PubMed Health⌕ Search

Biomedical subjects

D F Bainton

Publications and source records attributed to D F Bainton.

At least 55 records · Page 3Linked to original sources
PubMed1990

Intracellular localization of pp60c-src in human platelets.

We have studied the intracellular distribution of the proto-oncogene product pp60c-src in human platelets by immunofluorescence microscopy and by immunogold electron microscopy on frozen thin sections. Virtually all of the immunoreactive pp60c-src was found to be associated with the plasma membrane and with membranes of the surface-connected canalicular system. This localization suggests that pp60c-src may relay signals from an as yet unidentified cell surface protein to intracellular targets.

Antibodies, Monoclonal↗
PubMed1990

Ultrastructural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener's granulomatosis.

To investigate the distribution of proteinase 3, the target antigen of anti-cytoplasmic antibodies (ACPA or C-ANCA), within the organelles of resting normal human polymorphonuclear leukocytes and monocytes, the authors used immunocytochemical techniques on thin frozen sections. To obtain valuable tools for immunolabeling, two murine monoclonal antibodies (MAbs) directed against the ACPA antigen were produced. In neutrophils, the authors observed immunogold label for the ACPA antigen, predominantly in myeloperoxidase-positive azurophil granules, and in smaller amounts on the plasma membrane. In monocytes, the ACPA antigen could be detected in small granules, which occasionally also contained myeloperoxidase, and rare labeling was found on the monocyte membrane. The finding that the ACPA antigen is expressed on the plasma membrane of neutrophils and monocytes, thereby becoming accessible to circulating autoantibodies, supports the supposition that ACPA are not only markers of disease activity, but also are involved in the pathogenesis of Wegener's granulomatosis.

Antibodies, Anti-Idiotypic↗
PubMed1990

In vivo defibrination results in markedly decreased amounts of fibrinogen in rat megakaryocytes and platelets.

Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.

Ancrod↗
PubMed1989

Purification and characterization of human lysosomal membrane glycoproteins.

Two human cell lysosomal membrane glycoproteins of approximately 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing approximately 0.05% of the total detergent-extractable protein. Both molecules were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was approximately 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membrane of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.

Amino Acid Sequence↗
PubMed1989

Incorporation of intravenously injected albumin, immunoglobulin G, and fibrinogen in guinea pig megakaryocyte granules.

In a previous study we provide evidence for a circuitous pathway by which circulating plasma proteins enter megakaryocyte granules by an endocytic mechanism and are returned to the circulation in platelets (1987. Proc. Natl. Acad. Sci. USA. 84:861-865). Horseradish peroxidase (40,000 mol wt) was injected into guinea pigs and its uptake into megakaryocyte organelles examined by electron microscopy and cytochemistry. In the present study we tested the ability of guinea pig megakaryocytes to take up intravenously injected albumin, IgG, and fibrinogen. We used two types of proteins to study the endocytic pathway: (a) heterologous human proteins, which were detected immunohistochemically using antibodies that do not crossreact with the native guinea pig counterparts; and (b) human and guinea pig proteins labeled with the small (250 mol wt), inert molecule, biotin, which were detected using an antibody against biotin. We detected all three of the injected proteins in bone marrow megakaryocytes in patterns identical to those of native counterparts. The injected protein consistently appeared in platelets 24 h later and was secreted in response to thrombin. We conclude that there are at least two mechanisms by which guinea pig megakaryocyte granules acquire proteins (a) endogenous synthesis, as demonstrated by others, and (b) endocytosis of plasma proteins synthesized by other types of cells.

Albumins↗
PubMed1989

GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies.

We used an immunoperoxidase procedure to examine the tissue distribution of the platelet alpha-granule membrane protein, GMP-140. In addition to its presence in megakaryocytes and platelets, GMP-140 antigen was found in vascular endothelial cells of diverse human organs, but it was not detected in other types of secretory cells. [35S]Cysteine-labeled human umbilical vein endothelial cells synthesized a GMP-140 molecule containing complex N-linked oligosaccharides similar to those previously demonstrated in platelets and the megakaryocytic HEL cell line. Using an immunogold procedure on frozen thin sections of endothelial cells, we found GMP-140 antigen to be localized to membranes of electron-dense storage granules. In double-label experiments there was colocalization of GMP-140 with vWf, indicating that these granules are Weibel-Palade bodies. When endothelial cells were stimulated with histamine, GMP-140 rapidly redistributed to the plasma membrane. Immunoassays of cell lysates indicated that, relative to total cell protein, less GMP-140 is present in human umbilical vein endothelial cells than in platelets. The restricted expression of GMP-140 in secretory granules of platelets and endothelium suggests that it has a specific function in the vascular system rather than a general role related to inducible secretion.

Cells, Cultured↗
PubMed1989

Human neutrophil gelatinase is a component of specific granules.

Previous investigators have proposed that gelatinase, a metalloproteinase found in neutrophils, is stored in a novel secretory compartment distinct from the two major granule populations, azurophilic and specific. To locate this proteinase in human neutrophils we reacted the cells for peroxidase and then applied monospecific polyclonal antibodies to human neutrophil gelatinase to immunolabel ultrathin frozen sections using an immunogold technique. Gelatinase was localized in a population of peroxidase-negative granules. Double-labeling experiments using antibodies against lactoferrin, a marker for specific granules, and gelatinase demonstrated colocalization of the two antigens in 80% of the specific granules. However, some granules immunostained with only the lactoferrin or gelatinase antibody. Similar techniques were used to examine precursor cells from bone marrow. In myelocytes both gelatinase and lactoferrin were present in large developing specific granules; however, some mature specific granules contained only lactoferrin. Thus, it is possible that lactoferrin synthesis begins earlier than gelatinase synthesis and that overlapping synthesis and segregation occurs during the myelocyte stage. These findings suggest that the main storage compartment of gelatinase is within the peroxidase-negative specific granules.

Blotting, Western↗
PubMed1989

Rapid fragmentation and reorganization of Golgi membranes during frustrated phagocytosis of immobile immune complexes by macrophages.

The authors have observed the rapid reorganization of the cellular membranes of macrophages during Fc receptor-mediated frustrated phagocytosis of immune complex-coated surfaces. As the macrophages spread, large, clear basal vacuoles and anastamosing tubules were formed, occasionally contiguous with the adherent surface. Coated vesicles also were observed. This process was accompanied by a rapid reorganization of the Golgi complex region of the macrophages, which was observed using trimetaphosphatase histochemistry and an antibody to a Golgi membrane antigen as markers. On contact of the macrophages with the immune complexes, the Golgi complexes, which were tightly clustered around the centrioles, dispersed into vesicles and reorganized near the basal surface. The Golgi cisternae swelled, fragmented, and decreased in number. Golgi membrane antigen was found in the large basal vacuoles and also associated with the adherent basal surface of the macrophages. This indicates that the Golgi complexes were reorganized, in part, by a direct recruitment of their membranes to the increasing basal surface area of the spreading macrophages. The changes in the structure of the Golgi complexes were reversible; by 2 hours, the complexes had recovered their normal organization, with an accompanying decrease in the number of large basal vacuoles. These data suggest that the dynamic interrelationship among the Golgi membranes, intracellular vacuoles, and the plasma membrane can be perturbed by membrane spreading on a nonphagocytosable surface.

Animals↗
PubMed1989

Directed exocytosis of secretory granules containing apolipoprotein E to the adherent surface and basal vacuoles of macrophages spreading on immobile immune complexes.

As macrophages spread on an immune complex-coated surface, large, clear basal vacuoles and numerous coated vesicles and tubules form rapidly and are occasionally contiguous with the adherent surface, creating a microcompartment between the immune complex-coated surface and the cell membrane. The present study explored the nature of this basal compartment by examining the distribution of a major secretory product of macrophages, apolipoprotein E (ApoE), and of a lysosomal enzyme, acid phosphatase, by enzyme and immunocytochemistry. Upon contact of the macrophages with the immune complexes, intracellular stores of ApoE were secreted rapidly in the first 10 to 20 minutes to the area of ligand-receptor interaction. ApoE filled the large basal vacuoles and was also found in invaginations on the adherent surface that were sealed to the influx of proteins and peptides from the bulk medium. In contrast, the contents of lysosomes were not redistributed to the basal compartment. By 2 hours most of the ApoE had appeared in the bulk medium, suggesting that the protein could move out of the basal compartment. These data suggest that specific ligand-Fc receptor interactions serve to target secretion by macrophages to selective focal areas of contact, and that there are also mechanisms for retrieval of material from these sites.

Animals↗
PubMed1989

Incorporation of a circulating protein into alpha granules of megakaryocytes.

In order to determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for uptake by cytochemistry and electron microscopy. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare bone marrow cells. In megakaryocytes, more than 50% of alpha granules contained HRP between 75 minutes and 7 hours after injection. At 24 hours, 25% of the megakaryocyte granules were peroxidase positive; less were so by 48 hours and none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. A precipitous drop in circulating platelet numbers was observed 45 minutes after injection. At this time, circulating platelets showed the tracer only on the platelet plasma membrane, and none in platelet granules. Platelet numbers increased to 35% by 7 hours and only the platelet granules contained HRP. These platelets secreted the HRP stored in granules in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. Our studies are the first to demonstrate an endocytic pathway by which megakaryocytes can incorporate a circulating protein into alpha granules. An important physiologic implication of this endocytic pathway is the possible origin of certain alpha granule proteins from plasma.

Animals↗
PubMed1988

Pitfalls in ecto-5'-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages.

We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5'-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killed Escherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5'-nucleotidase onto the adhering particle was fact or artifact, we localized 5'-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5'-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.

5'-Nucleotidase↗
PubMed1988

HL-60 cells have abnormal myeloperoxidase transport and packaging.

An unusual distribution of myeloperoxidase has been observed in HL-60 cells using enzyme cytochemistry and electron microscopy. Its localization in these cells differed in two ways from its previously observed distribution in normal human promyelocytes developing in vivo. First, the majority of Golgi cisternae and vesicles of HL-60 cells did not contain detectable peroxidase, whereas the rough endoplasmic reticulum (RER), granules, and dilated vacuoles were positive. This suggests that there is a block in vesicular transport from RER to the Golgi complex, or in fusion of the vesicles with Golgi membranes. Second, the peroxidase-positive granules that were formed in vitro varied greatly from those formed in vivo as follows: (1) their limiting membrane was narrower, (2) their contents were less concentrated, and (3) they resemble autophagic vacuoles rather than normal storage granules because they contained numerous membranes. These findings suggest that information gleaned from HL-60 cells about the biosynthetic pathway of myeloperoxidase and the formation of granules should be interpreted with caution.

Biological Transport↗
PubMed1987

Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils.

Previous studies have suggested that the leukocyte adhesion proteins Mac-1 and p150,95 are stored in a latent intracellular pool in neutrophils, and cellular fractionation studies have shown that Mac-1 is localized primarily in the peroxidase-negative specific granules. To determine the subcellular location of leukocyte adhesion receptors (LAR), we used immunocytochemical techniques on frozen thin sections of human blood leukocytes that had been incubated for peroxidase to mark the peroxidase-positive azurophil granules. To enhance the sensitivity of detection, polyclonal antibodies against immunoaffinity-purified p150,95 were raised in rabbits and absorbed with leukocytes from a patient deficient in this protein. The antiserum reacted with p150,95 and two other antigens with the same beta subunit, Mac-1 and lymphocyte function-associated antigen 1 (LFA-1). In neutrophils, we observed immunogold label for LAR predominantly on the membranes of peroxidase-negative granules, and in smaller amounts on the plasma and perinuclear membranes. In double-label experiments, there was colocalization of LAR with lactoferrin in some of the peroxidase-negative granules. We conclude that the latent pool of LAR resides in the membranes of peroxidase-negative granules. A significant increase in label on the plasma membrane of neutrophils stimulated with PMA is consistent with secretion of LAR to the exterior of the cell during degranulation. While LFA-1 appears very early in neutrophil maturation, it is becoming clear that Mac-1 and p150,95 are upregulated from an intracellular storage pool of peroxidase-negative granules that appear during the myelocyte stage of differentiation. Further studies are indicated to determine the significance of these proteins on the plasma membrane of two other granulocytes, eosinophils and basophils.

Antigens, Surface↗
PubMed1987

Incorporation of a circulating protein into megakaryocyte and platelet granules.

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.

Animals↗
PubMed1987

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering.

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

Adenosine Diphosphate↗
PubMed1987

Auer rod-like inclusions in prolymphocytic leukemia.

Auer rod-like inclusions (ARLI) can be seen in B cell malignancies but are very rare in prolymphocytic leukemia (PLL). Prolymphocytes with ARLI from a patient with PLL were examined using cytochemical and immunochemical techniques and transmission electronmicroscopy (EM). The lymphoblastic cells gave a positive reaction with antibodies to the gamma heavy chains and kappa light chain. On EM the ARLI were 1-2 micron long, consisting of election-dense crystalline matrix, with a periodicity of 60 A and contained within a single unit membrane. The production of IgG by PLL cells with ARLI has not been previously reported. It is emphasized that in PLL the lymphoblasts can have inclusions resembling Auer rods. Cytochemical and immunochemical studies should be done to avoid a misdiagnosis of acute nonlymphoblastic leukemia.

Aged↗
PubMed1987

Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface.

Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte's ability to adhere and diapedese.

Animals↗