Armin Maidhof

EXTRACELLULAR DOMAINS OF THE BRADYKININ B2 RECEPTOR INVOLVED IN LIGAND BINDING AND AGONIST SENSING DEFINED BY ANTI-PEPTIDE ANTIBODIES

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) efficiently interfered, in a concentration-dependent manner, with agonist and antagonist binding and vice versa. Antibodies to extracellular domain 4 (third loop) blocked binding of the agonist but not of the antagonist, whereas antibodies to extracellular domains 1 and 2 or to intracellular domains failed to block ligand binding. Antibodies to ectodomain 3 competed with agonistic anti-idiotypic antibodies for B2 receptor binding. Further, affinity-purified antibodies to the amino-terminal portion of extracellular domain 3 transiently increased intracellular free Ca concentration and thus are agonists. The Ca signal was specifically blocked by the B2 antagonist HOE140. By contrast, antibodies to the carboxyl-terminal segment of extracellular domain 4 failed to trigger Ca release. The specific effects of antibodies to the amino-terminal portion of extracellular domain 3 suggest that this portion of the B2 receptor may be involved in ligand binding and in agonist function.

Action of 1-β-d-Arabinofuranosylcytosine on mammalian tumor cells—1.

Abstract Synchronized mouse lymphoma cells were treated with ara-C- 3 H at the beginning of the S-phase with twice the ED 50 concentration. In DNA isolated from these cells ara-C could be traced. It could be shown that the radioactivity in nucleotide-and nucleoside fractions obtained after enzymic digestion of ara-C-DNA was due for more than 87% to ara-C- 3 H. The radioactivity of ara-C- 3 H can be almost quantitatively attributed to cytosine and arabinose. An incorporation of ara-C into RNA could not be detected.

Inhibition of intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex from human breast cancer cells by the marine sponge metabolite (+)-aeroplysinin-1

1. (+)-Aeroplysinin-1, a naturally occurring tyrosine metabolite from the marine sponge Verongia aerophoba, was found to inhibit the phosphorylation of lipocortin-like proteins by a highly purified preparation of the epidermal growth factor (EGF) receptor-tyrosine protein kinase complex from MCF-7 breast carcinoma cells. 2. (+)-Aeroplysinin-1 blocked the EGF-dependent proliferation of both MCF-7 and ZR-75-1 human breast cancer cells and inhibited the ligand-induced endocytosis of the EGF receptor in vitro. 3. Treatment with aeroplysinin-1 in the concentration range at 0.25-0.5 microM resulted in a time- and dose-dependent total tumor cell death in vitro. 4. At a 10-fold higher concentration the compound did not reveal any cytostatic activity in normal human fibroblasts. 5. From these data we conclude that (+)-aeroplysinin-1 represents a compound which displays a strong anti-tumor effect on EGF-dependent tumor cell lines.

Probing human β1- and β2-adrenoceptors with domain-specific fusion protein antibodies

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta1- or beta2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta1- or beta2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one ([3H]CGP 12 177), indicating a specific interaction with the native receptor. In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of [3H]CGP 12 177. Affinity purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy. Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying beta-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

Polyclonal antibodies to mannan from yeast also recognize the carbohydrate structure of gp120 of the AIDS virus: an approach to raise neutralizing antibodies to HIV-1 infection in vitro.

This study initiates a new method of developing an antigen which might be useful in the prevention of HIV-1 infection. Using a mannan preparation from Saccharomyces cerevisiae neutralizing antiserum was raised in rabbits which prevents HIV-1 infection in vitro up to a titre of 1:128. The corresponding antibody preparation neutralized the in vitro infectivity down to a concentration of 5 micrograms/ml. Analytical studies suggest that the antibodies are directed against the mannose residues of the HIV-1 glycoprotein (gp) 120 and its precursor gp 160.

Influence of the antileukemic and anti-human immunodeficiency virus agent avarol on selected immune responses in vitro and in vivo

The effect of the antileukemic and anti-HIV agent avarol on the lymphoid system was studied both in vitro and in vivo. Radioactively labelled avarol ([3H]-dihydroavarol) was found to accumulate in vitro in the cytoplasmic compartment primarily of T-lymphocytes and not of B-lymphocytes. Avarol increased significantly the IgG and IgM production by cultures of human lymphoid cells (unseparated) in vitro and slightly the number of plaque forming cells in vivo in spleen of mice. Moreover, a pretreatment of mice with avarol resulted in a higher [3H]-dThd incorporation rate in both macrophage-containing and macrophage-depleted lymphocyte cultures in vitro. The stimulatory influence of avarol on humoral immune responses is not accompanied by a change of the antibody-mediated hypersensitivity reaction, as measured by the Arthus reaction. No significant influence of avarol on the cellular immune system in vivo (rats or mice) was found, as taken from studies on delayed-type hypersensitivity reactions to sheep red blood cells and to oxazolone. The in vitro and animal data indicate that avarol combines useful properties (anti-HIV efficiency in vitro and augmentation of humoral immune responses) to consider it as a potential anti-AIDS agent.

Histoincompatibility reactions in the hydrocoral Millepora dichotoma

The xenogeneic- and allogeneic immunological specificity of the hydrocoral Millepora dichotoma has been investigated. Xenogeneic histoicompatibility reactions have been observed between this hydrocoral and a series of species belonging to the Demospongiae and to the Anthozoa (both Hexacorallia and Octocorallia). The xenogeneic histoincompatibility reactions proceed in the following sequence: (a) Species-unspecific sensitization; (b) necrosis formation, which is very likely due to an autolytic process; (c) callus formation, due to an hyperplastic growth of stolons; and (d) formation of a contact barrier in form of a barrier layer or a restored stolonial layer. Allogeneic histoincompatibility reactions are restricted to those regions of the coral which are interspersed with polyps; allogenic fusion is observed between branches, with a high density of polyps and the “foot”-region, which is characterized by a low polyp density.

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: A target for potential therapeutic agents

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.

Distamycin: An inhibitor of DNA-dependent nucleic acid synthesis

Summary Distamycin A (DST-A) strongly inhibits cell proliferation of mouse lymphoma cells. The unbalanced growth of the cells observed during the DST-A caused inhibition cannot be linked to a cytotoxic effect. The DST-A effect on cell proliferation may be quenched by incubation with native DNA, but not with RNA. The two DST derivatives DST/4 and DST/5 show a high degree of inhibition of cell proliferation. DST-A inhibits the 4 different DNA-dependent DNA polymerases as well as the 3 DNA-dependent RNA polymerases to the same high extent, while inhibition of the enzymatic RNA-mediated DNA and RNA synthesis amounts to only 1.5% of it. Kinetic studies have shown that enzymatic DNA-dependent nucleic acid synthesis is inhibited in a competitive way.

Potentiation of the effectiveness of bleomycin by A·T-specific DNA ligands in vitro as well as in vivo

Summary The anti-tumor drug bleomycin reduces the activity of DNA-dependent DNA polymerase in isolated enzyme system as well as the cell proliferation by fragmentation of the DNA. These two test systems were used to determine the effect of the A·T-specific DNA ligands, steffimycin B and Hoechst 33258, on the activity of bleomycin. It has been found that both the inhibitory effect of bleomycin on the DNA-dependent DNA polymerase and the one on cell proliferation are potentiated when bleomycin is given in combination with steffimycin B or with Hoechst 33258.