Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin-1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein.

We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.

Inhibition of corticotropin releasing factor (CRF)-induced adrenocorticotropin (ACTH) secretion in pituitary corticotropic cells by substance P.

Substance P (SP) is one of the three distinct peptides of tachykinin system which possess a common spectrum of biological activities including a modulation of stress. It is assumed that the anterior pituitary is one possible target of SP in attenuation the stress response. Therefore the interaction between the hypothalamic stress hormone corticotropin releasing factor (CRF) and SP was investigated in AtT20/D16v-cells, a cellular model derived from a pituitary tumor. CRF stimulates the release of ACTH from AtT20/D16v cells in a concentration dependent manner. SP (1 microM) was able to abolish the CRF (100 nM)-induced ACTH release. In the same way SP inhibited the CRF-induced accumulation of cyclic adenosine monophosphate (cAMP), indicating that SP influenced the signal transduction pathway of CRF receptor activation. Thus, a direct inhibition of the CRF-mediated stress response by SP at the level of anterior pituitary seems to be likely.

Effects of long term treatment with corticotropin releasing factor on corticotropic tumor cells in vitro.

hCRF inhibits proliferation of corticotropic tumor cells cultivated in serum-reduced medium via interaction with CRF-receptors. This effect was attenuated by the specific antagonist hCRF (9-41), but not by a variety of substances which are inhibitors of cAMP production or cAMP-dependent kinases, suggesting that the effect was not mediated via cAMP. The growth inhibiting effect of hCRH was developed after 4 h incubation, a longer hCRF treatment did not change the effect observed after 4 h. Simultaneously, after hCRF treatment for 4 days the cells were insensitive for ACTH release by hCRF stimulation despite of an increase in the number of secretory granules. The results show that the inhibition of proliferation of pituitary tumor cells by hCRF seems to be a rapid receptor-mediated process connected with morphological changes, but not mediated via activation of adenylate cyclase.

Expression and characterization of the substance P (NK1) receptor in the rat pituitary and AtT20 mouse pituitary tumor cells.

Although substance P is known to take part in the regulation of the anterior pituitary, no conclusive evidence for the expression of the tachykinin NK1 receptor has been found yet in the pituitary or pituitary derived cells. With the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the low abundant transcripts of the NK1 receptor in the rat pituitary and in the AtT20 cell line (clone D16v). Furthermore, the functional expression of the NK1 receptor in AtT20 cells was confirmed by activation of the phosphatidylinositol-calcium second messenger system when the cells were treated with substance P. In addition, binding studies also indicated the functional expression of this receptor in AtT20 cells. Thus we provide the first evidence that the NK1 receptor is expressed in AtT20 cells and the rat pituitary.

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells.

Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le(y), Le(x), sialosyl-Le(a), precursor of Thomsen Friedenreich antigen (Tn), but not Thomsen-Friedenreich antigen and sialosyl-Tn]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2+ cells develop from the basal cell layer of the mammary gland.

Patterns of expression of tumor markers on non-transformed human mammary epithelial cells cultured in vitro.

We have examined the expression of 7 well defined tumor markers/tumor associated antigens (H type 2, X, Y, sialyl-Lea, CEA, MAM-6, and Tn) and a tumor associated antigen defined by a new own monoclonal antibody on non-transformed human epithelial cell lines derived from reduction mammoplasties by means of immunocytochemistry with monoclonal antibodies. Two cell types are discernible: slowly or non-proliferating, lumenal derived (I), and proliferating, stem cell-like, basal cell-derived cells (II). Five out of the 8 tumor markers were expressed on type I cells, and all 8 on type II cells. The number of positive cells varied considerably from a few to 100 per cent depending on the individual markers. The observed patterns proved to be characteristic and reproducible; they appear to reflect the developmental stage of the cells cultured in vitro rather than a direct influence of the culture conditions.

Proliferation of cultivated cells from normal, preneoplastic and neoplastic human breast tissue--preliminary report.

The success of cultivation of epithelial cells from normal, neoplastic and preneoplastic tissues is reviewed. MCDB 170 medium with special supplements is useful for epithelial cell culture. The epithelial character was demonstrated by the reaction with 3 different monoclonal antibodies. Most cell populations from preneoplastic and neoplastic tissues were identified as fibroblasts. Cell proliferation as measured by cell doubling time, labeling index, flow cytometry did not differ between the tissue types examined. Only 3H-thymidine uptake showed an increased tendency in cells from preneoplastic and neoplastic tissues. The use of normal human breast epithelial cells for molecular biological studies is recommended.

Direct comparison of electric field-mediated and PEG-mediated cell fusion for the generation of antibody producing hybridomas.

As an alternative to polyethylene glycol (PEG), electric field pulses offer, in theory, fusion conditions whose parameters are better controllable. In 1985 (1) we reported on the successful generation of hybridoma clones by means of electrofusion performed in a batch-type manner similar to that usually employed with PEG, and applicable to any type of antigens. Here we summarize the results of a series of fusions performed since then in which both electric field and PEG induced fusion were directly compared. Different types of antigens were used. Electrofusion resulted in a 3.8 to 33.0 times higher yield of hybridomas per unit number of spleen cells. Moreover, hybridomas grew more vigorously after fusion and, therefore, were earlier visible. Other parameters examined revealed no differences between hybridomas generated by either method.

A new monoclonal antibody (A46-B/B10) highly specific for the blood group H type 2 epitope: generation, epitope analysis, serological and histological evaluation.

A monoclonal antibody recognizing the blood group H type 2 antigen has been obtained from a BALB/c mouse immunized with MCF-7 (human mammary carcinoma) cells. The specificity of this antibody (A46-B/B10, IgM, kappa) has been identified by haemagglutination tests, immunohistochemistry, binding inhibition studies, and absorption experiments performed with synthetic oligosaccharides. The antibody is virtually nonreactive with H type 1 antigen or with closely related type 2 structures (e.g., Y antigen). A46-B/B10 strongly agglutinates human erythrocytes according to the amount of H substance expressed and can, therefore, easily discriminate between blood groups A1 and A2 as well as A1B and A2B (A1 and A1B are not or only weakly agglutinated). In immunohistochemistry, this antibody seems to provide a highly specific reagent for a restricted number of carcinomas and epithelial lineages in tissue sections and in vitro.

[Results of ultra-high streptokinase (Awelysin) doses in patients with thrombophlebitis]. / Ergebnisse einer ultrahohen Streptokinasedosierung (Awelysin) bei Patienten mit Phlebothrombose.

The results of 6-hour fibrinolysis with ultra-high streptokinase doses in 30 patients with peripheral venous thrombosis were presented. In 33.3% of the patients complete, and in 40% partial repatency was attained, while no repatency occurred in 20%. Deterioration was observed in two patients. The most beneficial effects were seen in postoperative and posttraumatic thrombosis. Bleeding complications did not occur. In one female patient treatment was discontinued due to bronchospasm.

[Thrombolytic treatment of peripheral arterial circulatory disorders with streptokinase]. / Thrombolytische Behandlung peripherer arterieller Durchblutungsstörungen mit Streptokinase.

The purpose of these investigations is to refer to the possibility of the reconstructive treatment of arterial obstructions by means of streptokinase. New application methods improved the chances of success of fibrinolysis. They run about 50%. The local application of smaller doses of streptokinase confines the contraindications to a certain extent. Obstruction in the pelvic area which are not older than three months and such ones in the femoropopliteal part up to 6 weeks are to be regarded as indication to the fibrinolysis. In the care of patients with arterial obstructive diseases importance should, therefore, be attached to the fact that thrombotic shifts should be recognized in time an the possibility of a fibrinolytic therapy is tested within the periods mentioned.

Monoclonal anti-cytokeratin antibody from a hybridoma clone generated by electrofusion.

Hybridomas producing mouse monoclonal antibodies to antigens of the human mammary carcinoma cell line, MCF-7, have been generated by electric field-mediated fusion at a frequency ten times higher than by polyethylene glycol. One of the monoclonal antibodies obtained recognizes a cytoskeletal structure restricted to epithelial cells and carcinomas with a distribution pattern resembling cytokeratin 19.

[Elimination of a paramyxo-type virus from oncornavirus-producing human cell lines (author's transl)]. / Eliminierung eines Paramyxovirus aus Oncornavirus-produzierenden menschlichen Zellinien

A virus of the paramyxo-type was eliminated from cell-free material of human oncornavirus-producing cell lines (PMF). After transmission of this paramyxovirus-free inoculum to a human permanent cell strain (Tu 197/Tr 1) oncornaviruses were permanently formed and no paramyxoviruses could be detected. The paramyxovirus-free, oncornavirus-producing PMF-39 cell line could be established after inoculation of the TU 197/Tr 1 line with cell-free material containing both oncorna- and paramyxovirus diluted 1 to 1000. A second way of elimination of the paramyxovirus was the treatment of cell-free material containing both viruses with antisera against paramyxovirus. In the Tu 197/Tr 1 line inoculated with such material only oncornaviruses were formed. The second paramyxovirus-free oncornavirus-producing cell line was designated PMF 50.

[Investigation of electrophoretical enzyme phänotypes glucose-6-phosphate dehydrogenase, red cell acid phosphatase, phosphoglucomutase and adenosine deaminase of permanent human cell lines (author's transl)]. / Untersuchung der elektrophoretischen Enzymphänotypen Glukose-6-Phosphatdehydrogenase, Saure Erythrozytenphosphatase, Phosphoglukomutase und Adenosindesaminase von 11 permanenten Menschzellinien

Isoenzymes of 11 cell lines were investigated by electrophoretical separation. All lines have been cultivated from tissues of white persons, all but one (Leuc. Th. B.) were phosphoglucomutase 1 and all were adenosine deaminase 1. Three out of 11 cell lines did show glucose-6-phosphate dehydrogenase (G6PD) type B as expected. Two out of 8 cell lines with G6PD A were distinguishable by their specific type of "red cell" acid phosphatase (SEP). We conclude that in vitro the electrophoretical G6PD phänotyp B changed to phänotype A. Further 4 lines had other peculiarities which are indicative to their originality, though they were G6PD A. Our investigations did show that G6PD may become type A if a cell line changes to permanent growth capacity in vitro. The enzyme marker G6PD A alone may not be valuated as an absolute evidence for contamination or mix up with He-La Cells.

Serological analysis of an oncornavirus (PMF virus) detected in malignant permanent human cell lines.

Immunodiffusion analysis of the PMF virus which was detected in malignant permanent human cell lines revealed positive reactions with antisera against the Mason-Pfizer monkey virus (MPMV). No cross-reactivity was demonstrated with murine leukemia virus (MuLV), rat leukemia virus (RaLV), hamster leukemia virus (HaLV), feline leukemia virus (FeLV), simian (woolly monkey) sarcoma virus (SSV-1) and mouse mammary tumor virus (MTV). The cross-reactive antigens of the PMF virus and the MPMV are considered as evidence for the human origin of the PMF virus.