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M M Mayer

Publications and source records attributed to M M Mayer.

At least 19 recordsLinked to original sources
PubMed1998

Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism.

Multiple cell types contribute to the pulmonary barrier including Type I and Type II alveolar epithelium. The objective of this research was to establish and characterize an in vitro model of Type II alveolar epithelium using the A549 human lung adenocarcinoma cell line. A549 cells form confluent monolayers with Type II characteristic morphology and tannic acid staining for typical lamellar bodies. A549 cells possess P450 IA1 and P450 IIB6 as determined by Western blots. Both CYPIA1 and CYPIIB6 P450 isozymes were determined to be functional with the fluorescent resorufin assay. Only the IA1 isozyme was observed to be inducible with selected polycyclic hydrocarbons. Uptake and transport experiments were carried out in cluster plates and in Snapwells. Cationized ferritin, a nonspecific absorbtive marker, was found to be taken up by the cells in a concentration-, time-, and temperature-dependent fashion. Lucifer yellow, a fluid-phase marker, was not internalized by the A549 cells. Transferrin, a representative receptor-mediated endocytic marker, was found to be taken up by the cells in a concentration-dependent and competitive fashion. Transport experiments involving fluorescein-transferrin also showed that A549 monolayers were polarized, with a greater amount of intracellular transferrin being transported out of the basolateral side of the cells. The experimental data agree favorably with literature for primary cultures of Type II pulmonary epithelial cells. These results indicated that the A549 cell line may be useful for the studying the metabolic and macromolecule processing contributions of alveolar Type II cells to mechanisms of drug delivery at the pulmonary epithelium.

Biological Transport↗
PubMed1986

Release of arachidonic acid and formation of oxygenated derivatives after complement attack on macrophages: role of channel formation.

Treatment of [3H]arachidonic acid ([3H]C20:4)-labeled, antibody-sensitized mouse resident peritoneal macrophages with rabbit serum complement, or C6-deficient rabbit serum + C6, caused hydrolytic release of incorporated [3H]C20:4 from phospholipids, followed by conversion to oxygenated derivatives. The C6 dose-response curve for release of C20:4 plus its metabolites was monotonic, which indicates dependence on channel formation, whereas the dose-response curve for lysis displayed multi-hit behavior. High-performance liquid chromatography demonstrated that the major radiolabeled products in the aqueous phase co-eluted with C20:4, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin E2. Kinetic studies of the release of 6-keto-PGF1 alpha, the major metabolite, displayed biphasic characteristics; a moderate amount of this prostaglandin was released before the onset of cell lysis. Experimental evidence obtained by freeze-thaw or by incubation of these cells with melittin or A23187 indicated that cell lysis does not necessarily result in the production of inflammatory mediators. Furthermore, when macrophages were treated with serum complement, it was apparent that the major part of the release was due to C5b-9 and not to the action of C5a. We conclude that release of C20:4 and its derivatives from complement-treated macrophages does not depend on cytolysis, but is a consequence of insertion and channel formation.

Animals↗
PubMed1985

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space.

We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.

Acyltransferases↗
PubMed1985

The relationship between channel size and the number of C9 molecules in the C5b-9 complex.

We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.

Animals↗
PubMed1984

Complement lysis of nucleated cells: effect of temperature and puromycin on the number of channels required for cytolysis.

We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37 degrees C, followed by prolonged incubation at 2 degrees C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30 degrees C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35-40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na+/K+-ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis.

Biological Transport, Active↗
PubMed1983

Size distribution and stability of the trans-membrane channels formed by complement complex C5b-9.

We have performed double marker sieving experiments with molecules ranging from ca. 0.5-3 nm dia. in order to evaluate the size distribution of the channels formed by complement in resealed sheep erythrocyte ghosts. Evidence is presented that marker release through the channels reached equilibrium between the ghosts and the extracellular fluid in a period of 3 hr and that the channels are stable at 37 degrees C for this period of time. Under these experimental conditions we have observed a differential in the endpoint release of inositol and sucrose, which indicates that some of the ghosts carried channels measuring between 0.7 and 0.9 nm dia. No differential was observed between release of sucrose and raffinose (0.9 and 1.1 nm mol. dia., respectively). Comparisons between sucrose and inulin (0.9 and 3 nm mol. dia, respectively) showed a difference in marker release. Also, there was substantial release of inulin, indicating the presence of channels above 3 nm in dia. Hence, the present data indicate formation of channels in three size ranges, namely, 0.7-0.9 nm, 0.9-3 nm and greater than 3 nm.

Animals↗
PubMed1983

Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics.

Lysis of nucleated cells by complement was studied to determine whether the lytic process by C5b-9 conforms to a one-hit mechanism as in the case of erythrocytes. Two nucleated cell lines, Molt 4 and U937, derived from human T lymphocytes and histiocytes, respectively, were employed as targets. The antibody-sensitized cells were used to develop the titration curves, measuring cell death as a function of limiting quantities of human C6 or C5,6 complex in the presence of an excess of other complement components. The cytolysis curves generated in both experiments were sigmoidal, in sharp contrast to the monotonic curves observed in lysis of erythrocytes treated similarly. The sigmoidal curves of cytolysis indicate a cooperative action of several molecules of C6 or acid-activated C5,6 complex, C(56)a. In contrast to the multi-hit characteristics of cytolysis, dose-response measurements of the release of 86Rb indicated that only one effective molecule of C6 per cell is required for assembly of a 86Rb-releasing channel. This divergence indicates that lysis requires formation of several channels or, alternatively, assembly of large channels that are formed by several molecules of C6. Because prior studies with erythrocyte ghosts have shown that only a single effective molecule of C6 is required for assembly of a transmembrane channel, regardless of size, we prefer to interpret the multi-hit characteristics of nucleated cell lysis as an indication of a multi-channel requirement, rather than channel enlargement.

Cell Line↗
PubMed1983

Macrophage-mediated cytotoxicity: role of a soluble macrophage cytotoxic factor similar to lymphotoxin and tumor necrosis factor.

Guinea pig peritoneal macrophages, when activated for cytotoxicity by the calcium ionophore A23187 or lipopolysaccharide, produce a cytotoxic factor [macrophage cytotoxic factor (M phi-CF)] that is not blocked by catalase or protease inhibitors. Fractionation of culture supernates containing M phi-CF by gel filtration revealed one peak of cytotoxic activity of Mr approximately 45,000, the same as guinea pig lymphotoxin (LT). Antiserum prepared against purified guinea pig LT completely neutralized the cytotoxic activity of M phi-CF. In addition, the cytotoxic factor in guinea pig tumor necrosis serum was found to have a Mr of 45,000 and was neutralized by anti-LT. Thus, M phi-CF is physicochemically and immunochemically similar to LT and tumor necrosis factor, if not identical. To investigate the role of M phi-CF in macrophage-mediated cytotoxicity, anti-LT was added to A23187- or lipopolysaccharide-activated macrophages before addition of L-929 target cells. In 10 of 16 experiments, the inhibition of macrophage-mediated cytotoxicity was 100%. In the others, cytotoxicity was blocked partially, the lowest inhibition being 49%. The effectiveness of inhibition appeared to be inversely related to the intensity of macrophage activation. These results indicate that M phi-CF plays a significant role in macrophage-mediated cytotoxicity but involvement of another mechanism cannot be excluded.

Animals↗
PubMed1983

Consequences of cell membrane attack by complement: release of arachidonate and formation of inflammatory derivatives.

Treatment of [3H]arachidonic acid [( 3H]C20:4)-labeled and antibody-sensitized Ehrlich ascites tumor cells with guinea pig or rabbit serum complement (C) released up to about 20 or 25% of the incorporated [3H]C20:4 into the aqueous phase as a consequence of C-induced hydrolysis of cellular phospholipid. The dose-response curve of release of [3H]C20:4 from Ehrlich ascites tumor cells, with respect to C, was approximately in the same range as the cytolytic response. In the case of [3H]C20:4-labeled and antibody-sensitized peritoneal mouse macrophages, treatment with C induced release of about 11% of the incorporated 3H as C20:4 and about 6% as prostaglandins, thromboxane B2, and hydroxyicosatetraenoic acids. C6- and C8-deficient rabbit and human sera, respectively, induced release of small amounts of [3H]C20:4 from Ehrlich ascites tumor cells and macrophages; these deficient sera also released traces of oxygenated derivatives from macrophages. Addition of purified C6 or C8 effectively restored release from both cell types, indicating that the terminal C proteins, up to and including C8, are required for the major part of the release. Our results do not rule out a possible requirement for C9.

Animals↗
PubMed1983

Elimination of complement channels from the plasma membranes of U937, a nucleated mammalian cell line: temperature dependence of the elimination rate.

We have studied the release of radiolabeled small markers from nucleated cells carrying complement channels in order to determine the life-span of these channels at various temperatures. U937 cells, a human histiocytic cell line, were labeled with 14C-aminoisobutyric acid or 86RbCl, and treated with sublytic doses of C to form transmembrane channels. The cells were then incubated at various temperatures, and the persistence of channels was evaluated by measuring the release of the intracellular markers through the remaining channels. The results indicate that the life-span of the C channels in the plasma membranes of these cells varies markedly with temperature. Thus, at 2 degrees C, the half-life of the channels was about 2 hr, whereas at 37 degrees C, the half-life was estimated to be approximately 1 min. The rapid elimination of the transmembrane channels from the plasma membranes of these nucleated cells contrasts sharply with the long persistence of C channels in the membranes of erythrocytes or erythrocyte ghosts. It is likely that the multi-hit requirement recently reported for lysis of nucleated mammalian cells by C is due, at least in part, to the rapid disappearance of channels.

Aminoisobutyric Acids↗
PubMed1982

On the lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by complement: dual role of C3b.

The efficiency of cytolysis by the terminal complement proteins C5b-9 can be markedly enhanced by C3b molecules bound on the target cell membrane (Hammer et al. 1976). This enhancement was shown to be proportional to the number of C3b molecules on the cell membrane. The present experiments have shown that the hemolytic efficiency of the complement membrane attack system is two to five times greater on paroxysmal nocturnal hemoglobulinuria erythrocytes (PNHE) than on normal human E. This difference is attribute to a derivative of C3, probably C3b, on PNHE since it was abolished by anti-C3 but not by anti-C2. The efficiency of C5b-9 to lyse PNHE was only partially decreased by C3b inactivator and beta 1 H, indicating that the C3b on PNHE is not readily inactivated by its regulatory proteins. Furthermore, cells from a single severely affected patient consumed 3-fold more C5b6 than normal human E yet concommitantly measured membrane fluidity was normal. From these observations we conclude that cell-bound C3b on PNHE serves two functions: (a) it increases the hemolytic efficiency of membrane attack components of the complement system; and (b) it provides sites for assembly of the alternative pathway convertases.

Complement C3b↗
PubMed1982

Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel.

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.

Animals↗
PubMed1982

Size of the transmembrane channels produced by complement proteins C5b-8.

It has been shown previously that erythrocytes can be lysed by complement proteins C5b-8, albeit at a much lower rate than C5b-9. We have now performed kinetic sieving experiments with resealed erythrocyte ghosts using sucrose (0.9 nm molecular diameter) and inulin (3.0 nm molecular diameter) as markers. We found that treatment of the ghosts with C5b-8 released sucrose, but not inulin. Addition of C9 to ghosts carrying C5b-8 dramatically increased the rate of sucrose flux and, in addition, caused release of inulin. Hence, unlike C5b-9 channels, those formed by C5b-8 measure less than 3 nm in diameter. Formation of C5b-8 channels was very slow compared with that of C5b-9 channels. Also, we found that about two-thirds of the C5b-8 ghosts did not have sucrose-releasing channels, but such channels were formed on reaction with C9.

Cell Membrane Permeability↗
PubMed1981

Effect of agents that produce membrane disorder on lysis of erythrocytes by complement.

To evaluate the effect of membrane lipid acyl-chain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alcohol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b-9. The enhancing effect by A2C is operative in the formation of erythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4, or Triton X-100 did not enhance lysis by C5b-9 appreciably, suggesting that enhancement of lysis by C5b-9 is not a general property of amphiphiles.

Complement C5↗
PubMed1981

Activation of the fifth and sixth component of the complement system: similarities between C5b6 and C(56)a with respect to lytic enhancement by cell-bound C3b or A2C, and species preferences of target cell.

Brief shift of purified C5 and C6 at 0 degrees C to pH 6.4, followed by immediate neutralization, results in the generation of a factor, designated C(56)a, that lyses erythrocytes together with C7, C8, and C9. We compared C(56)a and C5b6 generated by an alternative-pathway convertase, with regard to their action on different target cells. We found tht C(56)a is similar to C5b6 in the following properties: 1) Together with C7, C(56)a forms a stable intermediate on either sheep or guinea pig erythrocytes. 2) Membrane-bound C3b, or A2C incorporated in the membrane, enhances lysis by C(56)a-9, as well as lysis by C5b6-9. We also found that the lysis of EC(56)a7 or EC5b67 intermediates by C8 and C9 depends on the species of the erythrocytes and the species of C8 and C9. Thus, lysis of sheep erythrocytes is more efficient with guinea pig C8 and C9 than with human C8 and C9. In the case of guinea pig erythrocytes, this relationship is reversed, i.e., these cells lyse more efficiently when human C8 and C9 are used. Enhancement of lysis by membrane-bound C3b or A2C does not abrogate this species incompatibility pattern.

Animals↗