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J P Atkinson

Publications and source records attributed to J P Atkinson.

At least 163 records · Page 9Linked to original sources
PubMed1984

Biosynthesis of the human C3b/C4b receptor during differentiation of the HL-60 cell line. Identification and characterization of a precursor molecule.

The C3b receptor (C3bR) of the human promyelocytic leukemia cell line (HL-60) was induced by incubating these cells with dimethylsulfoxide (DMSO) or retinoic acid. A majority of differentiated (DMSO- or retinoic acid-treated) but not undifferentiated cells formed rosettes with C3b-coated erythrocytes and were morphologically mature granulocytes. HL-60 cells were surface- or biosynthetically labeled and then solubilized in 1% Nonidet P-40 in the presence of multiple protease inhibitors. The C3bR was isolated either by immunoprecipitation with anti-C3bR antibodies or by affinity chromatography with hemolytically inactive components in which the internal thioester bond within the alpha-chain was cleaved (iC3)- or iC4-Sepharose. Autoradiographs of NaDodSO4-polyacrylamide gels indicated that the surface-labeled C3bR on the differentiated cells had an Mr of 210,000 (nonreduced) or 240,000 (reducing conditions). The bulk (approximately 85%) of the radiolabeled material that was isolated from biosynthetically labeled cells co-migrated with the surface-labeled band. A small fraction (approximately 15%) of the biosynthetically labeled material that was isolated by affinity chromatography or immunoprecipitation had an Mr of 188,000, which did not correspond to any surface-labeled band. This putative precursor molecule was characterized by pulse-chase experiments and by analysis of its carbohydrate. In pulse-chase (15-min pulse) studies of differentiated cells, only the 188,000-mol wt molecule was detected at 0 h. By 2 h, greater than 80% of counts had chased from the 188,000 to the 210,000-mol wt molecule. Treatment of these two molecules with endoglycosidases indicated that the 188,000-mol wt molecule possessed high mannose oligosaccharides, while the mature C3bR had complex oligosaccharides. We conclude from these data that the 188,000-mol wt molecule is a precursor of the C3b receptor of HL-60 cells. Other experiments indicated that the half-maximal time for newly synthesized receptor to attain an Mr of 210,000 was 45 min, and that the t1/2 for the disappearance of the receptor on the surface of differentiated HL-60 cells in tissue culture was approximately 10 h. The ability to observe the induction of the C3b receptor as the HL-60 cell line differentiates is an instructive model system to study the biosynthesis of a human integral membrane receptor glycoprotein.

Cell Differentiation↗
PubMed1984

Functional studies on the secreted form of human C4 (C4s), two incompletely processed two-subunit C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and pro-C4.

The functional properties of the secreted form of C4 (C4s), which has a Mr approximately 5000 greater than the predominant C4 molecule found in plasma (C4p), two incompletely processed two-chain C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and the extracellular C4 precursor (designated pro-C4(E)] were evaluated. All four molecules are secreted in parallel by a human hepatoma-derived cell line (Hep G2). Secretion of hemolytically active C4 is linear up to approximately 12 hr, peaks at 24 hr, and then progressively decreases over the next 48 hr. This loss of C4s functional activity parallels the proteolytic conversion of C4s to C4bs. To compare the hemolytic efficiencies of C4s and C4p, a solid-phase competitive radioimmunoassay was developed to permit measurement of the small quantities of C4 antigen in these cultures. The hemolytic efficiencies of C4s and C4p were similar. These results indicate that extracellular processing of C4s to C4p does not modulate the hemolytic activity of the molecule. Consistent with their ability to bind methylamine, both the alpha s-chain and the alpha - gamma subunit undergo denaturation-induced autolysis. The extracellular and intracellular pro-C4 molecules are also sensitive to autolytic cleavage. Interestingly, the beta - alpha subunit is resistant to autolysis. In experiments in which C4s and C4p were cleaved by C1-s to C4bs, C4(beta - alpha + gamma), C4(beta + alpha - gamma), and pro-C4(E) were resistant to C1-s cleavage and thus hemolytically inactive relative to C4s. These data indicate that processing of C4 to a three-chain structure is required to provide the proper conformation for efficient activation by C1.

Animals↗
PubMed1983

Structural heterogeneity of the C3b/C4b receptor (Cr 1) on human peripheral blood cells.

In these studies CR 1 polymorphism previously demonstrated on erythrocytes (E) was also found on CR 1-bearing peripheral blood leukocytes including polymorphonuclear (PMN), eosinophils, monocytes, and B lymphocytes. However several cell-specific differences in CR1 were found: (a) an approximately 5,000-dalton increase in CR 1 on PMN and eosinophils, (b) unequal band intensity among heterozygotes suggests that there is preferential expression of 220,000- or 225,000-dalton receptors on leukocytes compared to E, and (c) "minor" bands, approximately 15,000 daltons larger than the major receptor molecule, were found on E but not on leukocytes. These observations constitute a unique example of heterogeneity of an integral membrane receptor.

B-Lymphocytes↗
PubMed1983

Esterification of monohydroxyfatty acids into the lipids of a macrophage cell line.

Cells of a mouse macrophage-like tumor cell line, J774.2, were incubated with 0.6 microM radiolabeled mono- and di-hydroxyfatty acids. Monohydroxyfatty acid products of the neutrophil and platelet lipoxygenase pathways (5-HETE, 15-HETE, and 12-HETE) were rapidly taken up (42-64% of the counts cell associated at 1 min) and esterified into triglycerides and phospholipids. 5-HETE and 12-HETE were found in triglycerides and distributed among phospholipid classes while 50% of added 15-HETE was esterified into phosphatidyl inositol. Treatment of phospholipids from cells incubated with 5-HETE, 12-HETE, and 15-HETE with phospholipase A2 resulted in release of the respective monohydroxyfatty acid. HHT, a monohydroxyfatty acid product of the cyclooxygenase pathway, was taken up and esterified more slowly than the lipoxygenase products. In addition, HHT was not released when the phospholipids from cells incubated with HHT were treated with phospholipase A2. LTB4, a dihydroxyfatty acid product of neutrophil lipoxygenase, was not taken up by J774.2 cells. The unique patterns of uptake and intracellular distribution of the different monohydroxyfatty acids suggests that the enzymes involved in the esterification of these compounds have substrate specificity and may also relate to the specific biologic effects of these compounds.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1983

Identification and partial characterization of the secreted form of the fourth component of human complement: evidence that it is different from major plasma form.

Immunoprecipitation of human C4 from plasma followed by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions revealed the expected alpha, beta, and gamma chains as well as a smaller quantity of a molecule containing an alpha chain (p98) approximately equal to 5,000 daltons heavier than the normal alpha chain. Further studies on p98 indicated that it covalently incorporated methyl-amine, was present at a concentration of approximately equal to 8% of the principal plasma form of the C4 alpha chain, and was found in highly purified C4 preparations. Hep G2, a human hepatoma-derived cell line, was found to secrete a C4 molecule in which the alpha chain had a molecular weight identical to that of the p98 protein found in plasma. The secreted C4 molecule possessed hemolytic activity. The 5,000-dalton difference in the alpha chain was localized to the COOH terminus and was attributed to an additional polypeptide. We propose that p98 is the alpha chain of the secreted form of C4, which is processed extracellularly by proteolytic cleavage to the principal C4 molecule found in plasma.

Complement C4↗
PubMed1983

Polymorphism of human erythrocyte C3b/C4b receptor.

The human erythrocyte receptor for the major activation fragments of the third and fourth components of complement (HuE-C3bR) was isolated from individual donors. Erythrocytes were surface labeled with 125I and solubilized in Nonidet P-40.HuE-C3bR was purified by using C3-Sepharose affinity chromatography and analyzed by autoradiography of NaDodSO4/polyacrylamide gels. Three distinct receptor patterns were demonstrated. Type a had a single major band with Mr of 190,000, type b had a single major band with Mr of 220,000, and type c had two major bands of Mr 190,000 and 220,000. In all three types, a minor band accounting for less than 25% of the total radioactivity was usually observed at a Mr 15,000 greater than that of each major band. Identical autoradiographic patterns were obtained by affinity chromatography using methylamine-inactivated C4-Sepharose or by immunoprecipitation of solubilized membranes with a monoclonal antibody against HuE-C3bR. All three types were distinct after reduction and alkylation, although the apparent Mr uniformly increased by approximately equal to 30,000. Characterization of HuE-C3bR types in 33 unrelated individuals demonstrated that 23 had type a, 1 had type b, and 9 had type c. Family studies provide evidence for transmission by two codominant alleles. Thus, in the normal population two alleles appear to control expression of HuE-C3bR phenotypes and account for the polymorphism of this integral membrane glycoprotein.

Complement C3↗
PubMed1983

Identification and structural characterization of two incompletely processed forms of the fourth component of human complement.

Immunoprecipitates of human C4 from EDTA-plasma were incubated with [14C]methylamine and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. In addition to finding label in the alpha-chains of the secreted (C4s) and predominant plasma (C4p) forms of C4, two additional molecules with apparent molecular weights of approximately 168,000 (p168) and approximately 125,000 (p125) covalently incorporated methylamine, indicating the presence of an internal thioester bond. These two molecules were present at a concentration of approximately 5% of total plasma C4 and were not immunoprecipitated by antisera to C3 or alpha 2-macroglobulin. A human hepatoma-derived cell line (HepG2), in addition to synthesizing C4s and small quantities of the polypeptide precursor of C4 (pro-C4), was found to secrete p168 and p125 at concentrations of 14 +/- 4.8 and 21 +/- 9.2% (mean +/- SD), respectively, of total secreted C4. These molecules were not found intracellularly. Both molecules were present on reduced, but not nonreduced, SDS-polyacrylamide gels. Chido (C4B) and Rodgers' (C4A) alloantisera precipitated the C4A and C4B variants of pro-C4, p168, p125, and C4s. Both tryptic and Staphylococcus aureus V8 protease peptide analyses showed homology between p168 and the beta- and alpha-chains and between p125 and the alpha- and gamma-chains. Partial NH2-terminal sequencing revealed that the beta-chain was NH2-terminal in p168 and that the alpha-chain was NH2-terminal in p125. Taken together, these data indicate that p168 and p125 represent uncleaved beta-alpha- and alpha-gamma-fragments of pro-C4, respectively. Thus, in most individuals, plasma C4 consists of five structurally distinct molecules, the single polypeptide precursor (pro-C4), the three-subunit secreted (C4s) and predominant plasma (C4p) forms of C4, and two incompletely processed two-subunit molecules with uncleaved beta-alpha- (p168) or uncleaved alpha-gamma (p125)-subunits. In addition, all five molecules are observed for both C4A (Rodgers) and C4B (Chido) structural genes.

Amino Acid Sequence↗
PubMed1983

Evidence that the functional responses of human neutrophils occur independently of transient elevations in cyclic AMP levels.

Exposure of human neutrophils to the tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) leads to a transient, 2-3 fold elevation of adenosine-3',5'-cyclic monophosphate (cAMP) that peaks at 5-15 seconds. This cAMP transient has been hypothesized as constituting an early activation event that may be responsible for subsequent functional responses. In order to evaluate the dependence of several FMLP-stimulated functional responses on elevated cAMP levels, we utilized 9-(tetrahydro-2-furyl)adenine (SQ 22,536), a putative inhibitor of adenylate cyclase. Pretreatment of cells with SQ 22,536 (1-1000 microM) caused dose-dependent inhibition of the FMLP (0.1 microM)-induced cAMP elevation (ID50 approximately 5 microM). Similar results were observed when cells were activated by the divalent cation ionophore A23187 (20 microM). At 1000 microM, a drug concentration which completely abolished the cAMP transient, SQ 22,536 had no effect on FMLP-stimulated superoxide radical (O2-) generation, granule enzyme release, or chemotaxis and only a modest inhibitory effect on A23187-induced O2- production. These studies strongly suggest that these FMLP- and A23187-induced responses occur independently of a transient elevation of cAMP and that, in intact human neutrophils, SQ 22,536 is a non-toxic inhibitor of adenylate cyclase.

Adenine↗
PubMed1983

Biosynthesis of C4 by mouse peritoneal macrophages. II. Comparison of C4 synthesis by resident and elicited cell populations.

Five elicited macrophage populations synthesized one-third to one-tenth as much hemolytically active C4 when compared with resident peritoneal macrophages. This decrease in functional C4 activity was not caused by inhibitors or protease activity in the elicited macrophage supernatants. Analysis of C4 antigen indicated a similar reduction in the elicited cells compared with the resident macrophages. A defect in precursor processing or secretion was deemed unlikely because the intracellular C4 precursor was appropriately reduced in the elicited cells. We postulate that mouse resident peritoneal macrophages are a pluripotent cell population with broad capabilities in regard to the initiation of the inflammatory response. In contrast, elicited or activated macrophages may be more specialized cells, with one manifestation of this being the "down" regulation of C4 synthesis.

Animals↗
PubMed1982

Genetic variation in glycosylation of the fourth component of murine complement. Association with hemolytic activity.

The molecular basis for variation in the Mr of the C4 alpha-chain of the fourth component of murine complement from several strains was investigated. All strains were capable of incorporating radiolabeled mannose into the alpha-chain of C4 from peritoneal macrophage cultures. In most cases, carbohydrate was found on both alpha-chain autolytic fragments produced by denaturation of the native C4. However, mice bearing the H-2 haplotypes w7, w16, and w19 produce C4 with no carbohydrate on the larger (COOH-terminal) autolytic fragment. This lack of carbohydrate is sufficient to cause the difference in Mr seen in the C4 alpha-chain from these mice. The chemical removal of all carbohydrate abrogated this difference. The levels of C4 antigen in the plasma of mice from these three strains are normal while the levels of hemolytic activity are reduced 60-80%. This suggests a strong role for carbohydrate in the functional properties of C4.

Animals↗
PubMed1982

Ligand binding specificity of a rabbit alveolar macrophage receptor for C3b.

We have recently reported the isolation from rabbit alveolar macrophages of a receptor which retained its ligand-binding activity for the third component of complement (C3) and for its major proteolytic derived activation fragment (C3b). The isolated receptor demonstrated a greater ability to bind C3b than an equimolar amount of C3. C3b differs from C3 in at least two ways: it is a proteolytic cleavage product of C3 and it lacks the internal thiolester bond of C3. We have analyzed the binding ability to isolated receptor to various C3 and C3b analogs and we demonstrate that the specificity of the C3b-C3b receptor interaction depends upon the lysis of the C3 thiolester bond and accompanying conformational change rather than upon proteolytic cleavage of the C3 molecule. Minimal, if any, binding of C3 with an intact thiolester bond to the isolated receptor was demonstrable.

Animals↗
PubMed1982

Stimulation of the respiratory burst in human neutrophils by crystal phagocytosis.

Exposure to monosodium urate crystals (MSU) stimulated the respiratory burst of human neutrophils as measured by increased O2 consumption and the generation of superoxide radicals (O(2)). From the comparison of data derived from nitroblue tetrazolium and cytochrome C reduction (two methods of detecting O(2) release), it appears tht O(2) production in response to MSU may be compartmentalized, i.e., occur predominantly in the intracellular space. After exposure to MSU, neutrophils from patients with chronic granulomatous disease lost viability at the normal rate; thus products of the respiratory burst are not likely to be responsible for cell death.

Cytochrome c Group↗
PubMed1982

5,8,11,14-Eicosatetraynoic acid (ETYA) inhibits binding of N-formyl-methionyl-leucyl-phenylalanine (FLMP) to its receptor on human granulocytes. A note of caution.

Binding of two biochemically chemotactic factors, FMLP and C5a, to their respective receptors on human granulocytes produces a transient several-fold increase in cAMP. To determine if arachidonic acid metabolites were responsible for the increased cAMP concentrations, two inhibitors of arachidonic acid metabolism, indomethacin and ETYA, were incubated with granulocytes prior to FMLP or C5A. ETYA, but not indomethacin, inhibited both the cAMP and superoxide responses to a similar degree. However, the mechanism of this effect was found to be through inhibition of the binding of FMLP to its receptor rather than through inhibition of arachidonate metabolism.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1982

Characterization of the Mr difference between secreted murine fourth component of complement and the major plasma form: evidence for carboxyl-terminal cleavage of the alpha chain.

The alpha-chain of murine fourth component of complement (C4) secreted by cells in vitro and in vivo has a Mr that is larger by approximately equal to 4,000 than that of the alpha-chain of the principal form of C4 in plasma. By using in vivo labeling of C4 with [35S]methionine, C4 was shown to be first synthesized with the higher Mr ("secreted") alpha-chain, which was then quickly processed (t1/2 approximately equal to 1 hr) extracellularly to the mature ("plasma") C4 possessing the lower Mr alpha-chain. Both forms of C4 were functional as assayed by the ability of their alpha-chains to be cleaved by the protease C1, to bind methylamine, and to undergo denaturation-dependent autolysis. When secreted C4 and plasma C4 were activated to C4b, the Mr difference of 4,000 was maintained in the alpha'-chains. The Mr difference was localized to the carboxyl-terminal autolytic fragment of the alpha-chain and was unaffected by the removal of carbohydrate. C4 from resident peritoneal macrophage cultures could be converted to the plasma form by incubation with heparin/plasma. This conversion could be blocked by EDTA or 1,10-phenanthroline. These data suggest that an enzyme, presumably a neutral proteinase present in mouse plasma, cleaves the carboxyl terminus of newly synthesized C4 alpha-chains, thereby creating the major form of C4 in plasma.

Animals↗
PubMed1982

Biosynthesis of C4 by mouse peritoneal macrophages. I. Characterization of an in vitro culture system and comparison of C4 synthesis by "low" vs "high" C4 strains.

C4 biosynthesis and secretion in mouse resident peritoneal macrophage cultures was examined by functional and antigenic analysis. The rate of secretion of functional and antigenic C4 decreased in parallel in short-term culture despite constant total protein secretion and increasing factor B secretion. Feedback inhibition and protease degradation did not account for the decreasing rate of C4 secretion. Despite a 10-fold difference in plasma C4 levels between high C4 (H-2d haplotype) and low C4 (H-2k haplotype) mouse strains, resident peritoneal macrophages from both strains synthesized and secreted similar amounts of antigenic and functional C4 in short-term culture. The C4 secreted by H-2d and H-2k macrophages was relatively stable, because it exhibited only approximately 2% loss of hemolytic activity per hour in culture. These data suggest that an unidentified environmental signal leads to the specific decrease in C4 synthesis and secretion by peritoneal macrophages and that a regulatory defect may not be present, at least in macrophages, to account for the low C4 levels in the H-2k haplotype.

Animals↗
PubMed1982

Structural and functional characterization of an incompletely processed form of murine C4 and Slp.

The properties of a 128,000-dalton polypeptide, corresponding to the uncleaved alpha- and gamma- chains of the fourth component of murine complement (C4), were studied. A fragment of similar size (137,000 daltons) from the structurally related sex-limited protein (Slp) was also found. These polypeptides have methylamine and iodoacetamide binding sites. suggesting that they, like the alpha-chains of C4 and Slp, possess an internal thiolester. In tryptic peptide map analysis, extensive homology is seen between the 128,000-dalton fragment and native C4 alpha- and gamma-chains. N-terminal sequences for this fragment and C4 alpha are identical, as are those for the 137,000-dalton Slp fragment and Slp alpha. Although the alpha-gamma fragment, like C4 alpha, undergoes denaturation-dependent autolytic cleavage, unlike C4 alpha, it cannot be cleaved by C1 This indicate that some of the properties of C4 do not require the native three-chain structure, whereas others do require it. These findings suggest that the alpha-gamma fragments represent an intermediate step in the processing of the C4 and Slp precursor polypeptides.

Animals↗