for both TNF-R55 and TNF-R75 was unaffected as judged by disappearance of mRNA after inhibition of transcription with actinomycin D. Thus the transcription of the TNF-R75 gene seemed to be enhanced by activation of protein kinase A. This enhancement was not dependent on de novo protein synthesis. Incubation with PMA did not affect the mRNA level of any of the TNF receptors. Both TNF-R55 and TNF-R75 mRNA showed a prolonged half-life after incubation with the inhibitor of protein synthesis cycloheximide, indicating superinduction of the genes. Our results demonstrate that the two TNF receptors can be regulated differently at the transcriptional level and that both transcriptional and posttranscriptional regulation occurs.

Base Sequence↗
PubMed1992

The receptors for regulatory molecules of hematopoiesis.

Proliferation and differentiation of hematopoietic cells are controlled by pleiotropic regulatory molecules. While the sequences of these factors are not related, their membrane receptors are restricted to two gene families with homologous domains. The members of the hematopoietic (or cytokine) receptor family (for erythropoietin, interleukins-2, -3, -4, -6 andu-7, granulocyte-macrophage and granulocyte colony-stimulating factor) are composed of multiple subunits necessary for high-affinity binding and cell signalling. Signal transducing mechanisms are largely unknown. The occurrence of variant signal transducers in different tissues could explain the pleiotropy of these regulatory molecules. Members of the receptor tyrosine kinase family bind dimeric forms of macrophage colony-stimulating factor, stem cell factor and platelet-derived growth factor leading to kinase activation and phosphorylation of many substrates involved in production of second messengers. Soluble forms (binding proteins) exist for members of both families. These may be proteolytic cleavage products of transmembrane receptors or naturally secreted products. Such binding proteins can potentially function as inhibitors in feedback regulation and in protection and transport of cytokines and would provide a rational therapy when cytokines are produced in excess. Knowledge of signal transduction mechanisms and of the three-dimensional structure of ligands and receptors can lead to the design of drugs with cell-specific effects.

Animals↗
PubMed1992

Calcitonin gene-related peptide (CGRP) activates human neutrophils--inhibition by chemotactic peptide antagonist BOC-MLP.

The effect of the neuropeptides substance P, neurokinin A and alpha-calcitonin gene-related peptide (CGRP) on human neutrophil granulocytes was investigated. Substance P induced secondary granule secretion at a concentration of 100 microM. CGRP induced a significant secretory response at 10 microM and thus appeared to be about 10 times more potent than substance P. Calcitonin and a fragment of CGRP, CGRP(8-37), had no effect on neutrophil degranulation. The chemotactic peptide antagonist BOC-MLP (100 microM) inhibited lactoferrin secretion mediated both by CGRP and chemotactic peptide FMLP almost completely, while secretion in response to tumour necrosis factor (TNF) was unaffected. Results from receptor binding studies showed that CGRP and N-formyl-methionyl-leucyl-phenylalanine (FMLP) do not compete for binding. This indicates that CGRP does not exert its effects by binding to the chemotactic peptide receptor. CGRP induced a rapid increase in the cytosolic-free calcium concentration and this increase was not, unlike that induced by FMLP, abolished by preincubation of the cells with pertussis toxin (1000 ng/ml). Therefore CGRP signal transduction in neutrophils appears to involve rapid changes in the cytosolic-free calcium concentration but not a pertussis toxin-sensitive G-protein. In summary, this is the first report to show that CGRP can directly activate neutrophil granulocytes, and this probably occurs via a cell surface receptor which is distinct from that of FMLP although both the CGRP and FMLP-mediated effects can be blocked by BOC-MLP.

Calcitonin Gene-Related Peptide↗
PubMed1992

Involvement of an Asn/Val cleavage site in the production of a soluble form of a human tumor necrosis factor (TNF) receptor. Site-directed mutagenesis of a putative cleavage site in the p55 TNF receptor chain.

Soluble forms of receptors for tumor necrosis factor (TNF) might be important for regulating the actions of TNF. Site-directed in vitro mutagenesis was employed to examine the processing of the p55 tumor necrosis factor receptor chain (TNF-R55) into soluble TNF-binding protein (TNF-R55-BP). An Asn/Val sequence close to the transmembrane region in TNF-R55 was indicated as a putative cleavage site for proteolytic processing. By mutagenesis, Asn and Val were replaced with Gly and Ala, respectively. Expression in Chinese hamster ovary (CHO) cells resulted in identical binding of ligand to mutated receptors as compared to receptors not subjected to mutagenesis. Turnover rates of receptors as judged by disappearance of TNF binding capacity after inhibition of de novo protein synthesis and downregulation in response to incubation with phorbol esters were also identical between wild-type and mutated receptors. However, mutations of the cleavage site resulted in a decreased spontaneous and phorbol ester-induced release of soluble receptor (TNF-R55-BP) which was almost abolished when both Asn and Val were mutated. Our results clearly demonstrate the importance of an Asn/Val sequence for proteolytic processing of the TNF-R55 into soluble TNF-R55-BP and indicate that phorbol ester-induced downregulation of the TNF-R55 may be dissociated from proteolytic cleavage of the receptor.

Animals↗
PubMed1990

Modulation of the constitutive gene expression of the 55 kD tumor necrosis factor receptor in hematopoietic cells.

The expression of the 55 kD human tumor necrosis factor (TNF) receptor gene was investigated. By use of a 1.2 kb cDNA we demonstrated a constitutive expression of a single 2.3 kb transcript in cell lines and fresh blood cells. The TNF receptor gene expression was not affected by phorbol esters, dibutyryl cAMP (dbcAMP), interleukin-1 (IL-1), interferon-gamma (IFN-gamma) or by TNF, agents known to modulate functional TNF receptors. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) showed a dose dependent inhibition of the TNF receptor gene expression. This inhibition is not mediated by cAMP, since neither dbcAMP nor forskolin had any effects on the expression of the 55 kD receptor gene. The results suggest that the effects of phorbol diesters, IL-1, IFN-gamma, TNF and dbcAMP previously observed on binding of TNF to cells are limited to posttranscriptional regulation of the 55 kD TNF receptor.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Cytokine and non-cytokine differentiating agents for myeloid leukemic cells.

Existing data suggest that normal maturation can sometimes be re-established in leukemia. A number of differentiation-inducing substances have been reported; these include retinoic acid, vitamin D3 and cytokines such as differentiation-inducing factor, tumor necrosis factor and lymphotoxin. Different agents obviously act by separate mechanisms to provide terminal maturation.

Binding Sites↗
PubMed1990

Characterization in vitro of a human tumor necrosis factor-binding protein. A soluble form of a tumor necrosis factor receptor.

Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammatory responses. A cysteine-rich, highly glycosylated 30-kD TNF-binding protein (TNF-BP) purified from urine may have a role in regulation because it protects in vitro against the biological effects of TNF. The cytotoxic effect of TNF on the fibrosarcoma cell line WEHI 164 was inhibited by 50% at a 10-fold excess of TNF-BP. The binding of TNF to the receptor was partially reversed after the addition of TNF-BP. Results from biosynthetic labeling of cells with 35S-cysteine followed by immunoprecipitation with anti-TNF-BP indicated that TNF-BP is formed and released at the cell surface by cleavage because no corresponding cellular polypeptide was observed. A cellular 60-kD polypeptide, which was immunoprecipitated with anti-TNF-BP, may correspond to the transmembrane TNF-receptor molecule and be the precursor of TNF-BP. Thus, TNF-BP appears to be a soluble form of a transmembrane TNF-receptor. Moreover our results demonstrate that the production of TNF-BP is increased when the TNF receptor is downregulated in cells by treatment with TNF or by activation of protein kinase C with phorbol esters. TNF-BP may be an important agent that blocks harmful effects of TNF, and, therefore, useful in clinical applications.

Cell Survival↗
PubMed1989

Recombinant lymphotoxin enhances the growth of normal, but not of chronic myeloid leukemic, human hematopoietic progenitor cells in vitro.

The effects of recombinant lymphotoxin (rLT) and tumor necrosis factor (rTNF) on the growth of clonogenic normal and leukemic hematopoietic cells were investigated. Two opposite and dose-dependent effects of rLT on normal CFU-GM were found. Low concentrations (5pM) did stimulate the growth, while higher amounts of rLT showed an antiproliferative effect. In contrast, the effect of rTNF was only an inhibition of growth in a dose-dependent fashion. In chronic myeloid leukemia (CML), the CFU-GM was resistant to the growth stimulatory effect of rLT. Furthermore, CML-cells were found to be more susceptible than normal CFU-GM to the antiproliferative effect of both rLT and rTNF. These results show that LT and TNF exhibit qualitative differences in the effects on hematopoietic cells and that CML-cells display an increased susceptibility for the cytostatic effects on LT and TNF.

Bone Marrow Cells↗
PubMed1989

Characterization of the receptor for lymphotoxin; a spontaneous internalization without recycling and ligand-induced downregulation in HL-60 cells.

The receptor system for lymphotoxin (LT) was investigated by use of the human promyelocytic HL-60 cell line. Utilizing subcellular fractionation, internalization of cell surface bound recombinant LT (rLT) and transfer to lysosomes followed by degradation was demonstrated. Binding of rLT to its cell surface receptor induced a downregulation of the receptor which was persistent for at least 6 h. Downregulation of the receptors from the cell surface by activation of protein kinase C with the diacylglycerol OAG was completely reversible but the recovery of the binding capacity was dependent on protein synthesis as it was inhibited by cycloheximide. Treatment with cycloheximide alone resulted in a loss of binding capacity with a half life of approximately 2 h, suggesting a spontaneous consumption of receptors. Affinity cross-linking revealed three ligand-receptor complexes with Mr of 85, 105 and 125 kDa. Because of a strong tendency for ligand polymerization these results suggest oligomer binding of the ligand to a single receptor molecule with an apparent Mr of 70 kDa. N-linked glycosylation constituted 4 to 5 kDa of the total molecular weight. In conclusion, we demonstrated a spontaneous internalization of the receptor for LT without recycling, and that ligand binding resulted in an irreversible downregulation of the receptor.

Asparagine↗
PubMed1987

Characterization of a relationship between the T-lymphocyte derived differentiation inducing factor (DIF) and lymphotoxin: a common receptor system for DIF, lymphotoxin and tumor necrosis factor downregulated by phorbol diesters.

Here we describe results which show that recombinant lymphotoxin (rLT), like the T-lymphocyte derived differentiation inducing factor (DIF), inhibited the clonogenic growth of some myeloid leukemia cell lines by concentrations of 1 to 30 pmol/l. Wild type HL-60 cells were resistant at these concentrations but responded with differentiation into monocyte-like cells at higher concentrations. An antigenic relationship between DIF and LT was indicated because a neutralizing monoclonal anti-LT antibody bound to and neutralized both differentiation and growth inhibitory effects of DIF. An activity, which cochromatographed with DIF during all purification steps, competed with binding of both rLT and recombinant tumor necrosis factor (rTNF) to HL-60 cells. By use of radioiodinated ligand, 2100 binding sites for rLT were detected on HL-60 cells with a Kd of 330 pmol/l. At 37 degrees C bound ligand was transferred to lysosomes, followed by degradation. rTNF and rLT were shown to compete for binding sites on HL-60 cells. Receptors for both rLT and rTNF were downregulated by activators of protein kinase C such as phorbol diester or diacylglycerol; the number of cell surface receptors decreased while the Kd remained unchanged. Our observations demonstrate a functional and antigenic relationship between DIF and LT and indicate that TNF, LT and DIF share binding sites on myeloid leukemia cells that are downregulated by activation of protein kinase-C.

Binding Sites↗
PubMed1986

The cytotoxic eosinophil cationic protein (ECP) has ribonuclease activity.

The eosinophil cationic protein (ECP) is a specific cytotoxic constituent of granules. In this work we demonstrated that ECP has a ribonuclease activity. Purified ECP was resolved by ion exchange chromatography into subfractions, which all showed ribonuclease activity. Another eosinophil granule protein, EPX, identical with eosinophil-derived neurotoxin (EDN) had a 125-fold higher RNase activity than ECP. ECP may exert its cytotoxic effects on parasites and cells because of its extreme basicity alone or it may be internalized and act by degrading mRNA.

Blood Proteins↗
PubMed1986

Differentiation induction in myeloid leukemic cells.

Work based on immortalised leukemic cell lines indicates that the maturation arrest in leukemia can be reversible. Successful differentiation induction would mean restoring the link between proliferation and differentiation. Human cell lines such as the promyelocytic HL-60 and the monoblastic U-937 can be induced to mature by incubation with a wide variety of agents, e.g. phorbol diesters, retinoic acid and 1,25-dihydroxycholecalciferol. In addition, mitogen-stimulated lymphocytes and some T-lymphocyte lines produce a polypeptide called the differentiation-inducing factor (DIF), which mediates maturation of HL-60 into macrophage-like cells with resulting proliferation inhibition. DIF also displays a primary growth inhibitory effect on certain subclones of the cell lines as well as on fresh clonogenic cells from patients with acute myeloid leukemia and on normal granulocyte-macrophage progenitors. Our data indicate that there is more than one way to induce differentiation in leukemia but final common pathways may exist. Complementary, synergistic, maturation effects are seen between some agents, which may become of clinical utility.

Cell Differentiation↗
PubMed1986

Effects of recombinant tumor necrosis factor on proliferation and differentiation of leukemic and normal hemopoietic cells in vitro. Relationship to cell surface receptor.

The clonogenic growth of myeloid leukemia cell lines was inhibited by recombinant tumor necrosis factor (rTNF) at 1-15 pM concentration. However, wild type (promyelocytic) HL-60 cells were highly resistant to growth inhibition, but responded with differentiation into monocyte-like cells at 100 pM rTNF. The clonogenic growth of fresh acute myeloid leukemia cells was inhibited by 50% at approximately 15 pM rTNF. The growth of normal granulocyte-macrophage progenitors (CFU-GM) was also inhibited (by 50 pM rTNF), as was the growth of erythroid progenitors (BFU-E) (by 150 pM rTNF). A synergistic antiproliferative effect was demonstrated between rTNF and recombinant interferon-gamma. Use of radioiodinated rTNF enabled us to detect 1,500-2,100 binding sites on myeloid cell lines at 4 degrees C with Kd of approximately 300 pM. At 37 degrees C, the transfer of bound ligand to lysosomes was followed by degradation, inhibited by NH4+. No correlation was observed between the number of binding sites or affinity at 4 degrees C and antiproliferative response to the addition of rTNF.

Cell Differentiation↗