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Biomedical subjects

A Mellors

Publications and source records attributed to A Mellors.

At least 37 records · Page 2Linked to original sources
PubMed1993

Epitopic structure of Tn glycophorin A for an anti-Tn antibody (MLS 128).

Glycophorin A was digested with glycoprotease (Pasteurella haemolytica) and the digest was fractionated by a combination of high-pressure column chromatographies to produce the glycopeptides GPA-1 to GPA-6. Sequence analysis of the glycopeptides revealed that two serine residues (Ser-14 and Ser-15) are not glycosylated, Thr-17 and Ser-19 being glycosylated instead, in disagreement with the accepted structure. The glycopeptides thus obtained were treated with sialidase and beta-galactosidase. The Tn antigenicity, as assayed by the binding to a monoclonal anti-Tn antibody (MLS 128), was found exclusively in the glycopeptides including three (cluster I) or four (cluster II) consecutive residues of GalNAc-Ser/Thr, whereas the glycopeptide (GPA-2) containing two nonconsecutive GalNAc-Ser/Thr residues had practically no Tn antigenicity. The immunoreactivities of GPA-1 and GPA-3, containing both clusters I and II, and GPA-4, containing cluster II, were 63% (calcd. 67%), 81% (calcd. 86%), and 50% (calcd. 50%), respectively, of the immunoreactivity of GPA-5 or GPA-6, containing cluster I (the average being taken as the basis), based on the reactivity per GalNAc residue. These results indicate that clusters I and II react with the antibody to the same extent. The structure consisting of three consecutive glycosylated Ser/Thr residues may be essential for Tn antigenicity in the light of previous results for ovine submaxillary mucin.

Amino Acid Sequence↗
PubMed1992

The glycoprotease of Pasteurella haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin.

HL-60 cells and neutrophils treated with the glycoprotease from Pasteurella haemolytica A1, an enzyme which is specific for O-sialoglycoproteins, were found to be incapable of binding P-selectin but still bound E-selectin. Comparative analysis of [35-S] cysteine labeled proteins from HL-60 cells by 2-dimensional electrophoresis indicated that two major proteins with M(r) 100 and 115 kd were significantly removed from cells which had been treated.

Antigens, CD↗
PubMed1992

Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.

Animals↗
PubMed1992

A neutral glycoprotease of Pasteurella haemolytica A1 specifically cleaves O-sialoglycoproteins.

A neutral metalloprotease with marked specificity for an O-sialoglycoprotein has been isolated from culture supernatants of Pasteurella haemolytica A1. The 35-kDa enzyme cleaves human erythrocyte glycophorin A, which is O glycosylated, but does not cleave N-glycosylated proteins or nonglycosylated proteins. Glycophorin A was cleaved when it was present in situ in erythrocyte ghost plasma membranes or when it was free in solution. The glycoprotease did not hydrolyze glycophorin A from which sialate residues had been removed by neuraminidase treatment. An immobilized preparation of the enzyme cleaved glycophorin A at several positions, with a major site of cleavage at Arg-31-Asp-32. The glycoprotease is inhibited by EDTA, citrate, and ascorbate, but inhibition appears to be due to the masking of metal ion activators rather than to their removal. The enzyme is not inhibited by phosphoramidon, an inhibitor of other bacterial neutral metalloproteases.

Ascorbic Acid↗
PubMed1992

Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells.

Our previous studies have shown that a unique glycoprotease from Pasteurella haemolytica specifically cleaves only proteins containing sialylated O-linked glycans. The hematopoietic progenitor cell antigen, CD34, which is heavily glycosylated with both N- and O-linked glycans, is readily cleaved by this protease. In this study, we demonstrate that the epitopes detected by five of the seven CD34 monoclonal antibodies are removed by the glycoprotease. The differential sensitivity of the CD34 epitopes to cleavage with either neuraminidase and/or glycoprotease establishes three classes of epitopes: 1) (class I) those identified by MY10, B1.3C5, 12.8, and ICH3 that are differentially affected by neuraminidase and removed by the glycoprotease; 2) (class II) the epitope detected by QBEND 10 that is removed only by the glycoprotease; and 3) (class III) those identified by TUK3 and 115.2 that are not removed by either enzyme. Cleavage of the 110-kd CD34 structure by the glycoprotease generates a major cell-bound fragment of about 75 kd, identified by the class III antibodies. We have also used the enzyme to improve the rapid recovery of CD34+ cells selected by immunomagnetic affinity techniques. In a preclinical model, we separated CD34+ KG1 cells with high yield (90%-95%) and high purity (94%-98%) from sham mixtures containing 50% CD34- cells. We also separated CD34+ blast cells from a patient in megakaryoblastic crisis of chronic myelogenous leukemia. In this case, the purity and yield were 93% and 94%, respectively. Enzyme treatment had no detrimental effect on cell viability, and the treated cells showed a normal quantitative expression and distribution of CD34 antigen as assessed with class III antibodies. We conclude that the P. haemolytica glycoprotease has potential to improve the isolation, from human bone marrow, of primitive hematopoietic cells that carry the CD34 antigen.

Antigens, CD↗
PubMed1992

Retention of progenitor cell function in CD34+ cells purified using a novel O-sialoglycoprotease.

We previously showed that the sialoglycoprotein, CD34, which is expressed on primitive human hematopoietic progenitor cells, is cleaved by a unique glycoprotease from Pasteurella haemolytica (P.h. glycoprotease). This proteolytic enzyme specifically cleaves glycoproteins rich in O-sialoglycans. Glycoproteins containing only N-linked glycans are not cleaved. Cleavage of the CD34 antigen results in the loss of epitopes detected by five of seven CD34-designated antibodies. In this study, we investigated the role of the P.h. glycoprotease in isolating CD34+ cells from unfractionated normal human bone marrow mononuclear cells (MNCs), and determined the effect of the glycoprotease on the proliferative capacity of the progenitor-enriched fraction. CD34+ cells were isolated from MNCs using immunomagnetic beads attached via a CD34 antibody whose epitope is susceptible to removal by the cleavage with the glycoprotease. Subsequent cleavage with P.h. glycoprotease for 30 min at 37 degrees C released the CD34+ cells from the beads with a recovery of up to 78%. Using a CD34 antibody whose epitope was not removed by the glycoprotease, up to 95% of the recovered cells expressed CD34. Compared to unseparated MNCs, the CD34+ cells showed the following enrichment of committed hematopoietic progenitors, as assayed in semi-solid media: CFU-GM, 45-fold; CFU-M, 13-fold; BFU-E, 26-fold and CFU-GEMM, 81-fold. Hematopoiesis was also studied in two-stage long-term bone marrow cultures in which the CD34+ cells were co-cultured over irradiated, allogeneic adherent layers. Output of CFU-GM over a seven week period from these cultures was similar to that from control cultures with autologous adherent-cell-depleted marrow MNCs. These data suggest that the loss of O-sialo-glycosylated peptide moieties from P.h. glycoprotease-released CD34+ cells neither affects the functional capacity of committed progenitors, nor impairs the proliferation of long-term culture-generating cells. The P.h. glycoprotease can be used to facilitate the isolation and recovery of functionally competent CD34+ cells at high yield and purity, without prior removal of other adherent cells. The ability to rapidly purify CD34+ cells using this non-cytotoxic enzyme has important implications for bone marrow transplantation as well as for gene transfer studies in vitro.

Antigens, CD↗
PubMed1991

Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene.

Pasteurella haemolytica serotype A1 secretes a glycoprotease which is specific for O-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pH1, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPH1. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPH1. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orfX, whose product is believed to function in the regulation of macromolecule biosynthesis.

Amino Acid Sequence↗
PubMed1990

Interrelationship between signals transduced by phytohemagglutinin and interleukin 1.

In the murine cell line LBRM-331A5, phytohemagglutinin (PHA) induces secretion of the T cell growth factor interleukin 2 (IL2). IL1 augments PHA-induced IL2 production. In this cell line, PHA stimulates a number of biochemical changes including phospholipid hydrolysis, increases in cytosolic free calcium [( Ca2+]i), membrane hyperpolarization, cytosolic alkalinization, and tyrosine phosphorylation of specific substrates. Using LBRM cells, we have studied the interrelationship between these events and the secretion of IL2. Increases in [Ca2+]i triggered by PHA or following addition of ionomycin result in membrane hyperpolarization but are not required for PHA-induced cytosolic alkalinization or tyrosine phosphorylation. Addition of IL1 to PHA-stimulated cells did not affect any of the biochemical parameters, although it significantly augmented PHA-induced IL2 secretion. Increasing [Ca2+]i with ionomycin did not trigger IL2 secretion, increases in cytosolic pH, or tyrosine phosphorylation in the presence or absence of IL1. Preventing increases in cytosolic pH did not alter PHA-induced changes in [Ca2+]i or membrane potential. These data are compatible with PHA including activation of phospholipase C and production of inositol phosphates resulting in both release of Ca2+ from internal stores and transmembrane uptake of Ca2+ as well as activation of protein kinase C. However, unlike other growth factor or mitogen-stimulated systems, the changes stimulated by PHA and IL1 in LBRM cells including IL2 secretion are not regulated by a pertussis toxin-sensitive G protein.

Adenosine Diphosphate Ribose↗
PubMed1989

The diacylglycerol analogue, 1,2-sn-dioctanoylglycerol, induces an increase in cytosolic free Ca2+ and cytosolic acidification of T lymphocytes through a protein kinase C-independent process.

In this paper, we demonstrate that low concentrations (0.5-2.5 microM) of 1,2-sn-dioctanoylglycerol (DiC8), a potent diacylglycerol used in many previous studies to probe the role of protein kinase C (PKC) in cell activation, cause cytosolic alkalinization of human, mouse and pig T lymphocytes through PKC-mediated activation of the Na+/H+ antiport. However, at higher concentrations (greater than or equal to 12.5 microM), the effect on cytosolic pH (pHi) is reversed, resulting in a marked cytosolic acidification, followed by a gradual return of pHi to baseline values. DiC8 also induces marked changes in cytosolic free calcium concentrations ([Ca2+]i), initially by releasing calcium from intracellular stores, followed by a net transmembrane influx of calcium. The DiC8-induced cytosolic acidification, the resultant return to baseline pH and the increase in [Ca2+]i are independent of activation of PKC. Unlike many other agents which increase [Ca2+]i, DiC8 does not induce phosphatidylinositol hydrolysis with the resultant production of inositol phosphates. Other compounds known to activate PKC, including the closely related diacylglycerol analogues, 1,2-sn-dihexanoylglycerol and 1,2-sn-didecanoylglycerol, phorbol esters and mezerein, did not induce changes in [Ca2+]i or cytosolic acidification in T lymphocytes. Thus the action of DiC8 on intact lymphocytes is different from that of phorbol esters and other diacylglycerols, and is specific to the length of the acyl chains. Because changes in [Ca2+]i are often associated with cell proliferation and cell differentiation, some effects of DiC8 on intact cells may be a consequence of changes in [Ca2+]i.

Animals↗
PubMed1989

Physiologic activation of protein kinase C limits IL-2 secretion.

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.

Animals↗
PubMed1989

The acquisition of lipids by African trypanosomes.

Bloodstream forms of African trypanosomes are dependent on their host for fatty acids, choline and other components of membrane lipids. The bulk of their choline requirement is met by their ability to take up lysophospholipids from the host tissue fluids. Trypanosoma brucei has ocyltransferose and phospholipase A(1) activities for the metabolism of exogenous lysophospholipids. The rate of uptake of lysophospholipids can be controlled by changes in the extrocellulor concentration of fatty ocyl-coenzyme A, and this control has potential for chemotherapy.

Journal Article↗
PubMed1988

A putative new growth factor in ascitic fluid from ovarian cancer patients: identification, characterization, and mechanism of action.

Ascitic fluid form ovarian cancer patients (n = 16), but not from patients with other cancers or with benign diseases, contains a growth-promoting activity which induces the proliferation of both fresh ovarian cancer cells (n = 5) and the ovarian cancer cell line HEY. The ascitic fluid growth factor(s) appears to signal cells through binding and activation of specific, saturable, high-affinity cell surface receptors. Incubation of fresh or cultured ovarian cancer cells with a partially purified preparation of ascitic fluid stimulates phosphatidylinositol turnover and increases cytosolic-free calcium. Each of these biochemical events has been implicated in the action of growth factors. Purified preparations of previously identified growth factors including epidermal growth factor, transforming growth factor-beta, tumor necrosis factor, platelet-derived growth factor, thrombin, insulin, interleukin-1, interleukin-2, vasopressin, angiotensin, alpha- and gamma-interferons, and fibroblast growth factor did not increase cytosolic-free calcium in either fresh ovarian cancer cells or HEY cells. Therefore, ascitic fluid appears to contain one or more previously unidentified growth factors which activate ovarian cancer cells through phosphatidylinositol hydrolysis and resultant changes in cytosolic-free calcium.

Ascitic Fluid↗
PubMed1988

Metabolism of phospholipids and lysophospholipids by Trypanosoma brucei.

African trypanosomes (Trypanosoma brucei brucei) rapidly metabolize exogenous 1-acyl-lysophospholipids by at least two routes: (1) hydrolysis by a phospholipase A1; (2) acylation by an acyl-CoA-dependent acyltransferase. In contrast to lysophospholipids, exogenous phospholipids are not rapidly metabolized by T. brucei. The acyltransferase (EC 2.3.1.23) converts exogenous 1-acyl lysophosphatidylcholine and exogenous acyl-CoA to phosphatidylcholine and CoA-SH. It is a membrane-bound enzyme and shows maximal activity within the first 2 min of exposure of trypanosomes to the exogenous substrates. The acyltransferase specificity for lysophospholipids is lysophosphatidylcholine greater than lysophosphatidylinositol greater than lysophosphatidylethanolamine greater than lysophosphatidate. Phosphatidylcholine enhances the enzyme activity towards lysophosphatidylethanolamine and lysophosphatidic acid. The preference for CoA acyl thioesters is oleoyl greater than palmitoyl greater than myristoyl greater than stearoyl greater than arachidonoyl, and this specificity distinguishes the protozoan enzyme from those of cells of mammalian hosts, which are specific for arachidonoyl-CoA. When the acyltransferase converts exogenous lysophosphatidylethanolamine to phosphatidylethanolamine, the latter is rapidly methylated to form dimethylphosphatidylethanolamine. There is also rapid hydrolysis of exogenous oleoyl-CoA by a thioester hydrolase in living trypanosomes, to yield free oleate and CoA-SH.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1986

Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. Possible relation to cell pH and volume regulation.

In lymphocytes, the Na+/H+ antiport can be stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by osmotic shrinking. Since TPA acts by stimulating protein kinase C, we undertook experiments to determine if protein phosphorylation also underlies the osmotic stimulation of the antiport. We found that at least one of the membrane polypeptides labeled in cells treated with TPA is also phosphorylated by hypertonic shrinking. In both instances phosphorylation is alkali labile and associated with serine and threonine residues. We tested the possibility that shrinking activates phospholipase C, thereby stimulating protein kinase C through release of diacylglycerol. No decrease in phosphatidylinositol 4,5-bisphosphate levels was detected in hypertonically treated cells. Moreover, the concentrations of inositol phosphates, including inositol trisphosphate, were not altered in shrunken cells. Thus, shrinking does not appear to activate phospholipase C. Whereas TPA induced intracellular redistribution of soluble protein kinase C, no such effect was detected in osmotically activated cells. It was concluded that osmotic stimulation of the Na+/H+ antiport is associated with activation of protein phosphorylation by a kinase that is similar, but not identical to protein kinase C. Experiments in Na+-free or amiloride-containing media indicate that phosphorylation is not a consequence of activation of the antiport.

Animals↗
PubMed1986

Interleukin 2 does not induce phosphatidylinositol hydrolysis in activated T cells.

Hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and myoinositol-1,4,5-trisphosphate is thought to be a primary event in the activation of cells by some growth factors, mitogenic lectins, and oncogenes. The mechanism whereby interleukin 2 (IL 2) binding to its receptor on activated T lymphocytes leads to cell proliferation has not been determined. Because the mitogenic has not been determined. Because the mitogenic action of IL 2 resembles that of some growth factors, the possible role of phosphatidylinositol breakdown in the activation of T cells by IL 2 was examined. In human or murine IL 2-sensitive cells, incubation with IL 2 did not alter the rate of turnover of phosphatidylinositol, phosphatidylinositol-5-phosphate, phosphatidylinositol-4,5-bisphosphate, or phosphatidylcholine in 32PO4-loaded cells. IL 2 also did not alter either the isotopic labeling of diacylglycerol or [3H]arachidonic acid release from cells. In addition, IL 2 did not alter the rate of formation of the phosphatidylinositol breakdown products myoinositol-1,4,5-trisphosphate, myoinositol-1,4-bisphosphate, or myoinositol-1-phosphate. In contrast, under similar conditions, IL 2 induced significant increases in [3H]thymidine incorporation and cell proliferation. Mitogenic lectins such as concanavalin A and phytohemagglutinin gave significant changes in isotopic labeling of phosphoinositols, diacylglycerols, and phosphatidylinositols, indicating that phosphatidylinositol hydrolysis induced by mitogenic lectins was detectable in the assay systems. IL 2, in contrast to other growth factors, does not appear to signal cells by increasing phosphatidylinositol breakdown.

Animals↗