Joachim Brenner

Effect of shock waves on suspended and immobilized L1210 cells

L1210 mouse leukemia cells have been exposed to different doses of shock waves generated by underwater spark discharge at 18 kV in an experimental lithotripter (XL1, Dornier). Histological and flow cytometric investigations revealed severe damage and a LD50 of about 420 shock waves when the cells were treated as suspensions. Cells immobilized in gelatine, however, were unaffected, indicating that secondary effects are responsible for the cellular damage. Possible mechanisms such as cavitation, jets, and shear forces are discussed.

The identification of Chrysochromulina and Prymnesium species (Haptophyta, Prymnesiophyceae) using fluorescent or chemiluminescent oligonucleotide probes: a means for improving studies on toxic algae

Chrysochromulina and Prymnesium are important bloom-forming organisms in marine and brackish waters, respectively. Both genera include toxic species, which are primarily implicated in fish kills. Previous analyses of small subunit (SSU) rDNA sequences from Chrysochromulina and Prymnesium spp. indicate that Chrysochromulina is paraphyletic. C. polylepis, which produced a spectacular, harmful bloom in 1988, is more closely related to toxic Prymnesium species than to most other Chrysochromulina species based on rDNA sequence comparisons. Signatures were identified in the SSU rRNA gene specific for a clade that comprised primarily toxic taxa (C. polylepis, P. parvum, P. patelliferum and P. calathiferum) and that recognized C. polylepis alone. Oligonucleotide probes complementary to these regions were designed, and their specificity tested using dot-blot hybridization on PCR products of the SSU rRNA gene from 28 strains of Chrysochromulina and Prymnesium. Whole-cell hybridizations were performed with FLUOS- as...

Holospora species infecting the nuclei of Paramecium appear to belong into two groups of bacteria

Summary Ten Holospora bacteria in various Paramecium species, namely P. novaurelia, P. caudatum, P. bursaria, P. woodruffi , and P. putrinum were found to be similar concerning their morphology and life cycle. However, four species (group 1): Holospora caryophila, H. bacillata, H. curvata , and a species newly found in P. putrinum differed from the other bacteria (group 2), among them H. obtusa, H. undulata, H. elegans, H. recta , as they had a different behavior during the division of the host nucleus; in group 2 the infectious forms were collected in the connecting piece of the dividing host nucleus and were then released into the medium, whereas in group 1 the infectious forms remained distributed over the whole nucleus during its division. The bacteria of the group 1 also were not labelled by in situ hybridization using an oligonucleotide probe hybridizing with the rRNA of bacteria of group 2. The data indicate that Holospora bacteria may at least belong into two groups.

Dinoflagellates from marine algal blooms produce neurotoxic compounds: effects on free calcium levels in neuronal cells and synaptosomes.

In this report, evidence is presented that the marine unicellular eukaryotic dinoflagellates can cause neurotoxicity very likely by an increase in intracellular free calcium ions ([Ca(2+)](i)). Determinations of the effects of culture supernatants from different clones of the dinoflagellate Alexandrium sp. isolated from algal blooms on the viability of rat primary neuronal cells revealed that all clones tested were toxic for these cells. In addition, all Alexandrium clones tested, except for A. ostenfeldii BAH ME-141, were found to be toxic for rat pheochromocytoma PC12 cells. No toxicity was observed for culture supernatants from Gonyaulax and Coolia monotis. Calcium ions are important in the process of apoptotic cell death; our studies revealed that the dinoflagellate supernatants from A. lusitanicum K2, A. lusitanicum BAH ME-091, and A. tamarense 1M caused an increase in [Ca(2+)](i) levels in both PC12 cells and primary neuronal cells. These dinoflagellate supernatants, as well as the A. tamarense ccmp 115 supernatant, were found to cause also an increase in free calcium concentration in isolated synaptosomes. Our results suggest that the neurotoxic effects of certain dinoflagellate supernatants may be associated with disturbances in [Ca(2+)](i) levels.

Production of tissue plasminogen activator (tPA) in two and three dimensionally growing cultures of Bowes melanoma cells.

Bowes melanoma cells synthesize more tissue plasminogen activator (tPA) in monolayer cultures than in multicell spheroids. Cellular production of tPA in these cells was measured during a cultivation period of 800 h. Without changing the cell culture assay, we were able to obtain monolayers, multilayers, and multicell spheroids (cell aggregates) by stirring microcarrier beads in 500‐mL spinner flasks operated at 50 rpm. Thus, the medium conditions in the liquid were similar for cells in monolayers and in multicell spheroids. Probes for measurements of intracellular and extracellular parameters were taken from the same culture at distinct times; therefore, their variations during cultivation can directly be compared. Because cells were cultured in an unregulated (with regard to pH, glucose, etc.) spinner flask, their concentration was kept below 106 cells/mL, thus avoiding too fast and too severe depletion of oxygen and other medium factors. Nevertheless, the tPA productivity decreased from 8 ng/h/106 cells (monolayer) to 4 ng/h/106 cells (multicell spheroids with microcarrier nucleus, 800 μm diameter), matching the decrease of total cellular protein. Due to medium depletion, the cell cycle distribution changed from 45% to 68% G1 cells in a characteristic way during growth of multicell spheroids. This is accompanied by changes in amino acids, glucose, lactate, and pH, which may account for the reduction of tPA productivity.

Zell- und Gewebsveränderungen durch eine Stosswellenbehandlung

Die extrakorporale Stoswellenlithotripsie kann seit Beginn ihrer klinischen Anwendung im Jahre 1980 bedeutende Erfolge bei der beruhrungslosen Nlerensteinzertrummerung vorweisen, was dazu fuhrte, das sie mittlerweile auch fur die Behandlung von Gallensteinen angewendet wird. Da offensichtlich wesentlich geringere Nebenwirkungen als nach Operationen auftreten, hat sich diese Methode weltweit rasch ausgebreitet und es ist nicht verwunderlich, wenn nunmehr auch uber eine Anwendungserweiterung in Richtung Tumortherapie spekuliert wird. Dazu mussen allerdings die zellularen Auswirkungen der Stoswellenbehandlung von Geweben genauer charakterisiert werden.

Bioproduktion in dreidimensional wachsenden Zellkulturen

Die biotechnologische Produktion von Substancen wie t-PA, monoklonalen Antikorpern, lnterleukinen, Wachstumsfaktoren, Interferonen und Hormonen sollte vorzugsweise mit Saugetierzellen erfolgen, weil Bakterien diese Substanzen nicht in ihrer nativen Form herstellen konnen. Die dazu benotigten grosen Mengen von Saugerzellen werden - je nach Zelltyp - in Monolayer- oder Suspensionskultur gezuchtet. Fur Monolayerkulturen mussen grose Oberflachen (Roller Bottles, Microcarrier) zur Verfugung gestellt werden, was viel Raum und Materlal beansprucht und damit kostenintensiv ist. In Suspension wachsen die Zellen voneinander isoliert, wodurch Zellkooperation unterbunden wird und interzellularer Informationsaustausch nicht mehr moglch ist. Es gibt jedoch eine weitere Moglichkeit, Eurokaryonten zu kultivieren, welche die Nachteile der beiden anderen Methoden aufhebt. Diese bisher vorwiegend in der Krebsforschung oder bei der Untersuchung von Regulationsmechanismen von Zellen verwendete Technik last die Zellen zu dreidimensional angeordneten kugelformigen Zellaggregaten - den Multizell-Spharoiden - heranwachsen.