Biomedical subjects

J T August

Publications and source records attributed to J T August.

At least 55 records · Page 3Linked to original sources

PubMed1986

Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2.

Several properties of the lysosomal membrane glycoproteins LAMP-1 and LAMP-2 have been analysed. Each molecule was strongly associated with lysosome membranes and was extracted only in the presence of detergent. Studies of the biosynthesis and processing of the glycoproteins showed that each contained a polypeptide core of approx. 43,000 Da as identified by use of tunicamycin and endoglycosidase H. Nascent glycoproteins pulse-labelled for 5 min with [35S]methionine were approx. 92,000 Da. These precursor molecules were processed in 30 min to highly heterogeneous mature glycoproteins of approx. 110,000 Da(LAMP-1) and 105,000 Da(LAMP-2). Concomitant with the increase in apparent Mr the molecules became endoglycosidase H resistant and acquired sialic acid residues, indicating that they were converted to complex-type oligosaccharides. The final maturation of the glycoproteins was blocked by monensin. Immunohistochemical analysis of tissues from Balb/c and Beige/J mice showed that the molecules were present on many types of cells, consistent with their presence in lysosomes. The patterns of tissue expression of LAMP-1 and LAMP-2 in the two mouse strains were the same except that the intensity of staining of LAMP-2 was less than that of LAMP-1. LAMP-2, but not LAMP-1, gave a decreased immunofluorescent staining intensity in transformed HaNIH as compared with NIH/3T3 cells. The marked similarities between the LAMP proteins raise the consideration of common functions, possibly associated with the high oligosaccharide content of the molecules.

Acetylglucosaminidase↗

PubMed1985

A high-performance liquid chromatographic procedure for the purification of mouse monoclonal antibodies.

High-performance liquid chromatography was applied to the purification of monoclonal antibodies from mouse ascites fluid. The method was based on anion-exchange chromatography using a TSK DEAE-5PW column and a gradient elution with 20 mM Tris, pH 8.5, and 20 mM Tris, pH 8.5, containing 2.0 M sodium acetate. The method can be applied to analytic or preparative scale separations. Purified immunoglobulins were isolated from samples of 20 to 100 microliter containing up to 19 mg total protein. The average recovery of total protein was 89 +/- 12%. Recovery of the immunoglobulins, based on recovery of immunological activity, was quantitative. In addition to separating the immunoglobulins from the other serum proteins, the various classes of IgG were resolved.

Animals↗

PubMed1985

Characterization of cell surface glycoproteins recognized by the granulocyte-specific monoclonal antibody, AHN-1.

Major surface-iodinated proteins of Mr 105,000 and 145,000 of normal human neutrophils are immunoprecipitated by a number of monoclonal antibodies (AHN-1 to AHN-6), which react specifically with granulocytes among peripheral blood cells and selectively inhibit phagocytosis. These proteins, and an Mr 60,000 component, were purified by monoclonal antibody affinity chromatography, molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Each of the three purified proteins was immunoprecipitated by all six antibodies. Nevertheless, tryptic peptide maps of the three proteins indicated that each was a distinct component. AHN-1 to AHN-6 also bound to glycolipid fractions of human neutrophils, and the binding of each antibody to human neutrophils was blocked by the carbohydrate sequences, lacto-N-fucopentaose III. The data indicate that a predominant antigenic determinant of human neutrophils is lacto-N-fucopentaose III, or related carbohydrates, present on three distinct proteins as well as glycolipids. At least one of these molecules appears to be involved in the process of phagocytosis.

Antibodies, Monoclonal↗

PubMed1985

Lysosome-associated membrane proteins: characterization of LAMP-1 of macrophage P388 and mouse embryo 3T3 cultured cells.

Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, has been identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anit-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromatography with 1D4B monoclonal antibody, yielding a homogeneous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the two purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [35S]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent Mr 92,000, and then converted to heterogeneous mature forms differing in average Mr from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [35S]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent molecular weight of the molecules or the differences between the various cell lines. beta-D-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.

Animals↗

PubMed1985

Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man.

The carboxyl-terminal one-third of the Epstein-Barr virus nuclear antigen (EBNA-1) encoded by the BamHI restriction fragment K was synthesized in Escherichia coli by use of a high-expression plasmid. The resultant 28-kDa EBNA fusion polypeptide, comprising 5-10% of the total soluble bacterial protein, was purified to apparent homogeneity by phosphocellulose and hydroxylapatite column chromatography. Both rabbit monospecific antibodies and mouse monoclonal antibodies against 28-kDa EBNA gave nuclear immunofluorescence staining on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines and recognized the appropriate intact EBNA polypeptide bands on immunoblots. An ELISA with the purified 28-kDa EBNA as antigen was used to quantitate anti-EBNA antibody in human serum samples. The ELISA method was approximately 100-fold more sensitive than the classical anticomplement immunofluorescence assay. Anti-EBNA antibody was detected in sera from 100% of normal individuals who were seropositive for the viral capsid antigen, and low anti-EBNA titers were detected in serum from most patients with acute infectious mononucleosis. The assay gave the expected pattern of titers in sera from patients with rheumatoid arthritis, Burkitt lymphoma, or nasopharyngeal carcinoma, thus confirming the validity of this purified reagent for assessing EBNA antibody status. Approximately 10% of normal individuals and rheumatoid arthritis patients had anti-EBNA titers as high as those seen in nasopharyngeal carcinoma patients. In these high-titer individuals, greater than 1% of the total IgG are antibodies that recognize 28-kDa EBNA, which indicates that the carboxyl-terminal domain of EBNA is highly immunogenic.

Amino Acid Sequence↗

PubMed1985

Identification of two lysosomal membrane glycoproteins.

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Animals↗

PubMed1985

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

Animals↗

PubMed1985

The human lymphocyte function-associated (HLFA) antigen and a related macrophage differentiation antigen (HMac-1): functional effects of subunit-specific monoclonal antibodies.

The structural and functional relations between the alpha- and beta-subunits of the human lymphocyte function-associated antigen (HLFA) and the human Mac-1 antigen (HMac-1) have been analyzed with the use of five monoclonal antibodies that react with these proteins. The specificities of these antibodies were examined by immunoprecipitation of proteins from 125I-labeled cells and purified HLFA and HMac-1 antigens. Three antibodies reacted with the Mr 95,000 common beta-subunit of the proteins, and also co-precipitated the Mr 175,000 HLFA alpha-subunit, the Mr 165,000 HMac-1 alpha-subunit, and a third polypeptide alpha-subunit of Mr 150,000. The other antibodies were specific to noncross-reactive epitopes present on the alpha-subunits of HLFA or HMac-1. These specificities were confirmed in sequential immunoprecipitation studies. Peptide mapping showed that the beta-subunits of HLFA and HMac-1 were identical, whereas the two alpha-subunits differed considerably. The HLFA alpha-subunit-specific monoclonal antibody inhibited phytohemagglutinin stimulation, the mixed lymphocytes reaction, cytolytic T lymphocyte-mediated killing, and tetanus toxoid stimulation, but did not affect natural killer cell-mediated killing or complement receptor type 3 function. The HMac-1 alpha-subunit-specific monoclonal antibody inhibited complement receptor type 3 function but had no effect on T cell or natural killer cell functions. Three monoclonal antibodies to the beta-subunit inhibited all functions tested, including T cell, natural killer cell, and complement receptor type 3 activities. The results suggest that the functions of the HLFA and HMac-1 molecules may be determined by the alpha-subunit, and that the common beta-subunit also bears functionally important epitopes.

Adult↗

PubMed1984

Redistribution of a major cell surface glycoprotein during cell movement.

The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not altered and the cells remained motile in the presence of the antibody. The uniform distribution of the direct immunofluorescence stain persisted for long periods (greater than 100 h), which indicates that the fluorescent monoclonal antibodies may be used to trace antigen surface distribution during cell functions. In motile cells, but not G0 or confluent cells, the degree of fluorescent staining decreased toward the leading edge; this gradient increased markedly during the time that the antibody was bound to the cells. However, the gradation was not seen with the lipid probe, dihexadecylindocarbocyanine. The antigen was "patched" only by the application of a second antibody directed to the rat monoclonal antibody and the relationships of these patches to the underlying cytoskeleton were characterized.

Animals↗

PubMed1984

Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function.

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell-cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.

Animals↗

PubMed1984

Antigenic analysis of hematopoiesis: II. Expression of human neutrophil antigens on normal and leukemic marrow cells.

The binding of five antineutrophil monoclonal antibodies AHN-1, -2, -3, -7, and -8, to normal and leukemic bone marrow cells was studied. AHN-7 bound to many granulocytic precursors, particularly myelocytes, and both lymphoid and blast cells in normal marrow, and to most but not all granulocyte-macrophage progenitors (CFC-GM). AHN-8 bound only to late (band and segmented) neutrophilic cells and not to CFC-GM. AHN-1, -2, -3 bound to morphologically identifiable neutrophil precursors, but not to (day-14) CFC-GM. Approximately half of nonlymphoid leukemia specimens were positive with AHN-1 or AHN-7; by contrast, lymphoid leukemia specimens were rarely positive. AHN-8 was rarely found on leukemic cells. These antineutrophil antibodies appear to detect distinct granulopoietic subsets and may be useful in the analysis of hematologic differentiation and in the subclassification of leukemias.

Animals↗

PubMed1983

Analysis of cell-surface protein changes accompanying differentiation of HL-60 cells.

The cell-surface proteins of HL-60 human promyelocytic leukemia cells have been compared to those of normal human neutrophils. Proteins of HL-60 cells surface labeled with 125I differed markedly from those of normal neutrophils, as shown by immunoprecipitation and polyacrylamide electrophoresis. Differentiation of HL-60 cells by treatment with dimethylformamide, trans-retinoic acid, or 12-O-tetradecanoylphorbol acetate did not modify the predominant surface-labeled proteins of HL-60 cells to produce a pattern similar to that of normal, mature neutrophils. However, the agents did induce greater quantities of minor cell-surface proteins immunoprecipitated by hyperimmune anti-human neutrophil serum. These immunoprecipitated proteins resembled several of the surface-labeled polypeptides of normal human neutrophils.

Cell Differentiation↗

PubMed1983

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells.

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.

Animals↗

PubMed1983

Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy.

Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

Animals↗

PubMed1983

Human granulocyte surface molecules identified by murine monoclonal antibodies.

We have investigated the nature of the antigens recognized by four classes of mouse anti-human monoclonal antibodies that characteristically reacted with neutrophilic granulocytes and their precursor cells, but not with monocytes or other normal hemopoietic cells. The antigenic targets of the majority (9/12) of the independently isolated monoclonal antibodies were present on two surface glycoproteins (Mr 145,000 and 105,000) and glycolipids. This antigen(s) was also detected on granulocyte precursor cells, including the bone marrow granulocyte/monocyte progenitor cells (CFU-GM). The same antigen(s) detected by these monoclonal antibodies was also present in non-hemopoietic cell lines (colon carcinoma and neuroblastoma). Three other antigens, defined by monoclonal antibodies AHN-8, L12.2, and L13.1 and present on granulocytes and their mid-late precursor cells, could not be identified as proteins but were detected in a protein-free glycolipid extract of these cells. The diversity of the antigens was confirmed by cross-competition experiments and by the identification of their different patterns of reactivity with cell lines and bone marrow cells.

Animals↗

PubMed1983

A human granulocyte-specific antigen characterized by use of monoclonal antibodies.

Hybridoma cell lines secreting monoclonal antibodies that bind to a surface antigen of human neutrophils have been prepared by fusion of mouse myeloma cells with spleen cells from mice immunized with human neutrophils. Several of the monoclonal antibodies (AHN 1-6) were specific for a neutrophil surface antigen and did not bind lymphocytes, monocytes, red blood cells, platelets, or basophils. All of the granulocyte-specific antibodies immunoprecipitated a polypeptide of 145,000 daltons and an isoelectric point of about 4.5 and other heterogeneous polypeptides of 105,000 daltons. These same components were the major lactoperoxidase-labeled proteins precipitated by hyperimmune mouse serum. The antibodies were further characterized for binding to several human myeloid leukemia cell lines and cells from patients with myeloid or lymphoid leukemia. All antibodies bound the HL-60, ML1, ML2, ML3, K562, and U937 myeloid leukemia cell lines. None of the antibodies bound the RPMI 6410 Raji, RPMI 8226, MOLT 4, or Daudi lymphoid cell lines. All of the hybridoma cell lines (AHN 1-6) produced IgM antibodies that were cytotoxic.

Antibodies, Monoclonal↗

PubMed1982

Murine cell surface glycoproteins. Identification, purification, and characterization of a major glycosylated component of 110,000 daltons by use of a monoclonal antibody.

A 110,000-dalton plasma membrane glycoprotein of mouse cells has been identified, purified, and characterized by use of a xenogeneic monoclonal antibody. The glycoprotein was a major component of the NIH/3T3 cell surface. It contained 15% of [3H]glucosamine incorporated into cell proteins and was composed of at least 16 isomorphic variants with apparent molecular weights from 100,000 to 120,000 and isoelectric points between 6.5 and 8.1. The glycoprotein constituted 0.1% of total cell protein, as measured by the yield of purified protein, and there were over 10(6) antibody-binding sites/cell. Immunoprecipitation from pulse-chase labeled cells showed that the 110,000-dalton glycoprotein was initially synthesized as a 92,000-dalton microsomal polypeptide which gradually was converted to the mature surface form. The turnover time of the surface form was at least 20 h. The antigenic determinant recognized by the monoclonal antibody was species-specific and nonpolymorphic. It was present in high concentration on most dividing murine cells in culture, whereas the concentration in normal mouse tissues differed widely. Among lymphoid tissues, antigen concentration was enriched in bone marrow as compared to spleen and thymus. Among nonlymphoid tissues, antigen concentration was 10-fold greater in kidney than in brain, liver, and skeletal muscle. The glycoprotein was purified without loss of antigenic activity by antibody affinity chromatography. The single step procedure yielded 3 mg of pure glycoprotein from 3 g of crude cell extract. The purification and characterization of this major membrane protein provide a basis for further study of its cell surface structure and function.

Animals↗

PubMed1982

Identification of mammalian sperm surface antigens: II. Characterization of an acrosomal cap protein and a tail protein using monoclonal anti-mouse sperm antibodies.

Monoclonal anti-mouse sperm antibodies have been produced by fusing mouse myeloma cells with spleen cells from rats immunized with epididymal sperm of C3H mice, Immunoprecipitation and immunoperoxidase techniques showed that one such monoclonal antibody, AMS IV-33, recognized a 200 000 dalton protein localized on the acrosomal cap of the sperm cell. Two other monoclonal antibodies AMS IV-54 and -76, reacted with a 68 000 dalton component on the surface of the sperm tail. Both antigenic targets were species specific and were present in about equal amounts on sperm from several different strains of mice. The tail protein was sperm specific, whereas the antibody reacting with the acrosomal cap protein also appeared to react somewhat with antigens present in other mouse tissues.

Acrosome↗