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1.
Transplant Proc ; 51(5): 1540-1544, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31155188

ABSTRACT

OBJECTIVE: The study focuses on the experience of family members of deceased potential donors in deciding to refuse donation when their loved one had expressed his or her wish in life not to donate organs and tissues for transplantation. METHOD: This is a qualitative study that uses social phenomenology as the theoretical reference, interviewing 8 family members of deceased potential donors. RESULTS: The family members' experiences were represented by the following categories: beliefs related to donation, fear in the face of the loved one's death, and the ethical dilemma of deciding to refuse the donation. The meaning of the refusal to donate was represented by the following categories: respect for the loved one's wishes and the family's peace of mind with the decision. CONCLUSIONS: The study shed light on the experience of family members of deceased potential donors in making the decision to refuse donation. The concerns that motivate refusal were elucidated and the meanings of the decision's intentionality were unveiled. The resulting knowledge about these families' experiences provides backing for experts in donation and transplantation who work in different realities, pointing to strategies for improving care for such family members.


Subject(s)
Family/psychology , Tissue Donors/ethics , Tissue and Organ Procurement/ethics , Decision Making , Female , Humans , Male , Morals , Qualitative Research
2.
Article in English | MEDLINE | ID: mdl-21209390

ABSTRACT

How a nuclear domain is formed at specific chromatin loci and maintained throughout multiple cellular divisions is a central question in the field of nuclear organization. Recent efforts have concentrated on understanding how a domain is set during development in a particular cell lineage and then how DNA replication and repair in interphase as well as chromosome dynamics in mitosis deal with chromatin states at specific loci to propagate functional organization. In the latter case, for each of these events, one must not only evaluate the impact in terms of the extent of the disruption and/or modification of chromatin but also determine how and when proper organization can be restored thereafter. Using heterochromatin at mouse pericentromeres as a model, we present how important advances have been made that open avenues for understanding mechanisms involved in de novo heterochromatin formation and its duplication during replication.


Subject(s)
Centromere/metabolism , DNA Replication/genetics , Gene Duplication/genetics , Heterochromatin/metabolism , Models, Genetic , Animals , Embryonic Development/genetics , Mice , NIH 3T3 Cells , S Phase
3.
Pathologica ; 100(5): 416-9, 2008 Oct.
Article in English | MEDLINE | ID: mdl-19253604

ABSTRACT

Multicystic peritoneal mesothelioma is a rare lesion occurring mainly in women in a reproductive age. Its pathogenesis is unclear. We report three cases of multicystic peritoneal mesothelioma in patients that were 28, 38 and 47 years of age (one male, two females). A history of abdominal surgery was reported in two cases. Explorative laparotomy was presumptive of a pseudomyxoma peritoni in two cases, and hyperthermic intraperitoneal chemotherapy was performed. Histological examination demonstrated multicystic lesions with mesothelial cells lining confirmed by immunohistochemical analysis. Unusual findings such as hyperplasia, hobnail features, cytoplasmic vacuolisation and papillary pattern were occasionally noted. The clinical presentation, pathogenesis and pathologic features including differential diagnosis of multicystic peritoneal mesothelioma are discussed.


Subject(s)
Mesothelioma, Cystic/pathology , Peritoneal Neoplasms/pathology , Abdominal Pain/etiology , Adult , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Appendectomy , Combined Modality Therapy , Dermoid Cyst , Diagnosis, Differential , Endometriosis/complications , Female , Humans , Hyperthermia, Induced , Intestinal Perforation/etiology , Laparotomy , Male , Mesothelioma, Cystic/complications , Mesothelioma, Cystic/diagnosis , Mesothelioma, Cystic/drug therapy , Mesothelioma, Cystic/surgery , Middle Aged , Neoplasms, Multiple Primary , Ovarian Neoplasms , Peritoneal Neoplasms/complications , Peritoneal Neoplasms/diagnosis , Peritoneal Neoplasms/drug therapy , Peritoneal Neoplasms/surgery , Peritonitis/etiology , Pseudomyxoma Peritonei/diagnosis , Remission Induction
4.
An Sist Sanit Navar ; 29 Suppl 2: 163-73, 2006.
Article in Spanish | MEDLINE | ID: mdl-16998525

ABSTRACT

The keratoplasty, or cornea transplant, is one of the oldest surgical techniques in opthalmology, whose indication are: 1) tectonic, in order to preserve corneal anatomy and integrity; 2) clinical, in order to eliminate the inflamed corneal tissue in cases refractory to medical treatment; 3) optical, in order to improve visual acuity; and 4) cosmetic, in order to improve the appearance of the eye. Improvements in technique and instruments, as well as in post-operative treatment and the means of preserving donated tissue, have improved survival of the grafts. The Pamplona Model of transplant coordination of the Virgen del Camino Hospital is considered to be original and unique in Spain. The logistics of this program include the protocol for detection and extraction of corneas as well as for keratoplasties.


Subject(s)
Corneal Transplantation , Adolescent , Adult , Aged , Aged, 80 and over , Child , Clinical Protocols , Corneal Transplantation/methods , Donor Selection , Eye Banks , Female , Graft Survival , Humans , Male , Middle Aged , Spain , Tissue Donors , Treatment Outcome , Visual Acuity
5.
An. sist. sanit. Navar ; 29(supl.2): 163-174, mayo-ago. 2006. tab
Article in Es | IBECS | ID: ibc-052133

ABSTRACT

La queratoplastia o trasplante de córnea es una de las técnicas quirúrgicas más antiguas de la oftalmología cuyas indicaciones son: 1) tectónicas, para preservar la anatomía e integridad corneal, 2) clínicas, para eliminar el tejido corneal inflamado en casos refractarios al tratamiento médico, 3) ópticas, para mejorar la agudeza visual y 4) cosméticas para mejorar el aspecto del ojo. El perfeccionamiento en la técnica y en el instrumental, así como en el tratamiento postoperatorio y en los medios de conservación del tejido donante han mejorado la supervivencia de los injertos realizados. El Modelo Pamplona de coordinación de trasplantes del Hospital Virgen del Camino (HVC) está considerado como original y único en España, y en la logística de este programa se incluye el protocolo de detección y extracción de córneas así como el de queratoplastias


The keratoplasty, or corena transplant, is one of the oldest surgical techniques in opthalmology, whose indication are: 1) tectonic, in order to preserve corneal anatomy and integrity; 2) clinical, in order to eliminate the inflamed corneal tissue in cases refractory to medical treatment; 3) optical, in order to improve visual acuity; and 4) cosmetic, in order to improve the appearance of the eye. Improvements in technique and instruments, as well as in post-operative treatment and the means of preserving donated tissue, have improved survival of the grafts. The Pamplona Model of transplant coordination of the Virgen del Camino Hospital is considered to be original and unique in Spain. The logistics of this program include the protocol for detection and extraction of corneas as well as for keratoplasties


Subject(s)
Humans , Corneal Transplantation/methods , Eye Banks/methods , Graft Survival , Tissue Donors/supply & distribution , Transplantation Conditioning/methods
6.
Nat Cell Biol ; 3(2): 114-20, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11175742

ABSTRACT

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.


Subject(s)
Cell Nucleus/drug effects , Centromere/metabolism , Enzyme Inhibitors/pharmacology , Heterochromatin/metabolism , Histone Deacetylase Inhibitors , Histones/metabolism , Hydroxamic Acids/pharmacology , Animals , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line , Cell Nucleus/enzymology , Cell Nucleus/metabolism , DNA/metabolism , Histone Deacetylases/metabolism , Humans , In Situ Hybridization, Fluorescence , Mice , Microscopy, Confocal , Protein Structure, Tertiary , Recombinant Proteins/metabolism
7.
Mol Cell Biol ; 20(13): 4562-71, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10848583

ABSTRACT

To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopus extract-dependent manner. U1 snRNA export requires a 5' monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.


Subject(s)
Carrier Proteins/metabolism , Karyopherins , Nucleocytoplasmic Transport Proteins , RNA, Small Nuclear/metabolism , RNA/metabolism , Receptors, Cytoplasmic and Nuclear , ran GTP-Binding Protein/metabolism , Animals , Biological Transport , Carrier Proteins/genetics , Female , Guanosine Triphosphate/metabolism , Humans , Hydrolysis , Methylation , Molecular Biology/methods , Mutation , RNA/chemistry , RNA Caps , RNA, Messenger/metabolism , RNA, Small Nuclear/chemistry , RNA, Transfer, Met/metabolism , Exportin 1 Protein
8.
EMBO J ; 16(16): 4839-50, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9305626

ABSTRACT

We have examined the in situ organization and nearest neighbours of the 'lamina-associated polypeptide-1' (LAP1), a type II membrane protein and a major constituent of the mammalian nuclear envelope. We show here that, during interphase, LAP1 forms multimeric assemblies which are suspended in the inner nuclear membrane and are specifically associated with B-type lamins. The LAP1-lamin B complex is distinct from analogous complexes formed by the 'lamina-associated polypeptide-2' (LAP2), another inner nuclear membrane protein, and includes a protein kinase. Upon nuclear envelope breakdown, LAP1 partitions with mitotic vesicles which carry nuclear lamin B. The LAP1 vesicles can be distinguished from fragments of the nuclear envelope containing LAP2 and exhibit a striking co-alignment with spindle microtubules. These observations suggest that the inner nuclear membrane comprises discrete territories which accommodate specific integral membrane proteins and are differentially disassembled during mitosis.


Subject(s)
DNA-Binding Proteins , Interphase , Membrane Proteins/metabolism , Mitosis , Nuclear Envelope/chemistry , Nuclear Proteins/metabolism , Spindle Apparatus/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Cell Line , Electrophoresis, Polyacrylamide Gel , Immunohistochemistry , Lamin Type B , Lamins , Membrane Proteins/immunology , Molecular Sequence Data , Nuclear Proteins/immunology , Peptide Fragments/chemistry , Phosphopeptides/metabolism , Protein Kinases/chemistry , Rats , Sequence Analysis , Spindle Apparatus/chemistry
9.
EMBO J ; 15(24): 7108-19, 1996 Dec 16.
Article in English | MEDLINE | ID: mdl-9003786

ABSTRACT

Morphological studies have established that peripheral heterochromatin is closely associated with the nuclear envelope. The tight coupling of the two structures has been attributed to nuclear lamins and lamin-associated proteins; however, it remains to be determined which of these elements are essential and which play an auxiliary role in nuclear envelope-chromatin interactions. To address this question, we have used as a model system in vitro reconstituted vesicles assembled from octyl glucoside-solubilized nuclear envelopes. Comparing the chromosome binding properties of normal, immunodepleted and chemically extracted vesicles, we have arrived at the conclusion that the principal chromatin anchorage site at the nuclear envelope is the lamin B receptor (LBR), a ubiquitous integral protein of the inner nuclear membrane. Consistent with this interpretation, purified LBR binds directly to chromatin fragments and decorates the surface of chromosomes in a distinctive banding pattern.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Chromatin/metabolism , Nuclear Envelope/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Binding Sites , Chromosomes , Cyclophosphamide , Doxorubicin , Microscopy, Electron , Nuclear Envelope/ultrastructure , Rats , Vincristine , Lamin B Receptor
10.
J Biol Chem ; 271(21): 12617-25, 1996 May 24.
Article in English | MEDLINE | ID: mdl-8647873

ABSTRACT

Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. In situ analysis by immunoelectron microscopy and examination of digitonin-permeabilized cells by indirect immunofluorescence show that p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, cycloheximide inhibition experiments reveal that the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with the p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane.


Subject(s)
Erythrocytes/metabolism , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Amino Acid Sequence , Animals , Erythrocytes/ultrastructure , Lamin Type B , Lamins , Membrane Proteins/chemistry , Microscopy, Immunoelectron , Molecular Sequence Data , Nuclear Proteins/metabolism , Protein Binding , Receptors, Cytoplasmic and Nuclear/chemistry , Turkeys , Lamin B Receptor
11.
Int Rev Cytol ; 164: 91-138, 1996.
Article in English | MEDLINE | ID: mdl-8575894

ABSTRACT

The intermediate filaments represent core components of the cytoskeleton and are known to interact with several membranous organelles. Classic examples of this are the attachment of keratin filaments to the desmosomes and the association of the lamin filament meshwork with the inner nuclear membrane. At this point, the molecular mechanisms by which the filaments link to membranes are not clearly understood. However, since a substantial body of information has been amassed, the time is now ripe for comparing notes and formulating working hypotheses. With this objective in mind, we review here pioneering studies on this subject, together with work that has appeared more recently in the literature.


Subject(s)
Intermediate Filaments/physiology , Organelles/physiology , Animals , Humans
12.
Article in French | MEDLINE | ID: mdl-9026511

ABSTRACT

OBJECTIVE: To search for a possible correlation between an effective marker of smoking and uterine and placental resistance during pregnancy. STUDY DESIGN: A prospective study was conducted at the Intercommunal Hospital Center of Montreuil, France: 81 healthy pregnant women underwent uterine and placental Doppler and cotidin blood assay, the best current test for smoking. RESULTS: This study shows a significative increase of utero-placental vascular resistances according with increased cotidine levels. CONCLUSIONS: The previously observed association between smoking and perinatal events most likely involves vascular resistance of uterus and placenta.


Subject(s)
Cotinine/blood , Pregnancy Complications/blood , Pregnancy Complications/diagnostic imaging , Smoking/blood , Ultrasonography, Prenatal , Adult , Biomarkers/blood , Female , Humans , Placental Circulation , Pregnancy , Prospective Studies , Vascular Resistance
13.
EMBO J ; 14(14): 3311-24, 1995 Jul 17.
Article in English | MEDLINE | ID: mdl-7628433

ABSTRACT

We have assessed the involvement of the nuclear lamins in nuclear envelope reassembly. Analysis of perforated mitotic cells shows that A-type lamins are partly cytosolic and partly chromosome-bound, whereas B-type lamins are associated with vesicular structures throughout cell division. Lamin B-containing vesicles appear to dock on vimentin intermediate filaments during prometaphase, but dissociate from the cytoskeleton and assemble around chromatin at later phases of mitosis. Mitotic vesicles isolated from prometaphase cells en bloc with vimentin filaments can specifically capture chromosomes. Efficient chromosome capturing requires cytosolic factors and a dephosphorylating environment. Urea-stripping of the vesicles abolishes binding to chromosomes. However, reconstitution of the stripped membranes with purified B-type lamins restores their ability to bind to chromosomes in a cytosol- and dephosphorylation-dependent fashion. Vesicles reconstituted with B-type lamins form membraneous 'crescents' on the surfaces of chromosomes, but, unlike native vesicles, do not fuse into large sheets. From these observations we conclude that the initial targeting of mitotic vesicles to chromosomes is dependent on B-type lamins and on factors present in the mitotic cytoplasm. Apparently, further recruitment of membranes and fusion of chromosome-bound vesicles onto chromatin involves non-lamin peripheral membrane proteins.


Subject(s)
Chromosomes/metabolism , Mitosis/physiology , Nuclear Proteins/metabolism , Organelles/metabolism , Vimentin/physiology , Animals , CHO Cells , Chromosomes/ultrastructure , Cricetinae , Cytosol/metabolism , Lamin Type B , Lamins , Nuclear Envelope/metabolism , Nuclear Envelope/ultrastructure , Phosphorylation , Protein Binding , Vimentin/ultrastructure
14.
J Cell Biol ; 123(6 Pt 1): 1491-505, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8253846

ABSTRACT

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.


Subject(s)
Intermediate Filaments/metabolism , Mitosis , Nuclear Envelope/metabolism , Vimentin/metabolism , Animals , Cell Compartmentation , Cell Line , Cricetinae , Fluorescent Antibody Technique , Immunohistochemistry , In Vitro Techniques , Lamin Type B , Lamins , Nuclear Proteins/metabolism , Phosphorylation
15.
Eur J Biochem ; 215(3): 825-31, 1993 Aug 01.
Article in English | MEDLINE | ID: mdl-8354288

ABSTRACT

Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21-kDa 125I-interleukin-6 (125I-IL-6) into 8-kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cell-proliferation bioassay. Degradation of 125I-IL-6 by plasma membranes was completely prevented by the serine-protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by alpha 1-protease inhibitor and antibody against cathepsin G. A similar incubation of 125I-IL-6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL-6 using cell-associated serine-protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8-kDa 125I-IL-6 fragments with purified U937 plasma membranes, led to a complete loss of IL-6 activity related to the transformation of the 8-kDa forms into a higher-molecular-mass complex (16 kDa). This complex was stable in SDS and 2-mercaptoethanol at 100 degrees C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non-ester bond between the 8-kDa 125I-IL-6-derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I-IL-6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8-kDa 125I-IL-6 peptide. The kinetics of complex formation (time dependence and plasma-membrane-concentration dependence), as well as its inhibition by a specific inhibitor of N-amino-peptidase activity, bestatin, suggest the participation of peptidyl-transferase activity in complex formation. Finally, a plasma-membrane fraction, corresponding to a molecular mass > or = 30 kDa, was able to convert the 8-kDa 125I-IL-6 forms into the 125I-labeled 16-kDa complex, suggesting that a > or = 30-kDa peptidyl-transferase enzyme catalyzes the reaction and provides the 125I-labeled 16-kDa peptide by dimerization of 8-kDa 125I-IL-6-derived intermediates. Further identification of the plasma-membrane-associated peptidyl transferase as a regulator of IL-6 proteolysis may be of physiological relevance for the control of IL-6 biological activity.


Subject(s)
Aminopeptidases/physiology , Cell Membrane/enzymology , Interleukin-6/antagonists & inhibitors , Serine Endopeptidases/physiology , Cells, Cultured , Humans , Hydrolysis , Interleukin-6/metabolism , Iodine Radioisotopes , Kinetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism
16.
Scand J Immunol ; 34(5): 585-95, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1947794

ABSTRACT

A novel method is described for the formation and purification of covalent complexes between the complement component C3b and an antigen (tetanus toxin, TT), using purified proteins in fluid phase. C3b is generated in situ by tryptic cleavage of C3 after co-precipitation of C3 and TT in the presence of polyethylene glycol. Various parameters were analysed to optimize complex formation; under conditions which minimized the formation of covalent C3b multimers, 30% and 8% respectively of C3b and TT were incorporated into covalent one-to-one complexes which were purified using gel filtration chromatography. The linkage was localized between the alpha' chain of C3b and either the H or L chain of TT; it required the in situ formation of C3b and was partially destroyed by 1 M hydroxylamine. Spontaneous dissociation of the complex could be partly avoided by HgCl2, a thiol reagent which inhibits the esterase-like activity of bound C3b. These findings suggest the involvement of the reactive carbonyl of nascent C3b with hydroxyl groups of TT. Such C3b-TT complexes provide a defined tool to analyse the influence of antigen-bound C3b on antigen addressing and intracellular processing by antigen-presenting cells.


Subject(s)
Antigens/immunology , Complement C3b/immunology , Tetanus Toxin/immunology , Animals , Antigen-Presenting Cells/physiology , Calcium/pharmacology , Electrophoresis, Polyacrylamide Gel , Humans , Hydrogen-Ion Concentration , Mercuric Chloride/pharmacology , Rabbits , Trypsin/pharmacology
17.
J Immunol ; 147(3): 921-6, 1991 Aug 01.
Article in English | MEDLINE | ID: mdl-1861080

ABSTRACT

Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.


Subject(s)
Cathepsins/isolation & purification , Cell Membrane/enzymology , Complement C3/metabolism , Amino Acid Sequence , Cathepsin G , Cell Line , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Molecular Sequence Data , Pancreatic Elastase/isolation & purification , Serine Endopeptidases
18.
Biochem J ; 261(2): 407-13, 1989 Jul 15.
Article in English | MEDLINE | ID: mdl-2775225

ABSTRACT

Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics.


Subject(s)
Complement C3/metabolism , Monocytes/immunology , Cell Line , Complement C2/biosynthesis , Complement C3/biosynthesis , Humans , Kinetics , Macrophages/immunology , Monocytes/drug effects , Tetradecanoylphorbol Acetate
19.
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