PubMed Health⌕ Search

Biomedical subjects

J P Paccaud

Publications and source records attributed to J P Paccaud.

At least 19 recordsLinked to original sources
PubMed1996

The N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) increases the lateral diffusion of complement receptor 1 (CR1/CD35) in human neutrophils; a causative role for oxidative metabolites?

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1-1 microM fMLF increased the diffusion constant (D) of CR1/CD35 to 167-228% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxon (PD, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

Catalase↗
PubMed1996

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

Animals↗
PubMed1996

Molecular and cellular mechanisms governing the ligand-specific and non-specific steps of insulin receptor internalization.

The surface events leading to insulin-induced internalization of its specific receptor can be subdivided in three major steps: the first step consists in the surface redistribution of the receptor from the villous to the non-villous region of the cell surface, it is ligand-specific, depends on kinase activation and phosphorylation of tyrosines 1146, 1150 and 1151, and consists in the relief of a constraint immobilizing the receptor on microvilli; the second step is characterized by the shift of the insulin-receptor complex in the plane of the membrane allowing it to get access to the nonvillous domain of the cell surface where internalization gates (clathrin-coated pits) are located; this stage is controlled, at least in part, by the transmembrane domain of the molecule and its flanking amino acids; the third step corresponds to the segregation of the insulin-receptor complex in clathrin-coated pits, this step is relatively non-specific and is governed by well defined signal sequences present in the juxtamembrane domain of the cytoplasmic segment of the b-subunit. These surface events are then automatically followed by the entry of the insulin receptor inside the cells through the formation of clathrin-coated vesicles, in its subsequent association with endosomes which acidic pH allows insulin dissociation from its receptor and the sorting of the receptor and the hormone in different directions: insulin is targetted to lysosomes to be degraded while the receptor is recycled back to the cell surface to be reused. This complex process does not seem to be involved in the transmission of the biological signal of the hormone. Nevertheless, it is initiated and controlled by insulin and results in the intracellular degradation of insulin and in the modulation of the number of surface insulin receptors. Thus, even if it does not directly participate in insulin signaling, insulin receptor internalization plays a crucial role in the control of insulin action.

Animals↗
PubMed1994

Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes.

Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CR1-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.

Adult↗
PubMed1993

Clathrin-coated pit-mediated receptor internalization. Role of internalization signals and receptor mobility.

Most signals controlling receptor-mediated endocytosis have been identified by alteration of sequences present in receptors normally internalized via clathrin-coated pits. In the present work we have reconsidered the factors that control internalization the other way around: i.e. by introducing potential internalization sequences in complement receptor 1 (CR1) which does not preferentially associate with clathrin-coated pits. The analysis of the internalization efficiency of NPxY related motifs generated by substituting His2010 and/or Glu2015 by either Phe or Tyr indicates that FxNPxY is the stronger promoter of endocytosis and that the signal efficiency depends on the presence of aromatic residues (including a tyrosine) at both ends of the -xNPx- motif. Moreover, CR1-tyr (substitution of Glu2015 for Tyr) internalization was superposable to that of a receptor composed of the extracellular and transmembrane domains of CR1 fused to the intracytoplasmic tail of the low density lipoprotein (LDL) receptor (including the FxNPxY motif) (CR1-LDL). When analyzed by fluorescence recovery after photobleaching, the surface mobility of CR1-LDL was decreased as compared with that of either CR1-tyr or CR1-wt, despite a similar association with clathrin-coated pits. The role of receptor mobility in internalization was confirmed by the observation that CR1-tl, with a deletion of the cytoplasmic tail, was more mobile and more efficiently internalized than CR1-wt.

Amino Acid Sequence↗
PubMed1993

Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p < 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p < 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p < 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (> 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p < 0.0001, and r = 0.60, p < 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p < 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.

Animals↗
PubMed1993

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Amino Acid Sequence↗
PubMed1992

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.

Animals↗
PubMed1992

Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation.

The role of insulin-induced receptor autophosphorylation in its internalization was analyzed by comparing 125I-labeled insulin (125I-insulin) internalization in Chinese hamster ovary (CHO) cell lines transfected with normal (CHO.T) or mutated insulin receptors. In four cell lines with a defect of insulin-induced autophosphorylation, 125I-insulin internalization was impaired. By contrast, in CHO.T cells and in two other CHO cell lines with amino acid deletions or insertions that do not perturb autophosphorylation, 125I-insulin internalization was not affected. A morphological analysis showed that the inhibition is linked to the ligand-specific surface redistribution in which the insulin-receptor complexes leave microvilli and concentrate on nonvillous segments of the membrane where endocytosis occurs.

Animals↗
PubMed1991

Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.

On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.

Autoradiography↗
PubMed1991

Immune complex binding efficiency of erythrocyte complement receptor 1 (CR1).

C3b-coated immune complexes adhere to the complement receptor 1 (CR1, CD35) on human erythrocytes. This multi-valent binding might be favoured by the known clustering of CR1 and by the multiple C3b-binding sites on each CR1. The size of the CR1 clusters correlates directly with the number of CR1/erythrocytes, and the different structural CR1 alleles bear between two and five C3b-binding sites. Using radiolabelled hepatitis B surface antigen-antibody complexes, we investigated whether CR1 numbers and structural alleles modulate the ability of erythrocytes to bind immune complexes, and assessed if any reorganization of immune complexes takes place at the erythrocyte surface after the initial binding reaction. The binding efficiency (immune complexes/CR1) correlated with CR1 number as determined by the maximal binding at 4 degrees C, the kinetics of binding at 37 degrees C, and the binding in the presence of excess immune complexes and of immune complexes of small size. Binding efficiencies were similar for erythrocytes with low CR1 from normal subjects and patients with AIDS or SLE. A monoclonal antibody blocking the C3b-binding sites (3D9) of CR1 interfered with binding efficiency at a lower concentration on cells bearing low CR1 numbers, suggesting that CR1 clustering is essential. The larger alleles of CR1 (DD and BB) were more efficient than AA alleles. The distribution of immune complexes, visualized by immunofluorescence, was heterogeneous on erythrocytes: about two out of three cells bore between one and 12 immune complexes. No visible immune complex reorganization took place after initial binding, as prefixed erythrocytes displayed the same immune complex distribution and number/erythrocytes as unfixed erythrocytes. The contribution of CR1 alleles in immune complex binding efficiency was confirmed by morphological analysis. These results demonstrate that immune adherence efficiency is the resultant of the CR1 clustering, as well as the particular alleles carried by erythrocytes. Moreover, there is little or no immune complexes surface reorganization after the initial binding reaction.

Antibodies, Monoclonal↗
PubMed1990

Exudation induces clustering of CR1 receptors at the surface of human polymorphonuclear leukocytes.

The complement receptor type 1 (CR1) surface distribution, density and immune adherence efficiency were determined in circulating PMN activated by fMLP, NAP-1/IL-8, TNF, GM-CSF and C5a, or exudate PMN harvested from skin-blisters. These observations were compared with those observed on resting peri-pheral blood PMN. PMN activators known to upregulate CR1 expression did not induce a significant increase in CR1 clustering, or immune adherence efficiency towards opsonized immune complexes. By contrast, increase in CR1 density at the surface of exudated PMN was accompanied by an increased clustering. This clustering was however insufficient to increase the binding efficiency for immune complexes. Eventually, CR1 expression of exudated neutrophil could not be increased further by stimulation with fMLP or PMA. These results indicated that clustering of CR1 on PMN may occur in vivo. Such reaction might determine the phagocytic potential of the cell for opsonized micro-organisms or debris. This clustering could not be attributed to one of the PMN activators tested.

Complement C1↗
PubMed1990

NAP-1/IL-8 induces up-regulation of CR1 receptors in human neutrophil leukocytes.

The effect of the neutrophil-activating peptide NAP-1/IL-8 on the expression of complement receptor type 1 (CR1) in human neutrophils was studied. NAP-1/IL-8 enhanced CR1 expression at concentrations between 10(-10) and 10(-8) M. The maximum increase with respect to unstimulated control cells was on average 2.3 fold. The effect was rapid: Half-maximum enhancement was obtained in 4 min and the plateau was reached in 15 min. The chemotactic peptide fMLP, tested for comparison, was effective between 10(-9) and 10(-7) M, showed a similar time course and a somewhat higher maximum effect (2.8 fold increase). The effect of NAP-1/IL-8 was prevented by pretreatment of the cells with B.pertussis toxin and desensitization was observed following restimulation. Stimulus combination experiments suggested that NAP-1/IL-8 mobilizes the same or a similar intracellular pool of CR1 receptors as fMLP or C5a.

Chemotactic Factors↗
PubMed1990

Difference in the clustering of complement receptor type 1 (CR1) on polymorphonuclear leukocytes and erythrocytes: effect on immune adherence.

Complement receptor type 1 (CR1) mediates the adherence of complement-reacted immune complexes (IC) to various blood cells. On the erythrocyte, CR1 are clustered, a distribution which favors efficient multivalent binding of C3b-coated IC. IC can also bind to CR1 expressed on polymorphonuclear (PMN) leukocytes. To evaluate the respective importance of these two cell types in immune adherence reactions, functional analysis of IC binding, as well as morphological studies of CR1 distribution at their surface were undertaken. At equal cell concentrations, resting PMN leukocytes bound the same percentage of IC as erythrocytes, despite expressing four times more CR1 at their surface. At equal CR1 concentrations, IC binding to resting or formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN leukocytes was always lower than to erythrocytes. The morphological counterpart of these differences was studied by label-fracture immunoelectron microscopy. On erythrocytes, almost 50% of the CR1 were distributed in clusters of greater than or equal to 3 units, while less than 15% were grouped in such clusters on the surface of PMN leukocytes. Activation of PMN leukocytes by fMLP increased the surface density of CR1, but the proportion of clustered CR1 remained unchanged. These observations suggest that the low responsiveness of PMN leukocytes towards C3b-coated IC may be due to the unaggregated state of CR1. In the circulation, erythrocytes might function as a "buffer" for PMN leukocytes, which would otherwise engage too swiftly in reactions with IC.

Antigen-Antibody Complex↗
PubMed1989

Two isotypes of human C4, C4A and C4B have different structure and function.

The two types of human C4, C4A and C4B, differ in their amino acid sequence and in their capacity to bind to different acceptor sites. C4B is more efficient than C4A in haemolytic assays; by contrast C4A binds preferentially to immune complexes. In assays comparing haemolysis to processing of immune complexes the two types of C4 differ more than fivefold. Thus, the classical pathway is a duplicated system that allows the formation of a C3 convertase on various substrates: this duplication may be of vital importance to eliminate invading microorganisms. In addition, the clinical observation of an increased incidence of homozygous C4A null alleles in systemic lupus erythematosus may be explained in part by defective processing of immune complexes in the absence of C4A.

Antigen-Antibody Complex↗