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Biomedical subjects

A Reboul

Publications and source records attributed to A Reboul.

At least 37 records · Page 2Linked to original sources
PubMed1984

Characterization of the C1q receptor on a human macrophage cell line, U937.

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.

Binding Sites↗
PubMed1983

Biosynthesis in vitro of complement subcomponents C1q, C1s and C1 inhibitor by resting and stimulated human monocytes.

The capacity of cultured human monocytes to synthesize and to secrete the subcomponents of C1 and C1 inhibitor was examined. Non-stimulated monocytes secreted C1q and C1s from day 5 of culture. C1s reached a plateau immediately at its maximum level, whereas C1q secretion increased progressively until the end of the second week. Between day 12 and day 25, C1q secretion remained nearly constant (1-15 fmol/day per microgram of DNA, depending on the donor), whereas C1s secretion decreased and even in some cases stopped. C1r and C1 inhibitor were not secreted in detectable amounts by these resting cells. Stimulation of monocytes by yeasts, immunoglobulin G-opsonized sheep red blood cells or latex beads did not modify consistently C1q and C1s secretion. Activation by conditioned media from mitogen-, antigen- or allogeneic-stimulated lymphocyte cultures increased C1q production from 2 to 7 times and re-activated C1s secretion. Under the same conditions of activation, C1 inhibitor was secreted (up to 300 fmol/day per microgram of DNA) and C1r became detectable in culture supernatants. Isolated human monocytes are thus able to synthesize the whole C1 subcomponents; C1, if assembled, could be protected from non-immunological activation by locally produced C1 inhibitor. Activated monocytes appear to be a good tool for studying the assembly of C1 subcomponents and the role of C1 inhibitor in this process.

Cells, Cultured↗
PubMed1982

A study of a covalent-like interaction between soluble nascent C4b and C4-binding protein.

In the classical pathway of complement, the interaction between C4b and C4bp can be considered as a control of the C3 convertase formation. Purified C4-binding protein (C4bp) interacts with soluble nascent C4b to form covalent-like complexes; the interaction is also possible with nascent C4b-like C4, but not with C4, C4b or C4b-like C4. Formation of the complexes upon incubation of C4bp, C4 and C1s appears to involve a single link between a subunit of C4bp and the alpha' chain of C4b, as observed by SDS-polyacrylamide gel electrophoresis in reducing conditions (160 000 dalton band). In non-reducing conditions, a mixture of C4b-C4bp complexes is observed as a function of the C4b:C4bp molar ratio, with apparent molecular weights differing by a value of 210 000 and reflecting different C4b-C4bp associations. A maximum of five molecules of C4b are bound per molecule of C4bp, which appears to consist of 10 subunits of apparent molecular weight 72 000. The link between C4b and C4bp is partially destroyed by 1 M hydroxylamine at pH 9.0; its formation is strongly inhibited by 3.5 mM hydroxylamine or 60 mM methylamine at pH 9.0. These findings suggest an ester or amide bond between the activated carboxyl group of the thioester bridge in the alpha' or alpha chain of nascent C4b or C4b-like C4 and a hydroxyl or amino group of C4bp. Thus, C4bp might compete with other C4b acceptors such as membranes or IgG.

Carrier Proteins↗
PubMed1979

Interaction of C1-inhibitor with the C1r and C1s subcomponents in human C1.

1. Insoluble IgG-ovalbumin aggregates were used to bind and activate C1 from human serum. The bound C1 provided a useful reagent for studying the interaction of C1 subcomponents with C1-inhibitor. 2. C1-inhibitor bound to both subcomponents (C1r and C1s in C1 and formed stable complexes of respective apparent molecular weights 197,000 and 185,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The binding reaction proceeded more readily with C1s than with C1r and was correlated with the inhibition of C1s esterase activity. 3. At physiological ionic strength, binding of C1-inhibitor to subcomponents C1r and C1s caused release of these subcomponents from the C1-immune aggregates complex, indicating that C1-inhibitor binding decreased the inter-subcomponent binding forces in C1. At low ionic strength, however, this release did not occur.

Complement C1↗
PubMed1977

Purification of proenzymic and activated human C1s free ofC1r. Effect of calcium and ionic strength on activated C1s.

1. A rapid method for the purification of the proenzymic and activated forms of C1s is presented. In the case of proenzymic C1s, di-isopropyl phosphorofluoridate (0.5--5 mM) is added at all stages of the purification procedure, which includes euglobulin precipation followed by DEAE-cellulose chromatography and affinity chromatography on anti-C1r IgG-Sepharose 6B. The final step completely removes contaminant traces of C1r and/or C1r, ensuring that the final preparation of C1s is stable in the proenzyme form and suitable for activation studies. 2. The apparent molecular weight of C1s and C1s determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis is 85 000 +/- 2000. Reduction followed by alkylation of C1s gives two fragments of apparent molecular weights 57 000 and 28 000. Results of N-terminal amino acid determination and labelling with di-iso[3H]propyl phosphorofluoridate are consistent with previous reports. 3. The influence of calcium and ionic strength on the structure and activity of C1s has been investigated. Calcium leads to a shift of the sedimentation coefficient from 4.3 to 5.6 S, whereas variation in ionic strength has no effect on this parameter. The thermal inactivation curve is profoundly modified both by calcium and ionic strength. In contrast, the esterase activity is only slightly influenced as judged from the absence of gross modification of Km and V.

Calcium↗
PubMed1977

Proenzymic C1s associated with catalytic amounts of C1r. Study of the activation process.

1. Proenzymic C1s isolated from human plasma by euglobulin precipitation and DEAE-cellulose chromatography is associated with trace amounts of C1r (0.5--1% on a molar basis). Incubation for 2 h at 37 degrees C leads to the proteolytic activation of C1s. The proteolysis is characterized by the sigmoidal appearance of C1s esterase activity and of the typical heavy (57 000-dalton) and light (28 000-dalton) fragments of C1s on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. 2. The C1s activation process observed is markedly temperature and concentration dependent, and the rate of activation is decreased by calcium and high ionic strength (I = 0.9). Diisopropyl phosphorofluoridate, benzamidine, polyanethol sulfonate and pentosane polysulphate inhibit the activation, which is also sensitive to C1-inactivator and anti-C1r IgC. From the kinetic experiments and from the inhibition characteristics, the activation of C1s can be attributed to the presence of C1r, which appears to undergo activation and then to activate secondarily C1s.

Calcium↗