Intravenously applied normal human immunoglobulin G (IgG) has anti-inflammatory effects in the treatment of autoimmune diseases. Systemic inflammation can originate from an overreacting amplification ...loop of the complement system. In blood, C3b2-containing complexes maintain complement amplification much better than the extremely short-lived C3b. Therefore, in patients with the complement-dependent autoimmune disease, dermatomyositis, we studied whether intravenously applied normal human IgG (IVIG) stimulated in vivo inactivation of these complexes. In the course of IVIG treatment, clinically effective in 6 of 8 patients, the concentration of C3b2-containing complexes dropped to 37% ± 14% (n = 6) of the pretreatment level when having infused 0.5 g IgG/kg body weight, increased marginally and in parallel to factor Bb thereafter until full-dose IgG was infused. By day 14 following infusion of 2 g IgG/kg body weight the concentration of C3b2-containing complexes was 66% ± 19%. The plasma concentration of C3 remained constant in myopathic or increased by 15% to 20% in amyopathic patients. In contrast to this, IVIG infusion was associated with consumption of up to 40% of plasma C4 at day 1 to 2 after completion of IVIG infusion. Thus, IVIG had an immediate and long-lasting attenuating effect on complement amplification in vivo, despite the fact that it induced classical complement pathway activation.
Human red blood cells were density separated on self-forming Percoll gradients. Redistribution of density fractionated red blood cells was studied by recentrifugation on self-forming Percoll ...gradients. A protocol that avoids centrifugation of red cells prior to removal of white cells and introduces EDTA before red cell pelleting completely avoided redistribution. Dense red cells separated according to this method were senescent on the basis of a biochemical and a physical criterion: the increase in the band 4.1a:4.1b ratio (Mueller, T., Jackson, C.W., Dockter, M.E. and Morrison, M. (1987) J. Clin. Invest. 79, 492-499) and the loss of maximum deformability. Characterization also included the relative content of two surface proteins (complement receptor 1, CR1 (Ripoche, J. and Sim, R.B. (1986) Biochem. J. 235, 815-821); decay accelerating factor, DAF) on density fractionated red cells. Unlike cytoplasmic proteins, these proteins face similar conditions, whether located on circulating reticulocytes or aging red cells. Both components were lost linearly within experimental errors with cell density and were lower by 60 and 40% in dense than light cells, respectively.
Naturally occurring anti-band 3 antibodies appear to have tissue homeostatic functions in the clearance of senescent red cells and in eliciting selective phagocytosis of oxidatively stressed red ...cells by mediating C3b deposition under conditions that favor the alternative complement pathway (Lutz, H. U., Bussolino, F., Flepp, R., Fasler, S., Stammler, P., Kazatchkine, M. D., and Arese, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7368-7372). They overcome the notoriously low affinities of naturally occurring antibodies by having affinity for C3 which renders these antibodies preferred targets of nascent C3b. Anti-band 3 antibodies preferentially formed covalently linked C3b-IgG complexes, when C3 was activated randomly by trypsin. IgG depleted of anti-band 3 antibodies had almost lost the ability to form C3b-IgG complexes. Likewise, anti-band 3 antibodies, but not anti-spectrin antibodies, preferentially formed C3b-IgG complexes on oxidatively stressed red cells in the presence of a 10(3)-fold excess of other serum IgG, when complement deposition was initiated by antibody binding in diluted serum. Moreover, anti-band 3 antibodies preferentially formed C3b-IgG complexes at a 10(5)-fold excess of other IgG on in vivo aging red cells, since C3b-IgG complexes from senescent red cells contained exclusively anti-band 3 antibodies with an affinity for C3. Thus, the low titer, low affinity naturally occurring antibody became functionally relevant by preferred generation of C3b-IgG complexes that can nucleate alternative complement pathway C3 convertases and represent the most effective opsonins (Fries, L. F., Siwik, S. A., Malbran, A., and Frank, M. M. (1987) Immunology 62, 45-51).
Treatment of erythrocytes with the thiolspecific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated ...erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3 antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.
Vesicles released from human E by Ca(2+)-loading, ATP-depletion, or storage are enriched in several glycosylphosphatidylinositol-anchored proteins such as acetylcholinesterase (AchE) and ...decay-accelerating factor (DAF). As a result of this, the remaining E are depleted of these proteins. We analyzed whether vesiculation induced by ATP-depletion in vitro was also responsible for a loss of C receptor 1 (CR1), which is a transmembrane protein arranged predominantly in small clusters. ATP-depleted E had lost 15.4% to 33.9% of their CR1. This loss was similar to that of AchE and DAF. The released vesicles contained CR1. The number of CR1 per band 3 protein was 1.7 to 2.7 that in the original E, indicating an enrichment of CR1 in vesicles. This enrichment was similar to that observed for AchE and DAF (1.83- and 2.6-fold, respectively). The capacity of the vesicles and the ATP-depleted E to bind C3b-coated immune complexes correlated with the CR1 number, suggesting that there was no preferential loss of CR1 clusters. Vesicles released from human E during C attack also contained CR1. In conclusion, in vitro aging induced by ATP-depletion is responsible not only for a loss of glycosylphosphatidylinositol-anchored proteins, but also of CR1. Whether vesiculation explains the loss of CR1 from aging E in vivo and from E of patients with SLE or AIDS remains to be studied.
An ELISA method is presented which is based on covalent binding of detergent-solubilized membrane proteins to surface-modified polystyrene plates (Chemobond plates). These plates carried 0.52-0.65 ...nmol of aldehyde groups per well (150 microliters) and allowed coupling of protein by Schiff base formation either at high pH and subsequent reduction with NaBH4 or by trapping reduced imines at pH 6-6.8 with cyanoborohydride. They bound 15 times the amount of normal plates. Sodium chloride (0.5 M) increased binding 2-3-fold. Binding was essentially resistant to elution by 1% sodium dodecyl sulfate. Reduction of uncoated plates with NaBH4 eliminated the high extent of binding. ELISA tests on Chemobond plates with a rabbit anti-band 3 antibody gave a ten-fold higher signal than plates to which band 3 protein was merely adsorbed. The use of an antigen-enzyme conjugate to detect bound antibody allowed to perform antibody binding and detection of bound antibody simultaneously in the presence of 0.05% Triton X-100. A competitive, one step ELISA system allowed determination of rabbit anti-band 3 antibodies in diluted serum with a sensitivity range of 0.02-0.4 microgram/ml.
The two oxygen‐bridged geminal frustrated Lewis pairs (FLP) tBu2P−O−AlBis2 (Bis=CH(SiMe3)2; 1) and tBu2P−O−Si(C2F5)3 (2) were reacted with the heterocumulenes PhNCO, PhOCN, PhNCS, CS2 and PhNSO as ...well as SO2. With isocyanate and cyanate, both 1 and 2, form addition products under formation of five‐membered rings. With CS2, isothiocyanate and sulfinylaniline, only 1 forms stable adducts, whereas 2 shows reactivity towards sulfinylaniline, but the product decomposed after a few minutes. The reaction of 1 with SO2 led to partial cleavage of the P−O−Al and Al−C units, as confirmed by X‐ray diffraction studies of a complex aggregate. The reaction of 2 with SO2 affords the 1,2‐addition product. All adducts were characterized by means of multinuclear NMR spectroscopy, X‐ray crystallography and CHN analyses.
Frustrated pairs’ different preferences: The geminal oxygen‐bridged frustrated Lewis pairs tBu2P−O−AlBis2 (Bis=CH(SiMe3)2) and tBu2P−O−Si(C2F5)3 show different reactivities towards heterocumulenes (PhNCO, PhOCN, PhNCS, CS2, PhNSO) and sulfur dioxide. Whereas the P−O−Al FLP forms stable adducts with all substrates except for SO2, the P−O−Si FLP does not form adducts with sulfur bound to silicon.
The structures of the three para‐substituted halotetrafluoropyridines with chlorine, bromine, and iodine have been determined in the solid state (X‐ray diffraction). The structures of these compounds ...and that of pentafluoropyridine were also determined in the gas phase (electron diffraction). Structures in the solid state of the bromine and iodine derivatives exhibit halogen bonding as a structure‐determining motif. On the way to an investigation of halogen bond formation of halotetrafluoropyridines in the solid state with the stronger Lewis base pyridine, co‐crystals of benzene adducts were investigated to gain an understanding of the influence of aryl–aryl interactions. These co‐crystals showed halogen bonding only for the two heavier halotetrafluoropyridines. In the pyridine co‐crystals halogen bonding was observed for all three para‐halotetrafluoropyridines. The formation of homodimers and heterodimers with pyridine is also supported by quantum‐chemical calculations of electron density topologies and natural bond orbitals.
Halogen‐bond formation in co‐crystals containing halogentetrafluoropyridines is markedly dependent on the type of halogen atom, the electronegativity of its substituent, and the halogen bond acceptor and it is less pronouncedly dependent on additional π–π stacking interactions.
Difluorothiophosphoryl isocyanate, F2P(S)NCO was characterized with UV/vis, NMR, IR (gas and Ar‐matrix), and Raman (liquid) spectroscopy. Its molecular structure was also established by means of gas ...electron diffraction (GED) and single crystal X‐ray diffraction (XRD) in the gas phase and solid state, respectively. The analysis of the spectroscopic data and molecular structures is complemented by extensive quantum‐chemical calculations. Theoretically, the Cs symmetric syn‐conformer is predicted to be the most stable conformation. Rotation about the P−N bond requires about 9 kJ mol−1 and the predicted existence of an anti‐conformer is dependent on the quantum‐chemical method used. This syn‐orientation of the isocyanate group is the only one found in the gas phase and contained likewise in the crystal. The overall molecular structure is very similar in gas and solid, despite in the solid state the molecules arrange through intramolecular O⋅⋅⋅F contacts into layers, which are further interconnected by S⋅⋅⋅N, S⋅⋅⋅C and C⋅⋅⋅F contacts. Additionally, the photodecomposition of F2P(S)NCO to form CO, F2P(S)N, and F2PNCO is observed in the solid Ar‐matrix.
All about isocyanates: Difluorothiophosphoryl isocyanate, F2P(S)NCO, was investigated using an experimental multimethod approach in the gaseous, liquid and solid state to elucidate its structure, vibrational behaviour and photodecomposition: spectroscopy (UV/Vis, NMR, IR, Raman) and gas electron as well as single‐crystal X‐ray diffraction and in addition quantum‐chemical calculations.
1,8-Substituted anthracene derivatives−monomers and photo-dimers−bearing alkynyl units were treated with the bulky aluminum hydride (SiMe3)2HC2AlH in hydroalumination reactions in order to ...investigate the influence of the substituents on the spacing between the alkynyl units. While 1,8-bis(trimethylsilyl)ethynylanthracene only resulted in the mono-hydroalumination product, 1,8-diethynylanthracene and its photo-dimer afforded the products of twofold hydroalumination. In the case of 1,8-diethynylanthracene, the product underwent a cis/trans-isomerization of one of the newly formed CC bonds. This process was examined via quantum-chemical calculations. The twofold hydroaluminated syn-photo-dimer of 1,8-diethynylanthracene was treated with various donor molecules to gain insights into its host–guest chemistry.
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