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1.
Neuropathol Appl Neurobiol ; 34(3): 296-305, 2008 Jun.
Article in English | MEDLINE | ID: mdl-17971073

ABSTRACT

Pineal parenchymal tumours (PPT) are rare neoplasms and there have been few in vitro studies. Their capacity for synthesizing and secreting melatonin has been only partially examined. We investigated the presence of messenger RNA (mRNA) encoding tryptophan hydroxylase (TPH), arylalkylamine N-acetyltransferase (AANAT), hydroxyindol-O-methyltransferase (HIOMT), three enzymes involved in melatonin synthesis, and c-myc, a tumoural marker, in 10 PPT, one papillary tumour of the pineal region (PTPR), cell cultures derived from four PPTs and from three other tumours of the pineal region, and in normal pineal gland. Moreover, protein expression of TPH was investigated in three PPT and PTPR. Quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry were used and the melatonin production by tumoural cells in vitro was analysed by radioimmunoassay. We showed that all the tumoural tissues and cells contained c-myc mRNA. mRNAs encoding TPH, AANAT and HIOMT were detected in all PPT, suggesting that tumour cells can synthesize melatonin. Only PPT expressed TPH protein. Cultured cells lost expression of transcripts throughout passages even if ultrastructural study revealed the presence of characteristic organelles in these tumoural cells. Nevertheless, the basal secretion of melatonin observed in one PPT culture is in favour of a maintained melatonin production and secretion by tumoural pinealocytes, but melatonin production was not stimulated by a beta noradrenergic agonist. Moreover, PTPR never expressed mRNA encoding TPH, AANAT and HIOMT. Our results may contribute to a better understanding of the biology of PTT and PTPR and may help to the diagnosis of these rare tumours.


Subject(s)
Brain Neoplasms/enzymology , Brain Neoplasms/pathology , Pineal Gland/enzymology , Pineal Gland/pathology , Pinealoma/enzymology , Pinealoma/pathology , Acetylserotonin O-Methyltransferase/biosynthesis , Adult , Aged , Arylalkylamine N-Acetyltransferase/biosynthesis , Cells, Cultured , Child , Child, Preschool , Female , Humans , Immunohistochemistry , Infant , Male , Melatonin/biosynthesis , Middle Aged , Proto-Oncogene Proteins c-myc/biosynthesis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Tryptophan Hydroxylase/biosynthesis
2.
J Neurooncol ; 43(2): 115-26, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10533723

ABSTRACT

We studied the effect of the treatment of a medulloblastoma cell line by human T cells derived soluble factors. Medulloblastoma is one of the more common aggressive solid neoplasms in children for which there is no adequate therapy. Cell lines established from such tumours may be helpful to test the effect of various molecules on cell proliferation. Previous studies have suggested that T cell-derived factors may be toxic for the medulloblastoma cell line Dev. Cytokines were thought to mediate this effect. In this paper, we described changes in morphology, survival and cell cycle induced in Dev cells cocultured with human T cell lines chronically infected with a retrovirus (HTLV-I) and known to secrete high level of cytokines TNF alpha, IL1alpha and IL6. Such cocultures resulted in the death of a part of Dev cells and in decreased proliferation of surviving cells, associated with morphological changes and increase in vimentin expression. Treatment with conditioned medium from infected Dev cells, containing virus induced cytokines, triggered the same effect. Reduction of these effects by TNF alpha deprivation of conditioned medium suggested that this cytokine may be implicated. Direct treatment of Dev cells with recombinant cytokines indicated that TNF alpha, but not IL1 or IL6, is associated with Dev cell alterations. TNF alpha was shown to induce the death of Dev cells by an apoptotic pathway. Furthermore, TNF alpha had a bimodal effect on the cell cycle of surviving Dev cells. These differential effects of such cytokines on medulloblastoma cells could be therefore of interest for immunotherapy of these tumours.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Cycle/drug effects , Cytotoxins/toxicity , Interleukin-1/toxicity , Interleukin-6/toxicity , Medulloblastoma/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/toxicity , CD4-Positive T-Lymphocytes/radiation effects , Cell Division/drug effects , Cell Line , Cerebellar Neoplasms/immunology , Coculture Techniques , Culture Media, Conditioned , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Cytotoxins/biosynthesis , Gamma Rays , Humans , In Situ Nick-End Labeling , Interleukin-1/biosynthesis , Interleukin-6/biosynthesis , Kinetics , T-Lymphocytes/radiation effects , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis
3.
Acta Neuropathol ; 95(5): 532-9, 1998 May.
Article in English | MEDLINE | ID: mdl-9600600

ABSTRACT

Using both tumor specimen and cultured tumor cells, we have studied the differentiation of a pineocytoma by light and electron microscopy (EM) and immunohistochemical demonstration of glial, neuronal and neuroendocrine markers. Only interstitial cells were labeled with anti-glial fibrillary acidic protein and anti-S100 protein antibodies. Synaptophysin, neurofilaments and tau labeling was found in cells forming the pineocytomatous rosettes. Some cells also bound the anti-tryptophan hydroxylase antibody (TPOH), but no staining was seen after application of anti-chromogranin A or S-antigen antibodies. EM provided evidence for neurosensory differentiation demonstrating the presence of vesicle-crowned rodlets, cilia (9+0) and fibrous filaments. In culture, tumor cells proliferated slowly and showed positive immunolabeling for vimentin and TPOH. Expression of mRNA coding for TPOH, serotonin N-acetyltransferase, hydroxyindole-O-methyl-transferase and c-myc was found in the tumor using reverse transcriptase-polymerase chain reaction. These results demonstrate neuronal differentiation of this pineocytoma and suggest that the neoplastic pineal cells are capable of synthesizing serotonin and melatonin.


Subject(s)
Brain Neoplasms/pathology , Pinealoma/pathology , Acetylserotonin O-Methyltransferase/metabolism , Arylamine N-Acetyltransferase/metabolism , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/ultrastructure , Cells, Cultured , DNA Primers , Female , Fluorescent Antibody Technique, Direct , Humans , Immunohistochemistry , Microscopy, Electron , Middle Aged , Pinealoma/metabolism , Pinealoma/ultrastructure , Polymerase Chain Reaction , RNA/isolation & purification , RNA/metabolism , Tryptophan Hydroxylase/metabolism
4.
Glia ; 23(1): 45-60, 1998 May.
Article in English | MEDLINE | ID: mdl-9562184

ABSTRACT

Gamma-Aminobutyric acid (GABA) transporters (GAT-1, GAT-2, and GAT-3) play a key role in the termination of GABA transmission and the regulation of extracellular GABA concentrations. In the present study, pharmacological, cellular, and molecular analyses provide evidence for a modulatory effect of serotoninergic neurons on the activity and expression of glial GABA transporters in the rat cerebellum. Degeneration of serotoninergic neurons after in vivo 5,7-dihydroxytryptamine (5,7-DHT) treatment resulted in a significant decrease (-27%) in [3H]-GABA uptake into cerebellar punches. This decrease probably occurred via inhibition of GAT-2 or GAT-3 activity since their inhibitor, beta-alanine, induced a decrease in [3H]-GABA uptake in punches of sham-operated rats (-28%), but not in punches of 5,7-DHT-treated rats, demonstrating that serotonin terminal degeneration had already impaired the beta-alanine-sensitive component of GABA uptake. In contrast, nipecotic acid, a preferential inhibitor of GAT-1, induced comparable decreases in [3H]-GABA uptake comparable in punches of 5,7-DHT (-38%) versus sham-operated rats (-37%). The decreases in GAT-1 (-16%), GAT-2 (-34%), and GAT-3 (-32%) mRNA levels after 5,7-DHT treatment (detected by quantitative RT-PCR) are consistent with a serotoninergic control of GABA transporter expression at the transcriptional level. The cellular distribution of GAT-2 and GAT-3 mRNA, shown by in situ hybridization, suggests a glial localization of these transporters in the cerebellum and demonstrated a preferential anatomical localization of GAT-2 mRNA in the granular layer and of GAT-3 mRNA in the deep cerebellar nuclei. A direct serotoninergic control of glial GABA uptake was further demonstrated in vitro since serotonin stimulated the activity and mRNA expression of the GABA transporters in cerebellar astrocyte cultures.


Subject(s)
5,7-Dihydroxytryptamine/toxicity , Carrier Proteins/metabolism , Cerebellum/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins , Nerve Tissue Proteins , Neuroglia/metabolism , Neurons/metabolism , Organic Anion Transporters , Transcription, Genetic , gamma-Aminobutyric Acid/metabolism , Animals , Animals, Newborn , Base Sequence , Carrier Proteins/biosynthesis , Cerebellum/pathology , DNA Primers , Female , GABA Plasma Membrane Transport Proteins , Male , Membrane Proteins/biosynthesis , Molecular Sequence Data , Nerve Degeneration , Neurotoxins/toxicity , Oligonucleotide Probes , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Serotonin Plasma Membrane Transport Proteins , Transcription, Genetic/drug effects
5.
J Neurosci Res ; 49(6): 750-8, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9335262

ABSTRACT

The levels of mRNAs coding for tryptophan hydroxylase (TPOH), the first enzyme in melatonin synthesis, have been investigated by quantitative reverse transcription of RNA, followed by polymerase chain reaction (RT-PCR), after stimulation of neonatal pineal organ cultures with Noradrenaline (NA). TPOH mRNAs were specifically amplified from various adult tissues, namely the pineal gland, raphe, retina, and kidney, but not the lung. PCR signals for TPOH were detected in the neonatal pineal gland in the absence of stimulation. Stimulation of neonatal pineal organ culture with 0.1 microM NA resulted in a significant increase (x2.5) in expression of TPOH mRNAs, whereas higher doses (1 and 10 microM) had no effect. All concentrations of NA enhanced melatonin secretion. Our results suggest that the level of TPOH mRNAs can be controlled by NA and that this effect might be implicated in the gene level regulation of the daily enzyme rhythm in the rat pineal gland.


Subject(s)
Norepinephrine/pharmacology , Pineal Gland/enzymology , Sympathomimetics/pharmacology , Tryptophan Hydroxylase/genetics , Animals , Animals, Newborn , DNA Primers , Gene Expression Regulation, Enzymologic/drug effects , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Male , Pineal Gland/drug effects , Polymerase Chain Reaction , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley
6.
Int Immunol ; 8(2): 211-9, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8671606

ABSTRACT

Murine mAb injected into patients behave as exogenous antigens and trigger a specific immune response characterized mainly by CD4+ T lymphocytes. They are recognized by T cells under a processed form and in a MHC class II-restricted fashion. IgG degradation which occurs in antigen-presenting cells (APC) has been studied in vitro. We have shown that partial reduction of this antigen is an early event which is significant for the generation of class II-restricted fragments presentable to antigen-specific T cells. Two different murine mAb were used as antigens and human monocytic U937 or antigen non-specific Epstein-Barr virus-transformed B cells as APC. Upon cellular internalization IgG are rapidly cleaved leading to 24-25 kDa fragments. One of the major and early events corresponds to partial reduction of IgG--the light chain is released from the intact molecule, heavy chains remaining bound together. Partial in vitro reduction of IgG followed by presentation by fixed B cells to specific T cells showed that only kappa light chain-specific T cell clones are stimulated, in contrast to heavy chain-specific T cell clones. The response to the kappa chains of specific T cells points to a significant role played by the early IgG partial reduction in the generation of kappa light chain class II binding fragments.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antigen Presentation , Antigen-Presenting Cells/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Antibody Specificity , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line, Transformed , Humans , Immunoglobulin kappa-Chains/metabolism , Intracellular Fluid/metabolism , Lymphoma, Large B-Cell, Diffuse , Oxidation-Reduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Cells, Cultured
7.
Eur J Immunol ; 24(7): 1620-6, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8026522

ABSTRACT

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigen-presenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,kappa and IgG2a,kappa murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and purified in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of kappa light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigen-presenting cells.


Subject(s)
Antibodies, Monoclonal/metabolism , Complement C3b/metabolism , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains/metabolism , Animals , Antigen-Antibody Complex/immunology , B-Lymphocytes/immunology , Cells, Cultured , Humans , Immunoblotting , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/immunology , Lymphocyte Activation , Mice , Radioligand Assay , T-Lymphocytes/immunology , Up-Regulation
8.
Mol Immunol ; 30(11): 1033-9, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8350873

ABSTRACT

Monoclonal antibodies used for diagnostic and therapeutic purposes behave as antigens when injected into patients. They are recognized by T cells in a processed form and in a major histocompatibility complex class II restricted fashion. Monoclonal murine IgG2a were used as a model to analyse the early phase of antigen processing in U937 cells. IgG2a prebound to cell surface Fc receptors were rapidly internalized in the cells. During internalization, they were proteolysed with a time-dependent intracellular accumulation of 26, 25, 24, 22 and 14 kDa fragments. Comparison of in vitro IgG2a proteolysis by U937 subcellular fractions or by purified cathepsin B and their intracellular processing indicated that a major cathepsin B like protease is responsible for IgG2a intracellular processing in endo-lysosomal compartments of U937 cells.


Subject(s)
Cathepsin B/physiology , Immunoglobulin G/metabolism , Endocytosis , Humans , Kinetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Lysosomes/metabolism , Tumor Cells, Cultured
9.
Neuroscience ; 52(4): 1069-79, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8450975

ABSTRACT

Constituent cells of medulloblastoma, the most common brain tumor occurring in childhood, resemble the primitive neuroepithelial cells normally found in the developing nervous system. However, mutational events prevent their further differentiation. We used the human T cell lymphotrophic virus type 1 to activate these deregulated immature cells by means of its transactivating protein Tax. Concomitant with viral infection was a decrease in cell proliferation characterized by inhibition of [3H]thymidine incorporation and in the number of cells in the G2/M phase of the cell cycle. Morphological changes suggested that medulloblastoma cells differentiated along the astrocytic lineage. The glial phenotype was confirmed by the induction of the glial fibrillary acidic protein and the glial enzyme glutamine synthetase. A direct viral effect and/or secondary effects to viral infection via paracrine/autocrine pathways could counterbalance the maturational defect in these medulloblastoma cells.


Subject(s)
Astrocytes/cytology , Cerebellar Neoplasms/pathology , Human T-lymphotropic virus 1/genetics , Medulloblastoma/pathology , Cell Cycle , Cell Differentiation , Cell Transformation, Viral , Gene Products, tax/metabolism , Glial Fibrillary Acidic Protein/analysis , Humans , Thymidine/metabolism , Tritium , Tumor Cells, Cultured , Vimentin/analysis , Viral Proteins/analysis
10.
J Biol Chem ; 267(13): 9053-8, 1992 May 05.
Article in English | MEDLINE | ID: mdl-1577743

ABSTRACT

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.


Subject(s)
Copper/metabolism , Tetanus Toxin/metabolism , Zinc/metabolism , Amino Acid Sequence , Binding Sites , Cations, Divalent , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Spectrometry, Mass, Fast Atom Bombardment
11.
Dev Biol Stand ; 77: 57-64, 1992.
Article in English | MEDLINE | ID: mdl-1426674

ABSTRACT

The bacterial extract IMOCUR is described as an in vivo stimulant of antibody production during animal testing and human clinical trials. Using a slightly modified procedure (13) dealing with in vitro immunoglobulin production by C57B1/6 mouse spleen cells, we have shown that IMOCUR potentiates spontaneous IgM production. In order to explore the putative relation between this in vitro activity and the current in vivo control test (stimulation of plaque-forming cell production after sheep red blood cell injection to Balb/c mouse), we have assayed 10 lyophilisates in vitro and in vivo before and after heat inactivation (80 degrees C, 7 days in a saturated water atmosphere). Results have shown that this treatment inhibits, respectively, totally and partially in vivo and in vitro activities. Thus the in vitro technique seems to be appropriate for the control of activity of the various batches of IMOCUR. Experiments are under way to clarify the mathematical correlation which may exist between the in vitro and in vivo experiments.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens, Bacterial/immunology , Bacteria , Biological Assay/standards , Cell Extracts , Hemolytic Plaque Technique/standards , Immunoglobulin M/biosynthesis , Animals , Cells, Cultured , Drug Stability , Female , Hot Temperature , Mice , Mice, Inbred BALB C/immunology , Mice, Inbred C57BL/immunology , Reproducibility of Results
12.
Biochem J ; 277 ( Pt 1): 47-51, 1991 Jul 01.
Article in English | MEDLINE | ID: mdl-1649603

ABSTRACT

Proteolysis of 125I-labelled tetanus toxin by subcellular fractions from an Epstein-Barr-virus-transformed B lymphoblastoid cell line, JY, was investigated. Fractions enriched in lysosomes and plasma membranes cleaved the toxin molecule at several sites, with a pH optimum of 5.5. N-Terminal sequence analysis of Mr-81,000, -45,000 and -35,000 proteolytic fragments indicated cleavage of the Asp-460-Leu-461, Asp-872-Glu-873 and Ile-1013-Thr-1014 peptide bonds, all sites located within the heavy chain of the toxin molecule. Additional sites near the C-terminus of the heavy chain, giving rise to low-Mr peptides, were implicated. The toxin light chain was more resistant to proteolysis. A similar pattern of fragmentation was observed with tetanus toxin biosynthetically radiolabelled with 14C-labelled amino acids, showing that the proteolysis was not an artifact caused by iodination. The proteolytic activity was inhibited by the serine proteinase inhibitor di-isopropyl phosphorofluoridate, thiol-blocking proteinase inhibitors N-ethylmaleimide and iodoacetamide, and by EDTA. These results represent a preliminary characterization of the processing in vitro of tetanus toxin by an antigen-presenting cell line.


Subject(s)
Tetanus Toxin/metabolism , B-Lymphocytes , Cell Fractionation , Cell Line , Cell Transformation, Viral , Electrophoresis, Polyacrylamide Gel , Herpesvirus 4, Human/genetics , Humans , Kinetics , Macromolecular Substances , Molecular Weight , Peptide Fragments/isolation & purification , Subcellular Fractions/metabolism
13.
Neurosci Lett ; 125(2): 101-6, 1991 Apr 29.
Article in English | MEDLINE | ID: mdl-1881585

ABSTRACT

This study was performed to determine whether neurons, where gamma-aminobutyric acid (GABA) and serotonin (5-HT) coexist, represent neuronal entities which can survive in vitro. In dissociated cultures from 18-day-old embryonic rat metencephalon, it was possible to develop glial and neuronal cells. Among the neurons, some of them, which contain glutamate decarboxylase or are capable of accumulating [3H]GABA are GABAergic; others, containing tryptophan hydroxylase or 5-HT are serotoninergic. By combining radioautography and immunocytochemistry, it was possible to observe neurons where 5-HT and GABA coexist. Cultures might be a suitable model to study the functioning (release or synthesis of both neurotransmitters) of neurons where two classical neurotransmitters coexist.


Subject(s)
Neurons/cytology , Pons/cytology , Serotonin/analysis , gamma-Aminobutyric Acid/analysis , Animals , Autoradiography , Biological Transport , Cells, Cultured , Embryo, Mammalian , Immunohistochemistry , Microscopy, Phase-Contrast , Neurons/metabolism , Pons/metabolism , Rats , Tritium , gamma-Aminobutyric Acid/metabolism
14.
Biochem J ; 263(1): 157-64, 1989 Oct 01.
Article in English | MEDLINE | ID: mdl-2481436

ABSTRACT

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon.


Subject(s)
Complement Activation , Complement C1/biosynthesis , Interferons/pharmacology , Lymphokines/pharmacology , Monocytes/metabolism , Amino Acids/analysis , Cell-Free System , Chromatography, Gel , DNA/analysis , DNA Probes , Electrophoresis, Polyacrylamide Gel , Humans , Interferon-gamma/pharmacology , Interferons/isolation & purification , Isoelectric Point , Lymphokines/isolation & purification , Precipitin Tests , Protein Biosynthesis , RNA/genetics , RNA/isolation & purification , Recombinant Proteins
15.
Behring Inst Mitt ; (84): 80-8, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2552984

ABSTRACT

Biosynthesis of C1r and C1s subcomponents has been studied using monocytes and macrophages, hepatocytes and hepatoma cell lines or fibroblasts. Both proteins have been detected in supernatants and cell lysates as proenzymic monocatenar molecules. C1r and C1s were secreted by stimulated monocytes and by Hep G2 cells, according to a 1:1 stoichiometry. Monocyte C1s secretion was enhanced by lymphokines, such as alpha- or gamma-interferon or by placental soluble factors. Expression of both proteins was coordinately modulated by a newly purified 14 kDa lymphokine at a pretranslational level. Data from in vitro RNA translation are discussed.


Subject(s)
Complement C1r/biosynthesis , Complement C1s/biosynthesis , Animals , Carcinoma, Hepatocellular/pathology , Cells, Cultured , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Growth Substances/pharmacology , Guinea Pigs , Humans , Liver/metabolism , Liver Neoplasms/pathology , Lymphokines/pharmacology , Macrophages/metabolism , Phagocytes/metabolism , Tumor Cells, Cultured/metabolism
16.
Biochem J ; 244(1): 117-21, 1987 May 15.
Article in English | MEDLINE | ID: mdl-3311024

ABSTRACT

The effects of proteolysis and deglycosylation on C1 inhibitor (C1Inh) were tested with respect to both its ability to form complexes with C1s and its capacity to block C1 autoactivation. Limited proteolysis of C1Inh by Staphylococcus aureus V8 proteinase, proline-specific endopeptidase or elastase generated a major high-Mr (approximately 86,000) fragment. In contrast with the fragment produced by elastase, which was inactive, the fragments resulting from V8 proteinase and proline-specific endopeptidase treatment retained activity. Deglycosylation with N-glycanase or O-glycanase, or both, had no major effect on the functional activity of C1Inh.


Subject(s)
Complement C1 Inactivator Proteins/metabolism , Complement C1s/metabolism , Electrophoresis, Polyacrylamide Gel , Endopeptidases , Glycoside Hydrolases , Humans , Macromolecular Substances , Peptide Fragments/analysis
17.
Biochem J ; 237(1): 93-8, 1986 Jul 01.
Article in English | MEDLINE | ID: mdl-3099750

ABSTRACT

The biosynthesis of C1 Inh (C1 inhibitor) was studied in a human hepatoma cell line (Hep G2) by metabolic labelling, immunoprecipitation with anti-(C1 Inh) serum, analysis on SDS/polyacrylamide gel slabs and fluorography. Two forms of C1 Inh are secreted by Hep G2: a minor form of Mr 90,000 and a major form of Mr approximately 100,000. The latter form is also found in small amounts intracellularly in co-existence with an 80,000-Mr form. Accumulation of the 80,000-Mr C1 Inh is favoured when the cells are labelled at 23 degrees C instead of 37 degrees C or when they are treated with monensin. In the presence of tunicamycin, a compound that blocks the formation of N-asparagine-linked oligosaccharide chains, a decrease in Mr of both secreted and intracellular major forms is observed, indicating that secreted and intracellular C1 Inh contain N-linked oligosaccharide units. The 100,000 Mr secreted C1 Inh is sensitive to endoglycosidase F but resistant to endoglycosidase H, and it incorporates [3H]galactose, [3H]glucosamine and [3H]galactosamine, indicating the presence of both N-linked oligosaccharides of the complex type and O-linked oligosaccharides. The intracellular C1 Inh contains N-linked oligosaccharide units of the high-mannose type as demonstrated by endoglycosidase H-sensitivity. The functional activity of C1 Inh during its biosynthesis was tested by studying its reactivity towards C1s. Both secreted and intracellular C1 Inh form covalent-like complexes with purified plasma C1s. The underglycosylated C1 Inh secreted in presence of tunicamycin is still reactive with purified C1s. These results clearly show that sugars are not essential for this inhibitory activity of C1 Inh.


Subject(s)
Complement Activating Enzymes/metabolism , Complement C1 Inactivator Proteins/biosynthesis , Liver/metabolism , Acetylglucosaminidase , Cell Line , Chemical Precipitation , Complement C1s , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases , Humans , Immunochemistry , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase , Monosaccharides/metabolism , Tunicamycin/pharmacology
18.
Biochem J ; 233(2): 559-64, 1986 Jan 15.
Article in English | MEDLINE | ID: mdl-3006672

ABSTRACT

The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.


Subject(s)
Complement Activating Enzymes/biosynthesis , Liver Neoplasms, Experimental/metabolism , Animals , Cell Line , Centrifugation, Density Gradient , Chemical Precipitation , Complement Activating Enzymes/immunology , Complement C1r , Complement C1s , Electrophoresis, Polyacrylamide Gel , Humans , Macromolecular Substances , Peptide Fragments/analysis
19.
FEBS Lett ; 190(1): 65-8, 1985 Oct 07.
Article in English | MEDLINE | ID: mdl-3876244

ABSTRACT

C1q, C1s and C1 Inh synthesized and secreted by human monocytes were characterized by SDS-PAGE. C1q is formed of three chains A (Mr approximately 35 000), B (Mr approximately 33 000) and C (Mr approximately 25 000) which are associated in two subunits A-B and C-C. It appears identical to C1q purified from plasma. C1s is secreted as a non-activated, monocatenar protein of Mr approximately 87 000 identical to proenzymic C1s from plasma. Secreted C1 Inh (Mr approximately 100 000) has a slightly higher Mr than purified plasmatic C1 Inh. Monensin treatment of the cells favours the intracytoplasmic accumulation of products at various glycosylation stages.


Subject(s)
Complement Activating Enzymes/biosynthesis , Complement C1 Inactivator Proteins/biosynthesis , Monocytes/immunology , Cells, Cultured , Chemical Precipitation , Complement C1q , Complement C1s , Cytoplasm/immunology , Electrophoresis, Polyacrylamide Gel , Humans , Immunochemistry , Monensin/pharmacology
20.
Biochem J ; 218(2): 547-55, 1984 Mar 01.
Article in English | MEDLINE | ID: mdl-6370241

ABSTRACT

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.


Subject(s)
Hyaluronan Receptors , Macrophages/immunology , Membrane Glycoproteins , Receptors, Complement/metabolism , Binding Sites , Carrier Proteins , Cell Line , Collagen/metabolism , Fluorescent Antibody Technique , Humans , Kinetics , Mitochondrial Proteins , Osmolar Concentration , Peptide Fragments/metabolism , Peptide Hydrolases/pharmacology , Receptors, Complement/drug effects
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