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At least 1,387 records · Page 77Linked to original sources
PubMed1989

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.

Antibodies↗
PubMed1986

Hemopexin is a developmentally regulated, acute-phase plasma protein in the chicken.

Identity has been established between chicken hemopexin and alpha 1-globulin "M," a plasma known for the hormone responsiveness of its synthesis in monolayer cultures of embryonic chicken hepatocytes (Grieninger, G., Plant, P. W., Liang, T. J., Kalb, R. G., Amrani, D., Mosesson, M. W., Hertzberg, K. M., and Pindyk, J. (1983) Ann. N. Y. Acad. Sci. 408, 469-489). Identification was based on immunological cross-reactivity, electrophoretic behavior on sodium dodecyl sulfate-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes. Electroimmunoassays were used to investigate plasma protein levels, particularly those of hemopexin, in the acute-phase response and embryonic development. Acute-phase plasma protein production, elicited by injection of chickens with turpentine, bore many similarities to the pattern of hepatocellular plasma protein synthesis produced in response to the addition of specific hormones in culture. The response of the stressed chickens included elevated levels of hemopexin and fibrinogen (5- and 2-fold, respectively) accompanied by a 50% drop in albumin. Hemopexin levels of developing chick embryos were measured for several days before and after hatching. Onset of hemopexin production occurred around the time of hatching, and was followed by a steep increase (more than 1000-fold over 4 days). Similarly, it was not until the 12th h of culture that hepatocytes isolated from both early and late stage chicken embryos began to produce hemopexin, although, from their initiation in culture, they secreted a number of other plasma proteins in quantity. After 12 h, hepatocellular output of hemopexin rapidly accelerated. This precocious induction ex vivo required no hormonal or macromolecular medium supplements. These observations indicate that the embryonic chicken hepatocyte culture system will provide a useful model for studying the regulation of hemopexin biosynthesis in hepatic development and the acute-phase response.

Acute-Phase Reaction↗
PubMed1987

Mechanisms of acetaminophen oxidation to N-acetyl-P-benzoquinone imine by horseradish peroxidase and cytochrome P-450.

Horseradish peroxidase rapidly catalyzed the H2O2-dependent polymerization of acetaminophen. Acetaminophen polymerization was decreased and formation of GSSG and minor amounts of GSH-acetaminophen conjugates were detected in reaction mixtures containing GSH. These data suggest that horseradish peroxidase catalyzed the 1-electron oxidation of acetaminophen and that GSH decreased polymerization by reducing the product, N-acetyl-p-benzosemiquinone imine, back to acetaminophen. Analyses of reaction mixtures that did not contain GSH showed N-acetyl-p-benzoquinone imine formation shortly after initiation of reactions. When GSH was added to similar reaction mixtures at various times, 3-(glutathion-S-yl)-acetaminophen was formed. The formation and disappearance of this product were very similar to N-acetyl-p-benzoquinone imine formation and were consistent with the disproportionation of 2 mol of N-acetyl-p-benzosemiquinone imine to 1 mol of N-acetyl-p-benzoquinone imine and 1 mol of acetaminophen followed by the rapid reaction of N-acetyl-p-benzoquinone imine with GSH to form 3-(glutathion-S-yl)acetaminophen. When acetaminophen was incubated with NADPH, oxygen and hepatic microsomes from phenobarbital-pretreated rats, 1.2 nmol 3-(glutathion-S-yl)acetaminophen/nmol cytochrome P-450/10 min was formed. Formation of polymers was not observed indicating that N-acetyl-p-benzoquinone imine was formed via an overall 2-electron oxidation rather than a disproportionation reaction. However, when cumene hydroperoxide was replaced by NADPH in microsomal incubations, polymerization was observed suggesting that cytochrome P-450 might also catalyze the 1-electron oxidation of acetaminophen.

Acetaminophen↗
PubMed1983

A novel ATPase in the chromaffin granule membrane.

The ATPase activity of the chromaffin granule membrane was found to differ profoundly from that of the mitochondrial inner membrane. First, mitochondrial ATPase is much more sensitive to low concentrations of dicyclohexylcarbodiimide than the chromaffin granule ATPase. Second, analysis of chromaffin granule preparations by immune replication revealed that mitochondrial contamination (as measured with an antibody against cytochrome c oxidase) could account for the presence of mitochondrial-type ATPase. Third, exposure of chromaffin granule preparation to sodium bromide removed the mitochondrial-type ATPase, but left over 70% of the total ATPase activity of the chromaffin granules intact. Finally, after solubilization of the chromaffin granule membranes with detergents, a novel ATPase could be separated from the mitochondrial-type ATPase. It is proposed that this novel ATPase represents most of the ATPase activity of the chromaffin granules and that the mitochondrial-type ATPase reflects contamination by mitochondria.

Adenosine Triphosphatases↗
PubMed1980

Uptake of aggregated immunoglobulin by the mouse kidney. I. Effect of endotoxin.

Endotoxin (ET) pretreatment of mice resulted in increased liver and spleen uptake and decreased circulating levels of aggregated IgC (AggIgG) compared to control animals. ET had no effect upon the uptake of AggIgG by lung or whole kidney cortex. However, immunofluorescent microscopy revealed increased localization of AggIgG in the mesangium of ET mice at 1 but not at 2 or 16 h after administration of AggIgG. These studies show that mesangial macromolecular uptake is not solely dependent upon blood levels, but appears to be influenced by unknown and probably complex variables.

Animals↗
PubMed1981

Role of the beta subunit in interaction of the eighth component of human complement with the membrane-bound cytolytic complex.

This study is concerned with the subunits of the eighth component of human complement (C8) and their role in facilitating specific incorporation of C8 into the membrane-bound cytolytic complex of complement. The noncovalently associated alpha-gamma and beta subunits of C8 were purified and examined for their ability to interact independently with the C8 binding site on C5b-7, the membrane-bound precursor of the cytolytic complex. Using erythrocyte-bound C5b-7 (EAC1-7), it was observed that native C8 and its isolated beta subunit have similar high affinities for this complex. Binding of beta to EAC1-7 was specific for the C8 binding site as evidenced by the fact that 1) nearly identical molar amounts of either C8 or beta were required to saturate C8 binding sites on EAC1-7; 2) pretreatment of EAC1-7 with saturating amounts of beta prevented binding of C8; and 3) pretreatment of EAC1-7 with saturating amounts of C8 prevented binding of beta. In related experiments, alpha-gamma alone had no affinity for EAC1-7, however, binding comparable to that exhibited by native C8 occurred if alpha-gamma was first incubated with an equimolar amount of beta. Other experiments showed that if EAC1-7 was first saturated with beta to produce EAC1-7(beta), alpha-gamma also bound to yield functional C8 on the cell surface. These results provide conclusive evidence that structural features of C8 which are essential for specific recognition by membrane-bound C5b-7 are contained in the beta subunit.

Cell Membrane↗
PubMed1976

Synthesis of functional aldolase tetramers in a heterologous cell-free system.

The present report describes the complete synthesis of a functional oligomeric enzyme in a heterologous cell-free system. Polysomal RNA from chicken skeletal muscle was used to direct the production of functional aldolase tetramers in wheat germ extracts. The aldolase product was (a) specifically precipitated with monospecific antibodies raised against pure muscle aldolase, (b) had the same subunit molecular weight (40,000) as that of native aldolase (as determined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate), (c) presumably contained a functional active site since it co-purified with authentic muscle aldolase upon substrate elution from phosphocellulose, and (d) had associated into tetrameric units (Mr=160,000) as shown by centrifugation in sucrose gradients. The present work suggests that, within the cell, post-translational processing of aldolase polypeptide chains is not involved in the formation of functional aldolase tetramers.

Animals↗
PubMed1976

Magnesium metabolism: a brief review.

The important role played by the magnesium ion in the body is not generally recognized. The action of numerous enzyme systems critical to cellular metabolism is regulated by it and it contributes importantly to macromolecular structure. Magnesium defiency occurs more often than is generally suspected; magnesium excess, though uncommon, is of special interest to the anaesthetist because it produces a curare-like effect on neuromuscular transmission. It is hoped that this brief review of magnesium metabolism will draw attention to its importance and relevance in everyday practice.

Animals↗
PubMed1994

FLOG: a system to select 'quasi-flexible' ligands complementary to a receptor of known three-dimensional structure.

We present a system, FLOG (Flexible Ligands Oriented on Grid), that searches a database of 3D coordinates to find molecules complementary to a macromolecular receptor of known 3D structure. The philosophy of FLOG is similar to that reported for DOCK [Shoichet, B.K. et al., J. Comput. Chem., 13 (1992) 380]. In common with that system, we use a match center representation of the volume of the binding cavity and we use a clique-finding algorithm to generate trial orientations of each candidate ligand in the binding site. Also we use a grid representation of the receptor to assess the fit of each orientation. We have introduced a number of novel features within this paradigm. First, we address ligand flexibility by including up to 25 explicit conformations of each structure in our databases. Nonhydrogen atoms in each database entry are assigned one of seven atom types (anion, cation, donor, acceptor, polar, hydrophobic and other) based on their local bonded chemical environments. Second, we have devised a new grid-based scoring function compatible with this 'heavy atom' representation of the ligands. This includes several potentials (electrostatic, hydrogen bonding, hydrophobic and van der Waals) calculated from the location of the receptor atoms. Third, we have improved the fitting stage of the search. Initial dockings are generated with a more efficient clique-finding algorithm. This new algorithm includes the concept of 'essential points', match centers that must be paired with a ligand atom. Also, we introduce the use of a rapid simplex-based rigid-body optimizer to refine the orientations. We demonstrate, using dihydrofolate reductase as a sample receptor, that the FLOG system can select known inhibitors from a large database of drug-like compounds.

Algorithms↗