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H Lemke

Publications and source records attributed to H Lemke.

At least 19 recordsLinked to original sources
PubMed1992

Cellular localizations and processing of the two molecular forms of the Hodgkin-associated Ki-1 (CD30) antigen. The protein kinase Ki-1/57 occurs in the nucleus.

The Ki-1 antibody not only detects a Hodgkin-associated membrane molecule of 120 kd (Ki-1/120 = CD30), but also reacts with an independently synthesized molecule of 57 kd (Ki-1/57) that only occurs intracellularly. Hodgkin's disease-derived cell lines L428 and L540 contain both Ki-1-reactive antigens, whereas others, e.g., U266/Bl myeloma cells, only express the intracellular Ki-1/57. The present immunoelectronmicroscopic analysis detected the Ki-1/57 antigen of U266/Bl cells not only in the cytoplasm, but also in association with the nuclear envelope, chromatin structures, and nucleoli. This Ki-1/57-specific type of labeling also was observed in L428 and L540 cells that, in contrast to U266/Bl cells, showed an additional staining of cell membranes and cytoplasmic vesicles. These results were confirmed by two independent methods: 1) cytocentrifuge preparations of isolated nuclei of L540 cells showed a spotted Ki-1-specific labeling, 2) immunoprecipitations demonstrated that the Ki-1/57, but not the Ki-1/120 antigen, was transferred into the nuclei of L540 and U266/Bl cells, whereas the Ki-1/120 antigen with its 90-kd precursor remained in the non-nuclei fraction of L540 cells.

Antigens, CD↗
PubMed1990

Protein kinase activity of the intracellular but not of the membrane-associated form of the Ki-1 antigen (CD30).

The Hodgkin-associated Ki-1 antigen (CD30) consists of a 120-kDa membrane-associated glycosylated phosphoprotein (Ki-1/120) and a 57-kDa non-glycosylated phosphoprotein (Ki-1/57) which only occurs intracellularly. Both molecules are phosphorylated at serine residues. An analysis of the peptide fragments resulting from staphylococcal V8-protease digestion of the Ki-1/57 molecule revealed identical bands irrespective of the cell source. Only a few bands of the Ki-1/57 digests appeared among the peptide fragments of the Ki-1/120 membrane antigen. The protein kinase activity was tested for both forms of the Ki-1 antigen. The Ki-1/120, devoid of the Ki-1/57 molecule, was immunoprecipitated from cell lysates of Hodgkin-analogous cell lines L428 or L540, which had been loaded with the Ki-1 or the Ki-1-analogous antibodies Ber-H2, HSR-1, -2 and -3 (method 1). These other antibodies reacted with the Ki-1/120, but not with the Ki-1/57 antigen. The latter, devoid of the Ki-1/120, was isolated from L540 cells after removal of the membrane form by method 1 or from U266/Bl myeloma or Raji Burkitt lymphoma cells which only contain the smaller form. Effects of non-specific adsorption were eliminated by various control precipitates. The Ki-1/57 intracellular antigen showed autophosphorylation and could phosphorylate histones as well. In contrast, a protein kinase activity of the membrane-associated Ki-1/120 could not be demonstrated.

Amino Acids↗
PubMed1989

Determination of cell-bound Fc receptors using a cell adherence test (CAT).

The determination of membrane-bound Fc receptors as rosette forming cells with antibody-coated erythrocytes (EA-RFC) is a tedious and time consuming method. Here, we describe a modification of the assay delineated by Eisenbarth et al. (J. Immunol. Methods (1980) 39, 387) for the detection of Fc receptors on cell surfaces. V-shaped microtiter plates were coated with an antigen (e.g., fluorescein isothiocyanate (FITC) coupled to bovine serum albumin (BSA] and subsequently with monoclonal anti-FITC antibodies of different isotypes. Cells to be tested were added, incubated for a short time or centrifuged immediately. Cells expressing FcR, became bound to the flanks of the V-shaped wells, whereas cells lacking the receptor were pelleted to the tip of the well. This cell adherence test (CAT) is independent of the agglutinating abilities of the antibodies, and since it is very fast and highly reproducible, large numbers of different cells can be screened for FcR expression. Anti-receptor antibodies can also be tested for their binding to FcR.

Animals↗
PubMed1989

The Hodgkin-associated Ki-1 antigen exists in an intracellular and a membrane-bound form.

The Hodgkin-associated Ki-1 antigen occurs in two different molecular forms. The 120-kDa membrane-associated form is a phosphorylated glycoprotein, which is derived from a non-phosphorylated intracellular 84-kDa apoprotein that is co-translationally N-glycosylated with a carbohydrate portion of 6 kDa. The other form of the Ki-1 antigen is a non-glycosylated phosphoprotein of 57 kDa which only occurs intracellularly. Both forms of the antigen are phosphorylated at serine residues. Enzymatic cleavage with sialidase reduced the 120-kDa membrane antigen by about 15 kDa, while its 90-kDa precursor and the 57-kDa intracellular form of the Ki-1 antigen remained unaltered. Pulse-chase experiments revealed that the 57-kDa and 90/120-kDa molecules are synthesized independently of each other. Four to eight hours after synthesis, the degradation of the 120-kDa molecule to a 105-kDa membrane-associated intermediate begins. This is further processed and appears in the cell supernate as a 90-kDa molecule. Hodgkin's disease-derived, Epstein-Barr virus-transformed cell lines and the acute T cell leukemia line MOLT-4 contain both forms of the Ki-1 antigen, whereas only the 57-kDa intracellular antigen is expressed in U266/B1 myeloma cells, in the Burkitt lymphoma cell lines Raji and Daudi and in acute promyelocytic HL-60 leukemia cells.

Amino Acids↗
PubMed1987

Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines.

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.

Antigens, Neoplasm↗
PubMed1979

Studies on the quantification of local tissue injury following intramuscular injection of aqueous solutions. VIII. The local lesion after application of turimycin in different preparations.

The increase in drug concentration in aqueous solutions is not responded to by a comparable enlargement of volume of necrosis after intramuscular injection. After application of TURIMYCIN the lesion is more voluminous than those caused by penicillin, lower than those following OTESOLUT and nearly equal to those caused by 5% WOFAPYRIN (phenylbutazone + aminophenazone 1:1).

Animals↗
PubMed1977

The role of fetal calf serum in the primary immune response in vitro.

The mode of action of 2-mercaptoethanol (2-ME) on the primary immune response in vitro was investigated. Fetal calf serum (FCS) was preincubated with 2-ME and lyophilized to remove free 2-ME. This 2-ME-treated FCS was able to substitute the function of adherent cells in the primary immune response against sheep red blood cells (SRBC) in vitro; Fractionation of 2-tme-treated FCS on a Sephadex G-100 column showed that 2-ME acted on a high molecular serum component which after activation, could substitute for macrophages. In order to obtain a humoral immune response against SRBC in vitro, spleen cells require selected FCS. These "good" sera could be distinguished from "deficient" sera by their higher content of this 2-ME-activated factor. The height of the in vitro immune response to SRBC was dependent on the amount of activated factor added to the culture medium. FCS normally required in the culture medium could be completely replaced by the factor-containing fraction without deleterious effect on the culture medium. The factor should be added to the spleen cells during the first 24 h of culture and remain there for 72 h in order to obtain an optimal immune response. The factor could be partially absorbed by spleen cells but not by SRBC. The relationship between macrophage, 2-ME, and FCS in eliciting an in vitro primary immune response is discussed.

Antibody Formation↗
PubMed1977

Growth regulation of a murine lymphoma cell line by a 2-mercaptoethanol or macrophage-activated serum factor.

A mouse lymphoma cell line has been established that is dependent for growth on the presence of an activated serum component. This growth factor is found in an inactive form in fetal bovine serum (FBS) and can be activated by 2-mercaptoethanol and by macrophages. A factor-containing fraction can be separated from FBS by Sephadex G-100 chromatography and once activated, can be used as a substitute for whole serum in the culture medium without adverse effect on the cell growth. The significance of these findings with respect to the study of the immune response and cancer research is discussed.

Animals↗
PubMed1977

Humoral primary immune response in vitro in a homologous mouse system: replacement of fetal calf serum by a 2-mercaptoethanol or macrophage-activated fraction of mouse serum.

The primary immune response in mouse spleen cell cultures against heterologous red cell antigens is dependent on the medium being supplemented with selected batches of fetal calf serum. Mouse serum itself is not able to support this response. The active immune response-supporting component in fetal calf serum seems to be a distinct factor (s), which has been partially purified by Sephadex G-100 filtration and termed MaSF-2-mercaptoethanol-activated serum factor. In this report it is demonstrated that MaSF is also present in mouse serum. For functional detection, mouse MaSF has to be separated from higher m.w. inhibitors, and has to be activated by 2-ME. After separation and activation mouse MaSF can support the primary immune response in a completely homologous in vitro culture system. Evidence is presented that MaSF can also be activated by macrophages. It is concluded that macrophages and 2-ME have the same mode of action in the primary immune response in vitro, i.e., induction of lymphocyte competence by activation of a serum factor.

Animals↗
PubMed1976

Polyclonal stimulation of lymphocytes by macrophages.

To analyze the interaction between macrophages and splenic lymphocytes with reference to time and concentration, the Mishell-Dutton system was divided into two experimental steps. Step 1 consisted of the cocultivation of spleen cells with various doses of macrophages for different periods of time, while in step 2 macrophages were removed, spleen cells transferred to fresh petri dishes and cultivated until plaque assay. Cocultivation of spleen cells with high doses of macrophages for 4--8 h markedly enhanced the DNA synthesis and plaque-forming cell (PFC) response of sheep red blood cell-stimulated and unstimulated cultures. A cocultivation longer than 24 h resulted in an inhibition of both DNA synthesis and PFC response of spleen cells. These studies suggest a nonspecific function of macrophages on proliferation and differentiation processes in antibody formation.

Animals↗
PubMed1976

The role of adherent cells in the immune response. Fibroblasts and products released by fibroblasts and peritoneal cells can substitute for adherent cells.

The primary immune response to sheep erythrocytes in adherent cell-depleted cultures was restored by adding a critical number of peritoneal cells. Complete substitution was achieved also with supernatants from allogeneic and syngeneic peritoneal cells. Both living fibroblasts and supernatants from fibroblast cultures were found to be highly efficient substitutes for adherent cells in both syngeneic and allogeneic systems. Supernatants from non-antigen-reated peritoneal cells and fibroblasts caused increased DNA synthesis and induction of polyclonal antibody synthesis in normal spleen cells. Thus, adherent cells need not function in the immune response by presenting antigen to the B cells via 'IgT' or by releasing signal-2 activity, which acts on lymphocytes that have already received signal 1.

Animals↗