Biomedical subjects

H Slegers

Publications and source records attributed to H Slegers.

At least 19 recordsLinked to original sources

PubMed1999

Isolation of plasma membranes from rat C6 glioma cells cultivated on microcarriers.

We have studied the feasibility of rat C6 glioma cell cultivation on microcarrier beads and the isolation of their plasma membranes from the beads. Cells were cultivated on Cytodex-1 microcarrier beads and the plasma membranes were subsequently isolated from confluent cell monolayers on the beads. This approach yielded approximately 4 x 10(6) cells/ml in a 1 L spinner vessel. Enzymatic assays indicated an 18-fold enrichment of plasma membranes isolated from the beads with minor contamination by other cell organelles. Assay for IGF-I receptor binding capacity revealed that 70% of the total receptor binding capacity could be recovered in the plasma membrane fraction isolated from the beads as compared with the receptor binding capacity of intact cells, demonstrating the functional integrity of the isolated membranes. Electron microscopy and immunofluorescence analysis indicated that the isolated plasma membranes were highly homogeneous with the majority exposing the cytoplasmic surface. Our procedure of C6 glioma cell cultivation on microcarriers and subsequent plasma membrane isolation, provides large quantities of homogeneous and metabolically active membranes which can be used to study receptor-mediated effects on cell proliferation and differentiation.

Animals↗

PubMed1999

An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma.

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

Adenosine Triphosphate↗

PubMed1998

Decrease in nucleoside diphosphate kinase (NDPK/nm23) expression during hematopoietic maturation.

The nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34(+) progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells.

Cell Differentiation↗

PubMed1998

Localization of extracellular superoxide dismutase in rat lung: neutrophils and macrophages as carriers of the enzyme.

Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to localize extracellular superoxide dismutase (EC-SOD) and its mRNA in rat lung before and after a lipopolysaccharide (LPS)- and hyperoxia-induced inflammation. In control rats, EC-SOD mRNA was synthesized in macrophages and in cells of the arterial vessel walls and the alveolar septa. The EC-SOD protein was mainly localized in plasma and on the apical side of the epithelial cells located near bronchus-associated lymphoid tissue (BALT). ISH did not reveal major changes in the distribution of EC-SOD mRNA upon induction of inflammation. In contrast, IHC demonstrated a progressive staining of the epithelium of the larger bronchi for the protein. Neutrophils and macrophages invading the lung showed an intensive staining for the EC-SOD protein concomitantly with a decrease of the enzyme in the plasma. Twenty-four hours after LPS stimulation only a spotty positivity remained on neutrophils in and between the alveolar spaces. In the bronchoalveolar lavage fluid (BALF), only macrophages showed a strong positivity for EC-SOD mRNA while the protein was detected in macrophages and neutrophils. Exposure to hyperoxia did not affect the distribution of EC-SOD mRNA and protein. The presented data demonstrated that in lung tissue the EC-SOD enzyme may have a protective function for activated macrophages, neutrophils, and lympoid tissue-associated epithelial cells.

Animals↗

PubMed1998

Expression of IGF-I and IGF-II receptors in rat C6 glioma cells as a function of the growth phase.

In the present study, we examined the specific binding of IGF-I and IGF-II to their receptors in C6 glioma cells taken during different growth phases in culture: phase A, early stage of the exponential growth (48 h after seeding); phase B, late stage of the exponential phase (96 h after seeding); phase C, confluent phase (at 144 h after seeding); and phase D, stopped at 48 h post-confluence. Scatchard analysis revealed higher Ka values for the IGF-IR during the exponential phases (A and B). The affinity of IGF-I for its receptor during the post-confluent phase (D) decreased to about half that at phase A (p < 0.01). Although lower at the later phase (D), the number of binding sites of the IGF-IR in the different tested growth stages in culture (A, B, and C) was not statistically different (p > 0.05). Conversely, the number of binding sites of the IGF-II/mannose-6-phosphate receptor appeared to increase during time in culture. The Ka values of the IGF-II/mannose-6-phosphate receptor decreased significantly during the culture time, phase D showing the largest decrease (50%) as compared with phase A (p < 0.005). These binding data suggest that IGF-I and IGF-II receptors are differentially expressed in rat C6 glioma cells in culture and are a function of the growth phase.

Animals↗

PubMed1997

Cyclic AMP-mediated induction of the glial fibrillary acidic protein is independent of protein kinase A activation in rat C6 glioma.

N6-O'2-dibutyryl cAMP (dbcAMP), N6-monobutyryl cAMP (N6-mbcAMP), 8-Chloro cAMP (ClcAMP), and O'2-monobutyryl cAMP (O'2-mbcAMP) were used to study glial fibrillary acidic protein (GFAP) induction in rat C6 glioma. With the exception of O'2-mbcAMP, these cAMP analogs induced GFAP after stimulation of cells with a concentration of 0.5-1 mM. Only dbcAMP and N6-mbcAMP increased the intracellular concentration of cAMP. Protein kinase A (PKA) activation is often proposed to be involved in GFAP expression in astrocytes. Ion-exchange chromatography indicated that protein kinase activity is associated with PKA type II in C6. dbcAMP, N6-mbcAMP, and ClcAMP upregulated the amount of cAMP-binding proteins approximately twofold. RI was upregulated in the cytosol and particulate fraction, whereas RII was not affected after stimulation with dbcAMP. Concomitant, the PKA activity decreased approximately 60% and 40% in the cytosol and particulate fraction, respectively. CREB is constitutively expressed in C6 and is downregulated after stimulation with dbcAMP. The membrane-permeable PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) did not suppress the induction of GFAP-mRNA and its translation into GFAP. On the contrary, depending on the time difference between H89 and dbcAMP addition to C6, GFAP synthesis could even be potentiated more than twofold. Experiments in the presence of cycloheximide showed that protein synthesis is necessary for GFAP transcription. Although all components of the PKA signal transduction pathway are present in C6, GFAP synthesis is not dependent on PKA activation but required the synthesis of an unidentified factor.

Animals↗

PubMed1997

Inhibition of nucleoside diphosphate kinase (NDPK/nm23) by cAMP analogues.

Nucleoside diphosphate kinase (NDPK/nm23) ATP/GDP phosphotransferase activity and serine autophosphorylation is inhibited by N6-mbcAMP, 8-ClcAMP and 8-BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N6-mbcAMP is a substrate of the enzyme. DbcAMP and 0'2-mbcAMP, cAMP analogues with a modified 0'2-ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra- and intracellular functions of NDPK/nm23 are discussed.

Animals↗

PubMed1996

Cyclic AMP-induced differentiation increases the synthesis of extracellular superoxide dismutase in rat C6 glioma.

The effect of membrane permeable cAMP analogues on the expression of extracellular superoxide dismutase (EC-SOD) was studied in rat C6 glioma. EC-SOD is constitutively expressed but stimulation with cAMP analogues still increased the EC-SOD transcription and the secreted SOD activity. The potency to enhance EC-SOD expression is correlated with the ability of the cAMP analogue to induce cAMP-dependent differentiation in C6. The increase in EC-SOD mRNA and in secreted activity depended on the concentration of the cAMP analogues and on the cultivation time. Twenty-four hours after addition of 0.5 mM N6, O'2-dibutyryl cAMP (dbcAMP) or N6-monobutyryl cAMP (N6-mbcAMP) EC-SOD mRNA expression increased approximately twofold, while stimulation for 68 h with 0.5 mM N6-mbcAMP or 1 mM 8-Chloro cAMP (ClcAMP) and 1 mM dbcAMP enhanced the mean secreted activity/cell three- and fivefold, respectively. O'2-monobutyryl cAMP (O'2-mbcAMP) did not affect EC-SOD synthesis. The enhancement in EC-SOD activity did not require activation of protein kinase A. ATP, TGF-beta, IFN-gamma, and LPS did not affect EC-SOD synthesis. The presented data point to a cAMP-dependent pathway for the enhanced expression of EC-SOD by glial cells in brain.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Lipopolysaccharide-enhanced expression of interleukin-6 in dibutyryl cyclic AMP-differentiated rat C6 glioma.

Rat C6 glioma synthesizes a low basal level of interleukin-6 (IL-6). Stimulation with 10 micrograms/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 mM N6,O2'-dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP-differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL-6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 micrograms/ml of LPS and 1 mM dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL-6 mRNA concentration and IL-6 immunoreactivity, whereas titration of the biological activity indicated a 100-fold increase in the secreted IL-6 activity. The enhanced secretion of IL-6 is correlated with the induction of differentiation. Chromatography on heparin-Sepharose and on DEAE-5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL-6-like activities could be neutralized by an anti-murine IL-6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.

Animals↗

PubMed1995

Cadherin-dependent cell aggregation is affected by decapeptide derived from rat extracellular super-oxide dismutase.

A synthetic HAV-containing decapeptide homologous to the amino acid sequence 44R-Q53 in rat extracellular superoxide dismutase B affects cadherin-dependent cell aggregation. Cell lines, some of them transfected, expressing different types of cadherins were tested using in vitro cell aggregation and cell dissociation assays. A concentration-dependent inhibition of aggregation by the EC-SOD-derived HAV-containing peptide was detected only in N-cadherin expressing cells. These results suggest the localisation and possible protective role of EC-SOD B for cells expressing N-cadherin.

Amino Acid Sequence↗

PubMed1995

Reduction of extracellular superoxide dismutase activity by decapeptide derived from FGF-receptor.

Several synthetic decapeptides containing an HAV tripeptide motif were tested for their ability to modulate the enzymatic activity of rat extracellular SOD, an enzyme which also contains an HAV motif. Out of nine decapeptides that were tested, only a FGF-receptor derived peptide was active as a negative modulator of enzyme activity. These results strengthen the thesis that HAV motifs are not only involved in homophilic interactions and suggest that soluble FGF-receptor molecules might moderate the activity of extracellular SOD.

Amino Acid Sequence↗

PubMed1994

Tyrosine phosphorylation of a M(r) 38,000 A/B-type hnRNP protein selectively modulates its RNA binding.

The M(r) 38,000 RNA-binding protein (P38) is the major component of translationally repressed messenger ribonucleoproteins in cryptobiotic gastrulae of the brine shrimp Artemia. Partial elucidation of the amino acid sequence of P38 reveals that it is homologous to A/B-type hnRNP proteins. This was confirmed by immunodetection with antibodies specific for A/B-type hnRNP proteins from Drosophila melanogaster. P38 can be phosphorylated in vitro by a src-related protein tyrosine kinase on multiple tyrosine residues located predominantly in the glycine-rich domain. Tyrosine phosphorylated P38 can be efficiently dephosphorylated by a specific protein tyrosine phosphatase (1B-like) and by protein phosphatase 2A activated by the phosphotyrosyl phosphatase activator. Tyrosine phosphorylation of P38 slightly influences its subsequent phosphorylation by casein kinase II. The latter phosphorylation site is located in the glycine-rich domain of P38. Two-dimensional gel electrophoresis resolves P38 into multiple isoforms which shift to more acidic pI values after phosphorylation by protein tyrosine kinase or casein kinase II. From nitrocellulose filter binding and UV cross-linking analysis, evidence was obtained that tyrosine phosphorylation of P38 impairs its binding to poly(A) but not to poly(U). This demonstrates the involvement of tyrosine residues in polynucleotide-specific RNA binding that can be regulated by phosphorylation/dephosphorylation.

Amino Acid Sequence↗

PubMed1994

Characterization of a rat C6 glioma-secreted follistatin-related protein (FRP). Cloning and sequence of the human homologue.

A protein was isolated from rat C6 glioma-conditioned medium and was biochemically characterized. The heparin-binding protein has a native molecular mass of 55-75,000 Da, a molecular mass of 40-48,000 Da under denaturing conditions, and a pI of 5.0-6.0. Based on the determined partial amino acid sequences, the full lenght cDNA encoding the rat and human proteins were cloned. The cDNA sequences identified the isolated rat and human protein as the homologue of a recently reported mouse osteoblast-transforming-growth-factor-beta 1-inducible protein, encoded by the TSC-36 gene [Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T. & Nose, K. (1993) Eur. J. Biochem. 217, 13-19]. Analysis of the human, rat and mouse amino acid sequences indicates that these proteins are highly conserved (> 92% sequence identity). Sequence similarities with follistatin and the follistatin-like domain of agrin are revealed. The relationship with follistatin and agrin points to possible common functions for the cloned follistatin-related proteins (FRP). The protein has no effect on the inhibitory action of transforming growth factor-beta 1, on CCl-64 cell growth.

Agrin↗

PubMed1993

Purification and sequence of rat extracellular superoxide dismutase B secreted by C6 glioma.

An enzyme which converts radical oxygen, produced by phorbol 12-myristate 13-acetate activated neutrophils, into nonluminescent products is secreted by rat C6 glioma. The enzyme was purified from chemically defined conditioned media and identified as an extracellular superoxide dismutase (EC-SOD). The purified enzyme is distinct from human EC-SOD C (Hjalmarsson, K., Marklund, S. L., Engström, A., and Edlund, T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6340-6344) by its elution from heparin-Sepharose at 300-400 mM NaCl, its pI of 6.1-7.2, and its native M(r) of 85,000 +/- 20,000. The rat EC-SOD is a dimer with a subunit M(r) of 34,000-36,000 and is extensively modified by post-translational processing. Although rat EC-SOD has a high sequence homology with the catalytic center and the polybasic heparin-binding site near the COOH terminus of human EC-SOD C, its NH2-terminal sequence and the sequences flanking the heparin-binding site differ substantially. The sequence of the isolated rat EC-SOD cDNA fully confirms the data obtained from amino acid sequence analysis. The amino acid sequence of the enzyme and its biochemical properties support its identification as the rat EC-SOD B.

Amino Acid Sequence↗

PubMed1993

Inhibition of casein kinase II by dinucleoside polyphosphates.

In our search for potential inhibitors of casein kinase II (CKII) in Artemia, we have shown that dinucleoside polyphosphates are a novel class of effectors for this ubiquitous protein kinase. P1,P4-di(guanosine-5')-tetraphosphate (Gp4G) is a better CKII inhibitor than P1,P4-di(adenosine-5')-tetraphosphate (Ap4A). The inhibition by both effectors is more potent when GTP is used as phosphate donor instead of ATP. The inhibition of CKII increases with the number of phosphates linking the guanosine/adenosine moieties (for n = 2-6). Ap4A does not compete with the protein substrate and causes an increase in the apparent KmATP and a decrease in the apparent VmATP, indicating a mixed type of inhibition with respect to ATP.

Amino Acid Sequence↗

PubMed1992

Synthesis of glial fibrillary acidic protein in rat C6 glioma in chemically defined medium: cyclic AMP-dependent transcriptional and translational regulation.

Glial fibrillary acidic protein (GFA) expression was induced in rat C6 glioma in chemically defined medium by the addition of N6, O2'-dibutyryl cyclic AMP (dbcAMP). Induction was dependent on the increase in intracellular cyclic AMP (cAMP), which was linearly correlated with added dbcAMP. Contrary to GFA mRNA synthesis, which can be obtained by cAMP-dependent and -independent pathways, translation of mRNA into GFA was observed only above a cellular cAMP concentration of approximately 0.2 fmol/cell. dbcAMP stimulation did not affect the vimentin concentration, which remained at a low level, but changed the cellular morphology from a bipolar to a stellate shape. A similar morphological change was observed after stimulation of C6 with lipopolysaccharide (LPS). However, LPS did not significantly increase the intracellular concentration of cAMP and the LPS-induced mRNA was not translated into GFA. Our results indicate that GFA synthesis is regulated at the mRNA level and at the translational level and that a cAMP-dependent mechanism determines the ultimate synthesis of GFA by a yet unknown mechanism.

Animals↗

PubMed1990

Hydrodynamic characterization of the major intrinsic protein from the bovine lens fiber membranes. Extraction in n-octyl-beta-D-glucopyranoside and evidence for a tetrameric structure.

The major protein from the bovine lens fiber cell membranes, the 26-kilodalton protein (major intrinsic protein (MIP26)), has been solubilized in n-octyl-beta-D-glucopyranoside and purified by gel filtration. The final preparation was free of detergent micelles. Gel electrophoresis in denaturing conditions has confirmed the purity of the protein sample. A s20,w of 5.55 S, obtained from analytical ultracentrifugation, and a D20,w of 3.62 x 10(-7) cm2 s-1, obtained from photon correlation spectroscopy, resulted in a molar mass of (176,000 +/- 15,000) g/mol for the protein-detergent complex using the Svedberg relation. The measured detergent content of 0.71 g of detergent/g of protein resulted in a calculated partial specific volume of 0.787 cm3/g for the protein-detergent complex and a molar mass of 103,000 g/mol for the protein moiety. This allowed us to conclude that the protein-detergent complex contains four copies of the MIP26 protein, which supports the suggestion that in vivo the MIP26 molecules cluster in tetramers to form a pore-like structure.

Animals↗

PubMed1987

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.

Adenosine Triphosphate↗