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B B Knowles

Publications and source records attributed to B B Knowles.

At least 109 records · Page 6Linked to original sources
PubMed1982

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells.

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.

Animals↗
PubMed1982

Identity of human epidermal growth factor (EGF) receptor with glycoprotein SA-7: evidence for differential phosphorylation of the two components of the EGF receptor from A431 cells.

A 165-kilodalton (kDal) surface glycoprotein encoded by human chromosome 7 (SA-7) had been characterized by using antisera raised against human chromosome 7-containing somatic cell hybrids. We now present evidence that SA-7 is the human receptor for epidermal growth factor (EGF) and that these antisera recognize human-specific determinants. The gene coding for the human EGF receptor is localized to the p12 to p22 region of chromosome 7. We have characterized the 145-kDal/165-kDal EGF receptor doublet of A431 cells after immunoprecipitation of radiolabeled cell extracts with these antisera. We find that a protein with endogenous kinase activity copurifies with the A431 receptor doublet and that both components of the doublet contain phosphotyrosine and phosphothreonine and the 165-kDal component contains phosphoserine as well. Further, although each component of the receptor doublet has an average pI of 7, both display charge heterogeneity and appear to have unique charge isomers. The relationship between the two components of the A431 EGF receptor is discussed.

Cell Line↗
PubMed1982

Synthesis and secretion of alpha 2-plasmin inhibitor by established human liver cell lines.

The site of synthesis of alpha 2-plasmin inhibitor (alpha 2-PI), a physiologic inhibitor of plasmin, is not known with certainty. We have studied the production and secretion of alpha 2-PI by three established human liver cell lines derived from hepatocellular carcinoma and hepatoblastoma (Hep G2, Hep 3B, and PLC/PRF/5). As measured by a specific radioimmunoassay, the titer of alpha 2-PI increased in the medium of Hep G2 and Hep 3B cells with time, but no significant amount of alpha 2-PI was found in the medium of PLC/PRF/5. There was no evidence for a significant intracellular pool of this protein. On immunodiffusion against anti-alpha 2-PI serum, alpha 2-PI secreted by Hep G2 (G2 alpha 2-PI) formed a simple precipitin line of complete identity with the alpha 2-PI present in plasma (plasma alpha 2-PI). G2 alpha 2-PI behaved similarly to plasma alpha 2-PI in Sephadex G-150 gel filtration, sucrose density-gradient centrifugation, and crossed immunoelectrophoresis. G2 alpha 2-PI inhibited plasmin activity instantaneously in a functional assay and formed a complex with plasmin demonstrable by crossed immunoelectrophoresis. De novo synthesis of alpha 2-PI was shown by the presence of specific immunoprecipitable radioactivity in the medium after 5 hr of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by NaDodSO4/polyacrylamide gel electrophoresis showed a single peak of radioactivity corresponding to Mr 68,000. These results indicate that the liver is a site of alpha 2-PI production.

Carcinoma, Hepatocellular↗
PubMed1982

Benzo[a]pyrene--DNA adduct formation in target cells in a cell-mediated mutation assay.

In order to analyze the adducts formed in V79 Chinese hamster target cells in a Syrian hamster embryo (HE) cell-mediated mutation assay, a procedure was developed in which the HE cells are differentially killed when the mixed cell suspension is treated with antiserum to Syrian HE cells and complement. The V79 cell suspension, separated from lysed HE cells by gradient centrifugation, is greater than 90% HE cell-free. When the carcinogen--DNA interaction products formed in these two cell types were analyzed after exposure to [3H]benzo[a]pyrene (B[a]P) for 24 h under the conditions of a cell-mediated mutation assay, the major B[a]P--DNA adducts in both cell types resulted from reaction of the anti and syn isomers of B[a]P-7,8-diol-9,10-epoxide with DNA. The amount of B[a]P bound/mg DNA in the target cells was only 30% less than in the activator cells, and the relative proportions of the adducts of the syn and anti isomers were similar in the two cell types. The target cells, which do not metabolize B[a]P, were also unable to metabolically activate B[a]P-7,8-diol to DNA-binding metabolites. Thus, the transfer of activated B[a]P metabolites from activator cells to the DNA of target cells is a relatively efficient process that appears to be independent of the relative reactivity of specific metabolites with cellular nucleophiles. The immunological cell separation procedure we describe can be adapted to the analysis of carcinogen--cell interaction products formed in cell-mediated assays in which other types of activator and target cells are used to measure either mutation or another biological endpoint.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Complement biosynthesis by the human hepatoma-derived cell line HepG2.

The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.

Carcinoma, Hepatocellular↗
PubMed1982

Immunohistochemical localization of murine stage-specific embryonic antigens in human testicular germ cell tumors.

Monoclonal antibodies raised against and/or recognizing stage-specific antigens on preimplantation mouse embryos and stem cells of murine teratocarcinoma were used to localize these antigens immunohistochemically on human testicular germ cell tumors. SSEA-1, the antigen found on mouse embryonal carcinoma (EC) cells and embryonic cells from the 8-cell stage embryo onward, including the fetal primordial germ cells, was detected on yolk sac carcinoma components of human tumors, but not on EC cells. SSEA-3, the antigen found on follicular ova, fertilized eggs, early cleavage stage embryonic cells, and visceral endodermal cells of the mouse embryo, but not on mouse EC cells, was detected on human EC cells. Both antigens were found on the cell surface of fetal testicular germ cells but not in the seminiferous tubules of adult human testes. These data point out differences between human and murine EC cells suggesting that human EC cells correspond developmentally to a less mature embryonic cell than the murine EC cells. The possible histogenesis of human germ cell tumors from primordial and/or fetal germ cells is briefly discussed.

Animals↗
PubMed1982

H-2Kb mutations limit the CTL response to SV40 TASA.

The cytotoxic T lymphocyte (CTL) responses directed towards SV40 tumor-associated specific antigen (TASA) in nine strains of spontaneously arising Kb mutant mice were analyzed. All nine mutants generated normal levels of H-2Db-restricted response, but the K-end-restricted CTL response varied. B6.C-H-2bm1 (bm1) did not produce K-end-restricted SV40 TASA-specific CTL upon immunization, and SV40-transformed bm1 cells were not lysed by intra-H-2 recombinant Kb [B10.A(5R)] CTL. Nonreciprocal cross-reactive lysis was seen between B6-H-2bm8 (bm8) and B10.A(5R). Strain B6-H-2bm8 mice produce highly specific Kbm8-restricted CTL that lyse SV40-transformed bm8 cells (Kbm8SV) but not B10.A(5R) target cells (K5RSV), although Kbm8SV targets can be partially lysed by B10.A(5R) CTL. The other seven Kb mutants cross-react with B10.A(5R). These experiments definitively show that genes mapping to the K and/or D region directly control the H-2-restricted CTL response to SV40 TASA.

Animals↗
PubMed1981

Characterization of the major apolipoproteins secreted by two human hepatoma cell lines.

Two newly described human hepatoma derived cell lines, Hep G2 and Hep 3B [Knowles, B. B., Howe, C. C., & Aden, D. P. (1980) Science (Washington, D.C.) 209, 497-499], synthesize and secrete into the culture medium most of the major plasma apoproteins (apoA-I, apoA-II, apoB, apoC-II, apoC-III, and apoE). The synthesized apoproteins were identified by direct two-dimensional gel analysis of the culture medium or by two-dimensional analysis following purification of the apoproteins by ultracentrifugation or immunoprecipitation. We found that the apoA-I synthesized by both of the hepatoma cell lines consists of two isoproteins designated 2 and 3 which are more basic than the major plasma apoA-I isoproteins designated 4 and 5. The apoE synthesized by both cell lines is composed mainly of an array of isoproteins with increasingly higher molecular weights and lower isoelectric points as compared to those of the major apoE isoproteins found in plasma. These precursors of apoE are converted to the major apoE isoproteins upon treatment with Clostridium perfringens neuraminidase and represent sialo apoE isoproteins. ApoA-II, apoC-II, apoC-III-1, and apoC-III-2 correspond to the protein forms present in plasma. The human hepatoma cell lines (Hep G2 and Hep 3B) provide a unique model for studies of the regulation of human apoprotein and lipoprotein synthesis and catabolism.

Apolipoproteins↗
PubMed1981

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line.

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.

Asialoglycoprotein Receptor↗
PubMed1981

A human cell-surface antigen defined by a monoclonal antibody and controlled by a gene on chromosome 12.

A murine monoclonal antibody 602-29, subclass IgG1, that recognizes an antigenic determinant expressed by most human cells is described. Immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis analysis indicate that the antigenic determinant is carried by a protein with an apparent molecular weight of 21,000. The antigen is expressed by human-mouse somatic cell hybrids, and analysis of segregants that have lost human chromosomes indicates that the gene controlling expression of the 602-29 antigen is on chromosome 12.

Animals↗