Probing human beta1- and beta2 -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 beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonis…

Development of a Peptide-Based Sandwich Elisa for Human Tissue Prokallikrein with No Cross-Reactivity from Mature Kallikrein

Human tissue prokallikrein is the enzymatically inactive zymogen of a serine proteinase involved in the liberation of vasoactive kinin peptides, and it is supposed that an impaired prokallikrein-to-kallikrein conversion is closely related to certain hypertensive and inflammatory disorders. Progress in understanding the biological role of the proenzyme has been limited by the absence of an accurate assay for the kallikrein precursor. We describe a sandwich enzyme-linked immunosorbent assay to measure human tissue prokallikrein using monospecific anti-peptide antibodies raised against propeptide derivatives. This method could detect a minimum concentration of 60 pg/ml prokallikrein and displa…

Synergistic effect of peplomycin in combination with bleomycin on L5178y mouse lymphoma cells in vivo.

Studying the treatment of NMRI mice with ip injections of bleomycin (BLM) for 5 days we found an approximate LD50 of 35 mg/kg; the toxicity of peplomycin (PEP) was slightly higher (LD50: approximately 25 mg/kg). The effect of the two drugs on growth of L5178y mouse lymphoma cells in NMRI mice was examined. BLM alone caused at a concentration of 2.5 mg/kg an almost complete inhibition of tumor cell growth; the same effect was determined with 1 mg PEP/kg. At these concentrations the drugs caused an increase of the survival time of 110% (BLM) or 104% (PEP). Given in combination, one-sixth of the optimal doses yielded an 100% increase of the median survival time. These results indicate a signif…

Clavines as antitumor agents. 3: Cytostatic activity and structure/activity relationships of 1-alkyl agroclavines and 6-alkyl 6-noragroclavines.

The cytostatic potential of twenty antibiotic agroclavines has been examined in the L5178y mouse lymphoma cell system. Twelve of these compounds are described for the first time. It is shown that the substituent at N-1 of agroclavine is very important whereas the substituent at N-6 is of less influence if it is not hydrogen. Incorporation studies in the presence of 1-propylagroclavine suggest that DNA synthesis in the lymphoma cells is inhibited. The effect on the corresponding [3H]thymidine incorporation in murine spleen lymphocytes is comparably low. Neither a significant change of mRNA efflux nor of DNA polymerase alpha and beta activities was caused. The mechanism of action seems to be …

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…

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 …

Biological Activity of the phenylethanol and its derivatives. Influence on isolated DNA nucleotidyltransferase and DNAase.

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…

Cytostatic Activity of Aeroplysinin-1 against Lymphoma and Epithelioma Cells

(±)-Aeroplysinin-1, an optically active 1.2-dihydroarene-1.2-diol. was isolated from the marine sponges Verongia aerophoba (+-isomer) and lanthella ardis (--isomer). For the experiments presented we used the +-isomer from Verongia aerophoba. Here we describe the hitherto unknown biological and pharmacological property of this compound to display pronounced anticancer activity against L5178y mouse lymphoma cells (ED50: 0.5 μm). Friend erythroleukemia cells (ED50: 0.7μm) , human mamma carcinoma cells (ED50: 0.3μm) and human colon carcinoma cells (ED50: 3.0 μm) in vitro. Furthermore, aeroplysinin caused a preferential inhibition of [3H]thymidine (dThd) incorporation rates in L5178y mouse lymph…

Structural features of the human bradykinin B2 receptor probed by agonists, antagonists, and anti-idiotypic antibodies

The human bradykinin B2 receptor belongs to the family of G-protein-coupled receptors. To characterize the receptor protein, we have solubilized the membranes of cultured human foreskin fibroblasts bearing the B2 receptor. Affinity cross-linking of the solubilized receptor with the labeled agonist, 125I-Tyr0-bradykinin, or the labeled antagonist, 125I-(4-hydroxy-phenyl-propionyl)-HOE140, revealed major bands of apparent molecular mass of 69 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and of 59 kDa under non-reducing conditions. A 1000-fold molar excess of each of the unlabeled ligands quenched the specific labeling suggesting that the agonist and the antagonist …