Browsing by Author "Hülser, Dieter F."

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Open Access
β-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures
(1993) Klünder, Irene; Hülser, Dieter F.
We have investigated whether three-dimensional cultivation of cells to multicell spheroids influences the expression of a transfected gene. Ltk- cells (mouse fibroblasts. thymidine kinase negative) have been transfected with a bacterial lacZ gene which was coupled to a β-actin promoter. The transfected cells synthesize β-galactosidase, a cytoplasmic enzyme which can easily be stained for histology with 5-bromo-4-chloro-3-indoxyl β-D-galactoside and for cytometry with fluorescein di(β-D-galactopyranoside). As we have shown with monolayer cells, β-galactosidase is produced independently of cell density, medium condition, and cell cycle. In multicell spheroids, however, the portion of producing cells was reduced from ~98% to ~2% within a week. This reduction is also independent of cell density, medium condition, and cell cycle. Nonproducing multicell spheroid cells, however, regained their ability to synthesize β-galactosidase within a few days when the cells were recultivated as monolayers. Since the lacZ gene was not lost, its expression might have been regulated by its β-actin promoter. We, therefore, investigated whether the endogenous synthesis of β-actin was similarly regulated. A correlation between the distinct reduction in β-galactosidase-producing cells and filamentous or total actin concentration was not unequivocally observed.
Open Access
β-galactosidase production in two- and threedimensionally cultivated cell cultures
(1991) Klünder, Irene; Hülser, Dieter F.
In these studies, we demonstrate that the threedimensional arrangement of the cells in spheroids influences the β-galactosidase activity of transfected ltk--cells. In contrast to cells grown in monolayer culture cells growing as spheroids lost their β-galactosidase activity within few days. The reduction of β-galactosidase activity in cells grown as spheroids was not caused by a lost of the lacZ-gene as can be seen from the recovery of the β-galactosidase production when cells were retransferred in monolayer culture. As we showed in cells cultivated as monolayer, β-galactosidase activity seems to be not impaired by gradients of nutrient supply or cell cycle. These measurements are confirmed by histological sections, where the highest β-galactosidase activity was found in the viable center of the spheroids.
Open Access
Arachidonovaja kislota obratimo blokiruet vysokopronicaemye mežkletočnye kontakty
(1994) Hülser, Dieter F.; Zempel, Günther; Reuss, Bernhard; Suhr, Dierk; Sarovskaja, Julija J.; Murav'eva, O. V.; Dunina-Barkovskaja, Antonina; Margolis, Leonid B.
The effect of arachidonic on intercellular coupling via gap junctions has been studied in BICR/M1R k cells - a mammary tumor cell line of the Marshall ratt. Arachidonic acid is shown to reversibly block both ionic and dye coupling in a dose-dependent manner. The cells recoupled after the washout with either serum- or albumin (essentially fatty acid-free)-containing solution. The intercellular pH decreased from 7,2 to 7,0 after arachidonic acid treatment; the same pH shift in the absence of arachidonic acid, however, had no effect on the junctional permeability. Flow cytometric measurments revealed an arachidonic acid-induced increase of the cytoplasmic free Ca 2+ concentration which was also reversible upon albumin treatment. Intracellular Ca 2+ or H+ are unlikely to be involved in the mechanism of the arachidonic acid effect on intercellular coupling: high resolution measurments using double whole-cell technique also show reversible blockage of the junctional conductance in the presence of arachidonic acid while the pipette solution was buffered with 10 mM HEPES and 10 mM EGTA to clamp intracellular calcium and proton concentrations. We suggest that arachiconic acid directly affects the gap junction channels, probably interfering with the lipid-protein interactions.
Open Access
Are biological effects of shock waves caused by free radicals?
(1991) Suhr, Dierk; Brümmer, Franz; Hülser, Dieter F.
Stone fragmentation in extracorporeal shock wave lithotripsy as well as accompanying tissue damage are attributed to cavitation. We attempted to demonstrate and localize the occurrence of cavitation in cell-free solutions and MGH-U1 cells using sensitive dyes for the detection of cavitation-generated free radicals.
Open Access
Autoradiography with the electron microscope: properties of photographic emulsions
(1968) Hülser, Dieter F.; Rajewsky, Manfred F.
In this chapter, some current techniques of autoradiography with the electron microscope will be discussed, as well as some properties of the three photographic emulsions most commonly used for this purpose, namely, Gevaert NUC 307, Ilford LA, and Kodak NTE.
Open Access
Bioelectric responses at fertilization : separation of the events associated with insemination from those due to the cortical reaction in sea urchin, Lytechinus variegatus
(1982) Hülser, Dieter F.; Schatten, Gerald
The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50-400 msec), but minor (≅10 mV), electrical transient that leads to an action potential and then both the Na+-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.
Open Access
Biological effects and physical characterization of shock waves by an XL-1 experimental lithotripter
(1989) Brümmer, Franz; Staudenraus, Joachim; Nesper, Martina; Suhr, Dierk; Eisenmenger, Wolfgang; Hülser, Dieter F.
Extracorporeal shock wave lithotripsy (ESWL) has become the clinical standard method for non-invasive disintegration not only of concrements in kidney and urinary tract but also of gallstones. Despite the widespread clinical use of ESWL, the mechanism of stone destruction is not yet really understood, but several possibilities (cavitation, shock wave reflection) are discussed. The final cause of various side effects is still under discussion. Nevertheless, during the last few years much effort was put into possible extensions of ESWL applications On the other hand , physical characterizations of shock waves are rarely reported and combined measurements of biological effects in vitro and physical characterization of the applied shock waves are not available. We, therefore, examined the influence of water temperature and gas content on the shock wave efficency in biological systems and determined several physical characteristics (pressure amplitudes, rise time etc.) of the shock waves under the same experimental conditions.
Open Access
Biological effects of shock waves
(1990) Brümmer, Franz; Bräuner, Thomas; Hülser, Dieter F.
Extracorporeal shock wave lithotripsy has become established worldwide as the method of choice for the treatment of nephrolithiasis and ureterolithiasis over the last 10 years. Although initial studies showed no damaging effects of the shock waves on organs and tissues, numerous recent reports have presented evidence for severe acute effects and chronic complications after shock wave treatment. The pathophysiological effects on kidneys and the histopathological effects on organs or tissues in man and animal, and also the effects on cells in culture and tumors are sumarized. Suspended and immobilized cell cultures were used to characterize and quantify the efficacy of shock wave. Extended applications of shock waves and possible modifications to shock wave generators are discussed.
Open Access
Biological effects of shock-waves
(1991) Bräuner, Thomas; Brümmer, Franz; Hülser, Dieter F.; Rassweiler, Jens J.
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Open Access
Biophysical characterization of gap-junction channels in HeLa cells
(1993) Eckert, Reiner; Dunina-Barkovskaja, Antonina; Hülser, Dieter F.
HeLa cells seem not to be junctionally coupled when probed with techniques such as Lucifer yellow spreading and/or ionic coupling measured with three inserted microelectrodes. When investigated with double whole-cell patch-clamp measurements, HeLa cells in monolayer cultures were electrically coupled in 39% of the cases with very low transjunctional conductances (average one to five open channels). These gapjunction channels had a single-channel conductance γ=26±6 pS and were voltage-gated with an equivalent gating charge ζ=3.1±1.5 for a voltage of half-maximal inactivation Uo=49±10 mV. The voltage-dependent component represents only 31±8% of the total junctional conductance. The voltage-insensitive conductance is characterized by a residual open probability po(∞)=0.34±0.12, which corresponds to a ratio Gmin/Gmax=0.50±0.12. Dissociation of monolayer cells into cell pairs yielded about 58% coupled cell pairs with no notably altered single-channel properties.
Open Access
Biophysical investigations of the in vitro effects of shock waves and ultrasound
(1993) Brümmer, Franz; Suhr, Dierk; Irmer, Ulrich; Bachleitner, Christoph; Hülser, Dieter F.
To investigate the interactions of ultrasonic waves with biological tissues, we developed and standardized several in vitro models. Using these systems - artificial stones, human erythrocytes, L1210 mouse leukemia cells, multicellular spheroids, cavitation assay - we are able to elucidate the mechanisms of interaction as well as the cause of clinically observed side effects.
Open Access
Bioproduktion in dreidimensional wachsenden Zellkulturen
(1989) Hülser, Dieter F.; Klünder, Irene; Brenner, Joachim
Die biotechnologische Produktion von Substancen wie t-PA, monoklonalen Antikörpern, lnterleukinen, Wachstumsfaktoren, Interferonen und Hormonen sollte vorzugsweise mit Säugetierzellen erfolgen, weil Bakterien diese Substanzen nicht in ihrer nativen Form herstellen können. Die dazu benötigten großen Mengen von Säugerzellen werden - je nach Zelltyp - in Monolayer- oder Suspensionskultur gezüchtet. Für Monolayerkulturen müssen große Oberflächen (Roller Bottles, Microcarrier) zur Verfügung gestellt werden, was viel Raum und Materlal beansprucht und damit kostenintensiv ist. In Suspension wachsen die Zellen voneinander isoliert, wodurch Zellkooperation unterbunden wird und interzellulärer Informationsaustausch nicht mehr möglch ist. Es gibt jedoch eine weitere Möglichkeit, 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 läßt die Zellen zu dreidimensional angeordneten kugelförmigen Zellaggregaten - den Multizell-Sphäroiden - heranwachsen.
Open Access
Cavitation-generated free radicals during shock wave exposure: investigations with cell-free solutions and suspended cells
(1991) Suhr, Dierk; Brümmer, Franz; Hülser, Dieter F.
Extracorporeally generated shock waves as used in lithotripsy of urinary and biliary stones exhibit side effects in vivo. Furthermore, these shock waves destroy eukaryotic cells during in vitro treatment in suspension. A possible cause of these damaging effects might be cavitation, the growth and collapse of bubbles in liquids exposed to tensile stresses. During the collapse, temperature inside these cavitation bubbles rises up to several thousand K, leading to the formation of free radicals. We demonstrated the occurrence of cavitation-generated free radicals by direct reaction with fluorescent dyes in solution after shock wave treatment and investigated the resulting cell killing by variation of the cellular antioxidative defense status. We present evidence for the generation of intracellular free radicals during shock wave treatment of suspended cells.
Open Access
Cell communication by periodic cyclic-AMP pulses
(1975) Gerisch, Günther; Hülser, Dieter F.; Malchow, Dieter; Wick, Ursula
At the surface of aggregating cells of the slime mould, Dictyostelium discoideum, two different sites interacting with extracellular cAMP are detectable: binding sites and cyclic-nucleotide phosphodiesterase. Both sites are developmentally regulated. An adequate stimulus for the chemoreceptor system in D. discoideum is the change of cAMP concentration in time, rather than concentration per se: long-term binding of cAMP causes only a short-term response. The system is, consequently, adapted to the recognition of pulses rather than to steady-state concentrations of cAMP. The cells are, nevertheless, able to sense stationary spatial gradients and to respond to them by chemotactic orientation. The possibility is discussed that they do so by transforming spatial concentration changes into temporal ones, using extending pseudopods as sensors.
Open Access
Characteristics of three nuclear emulsions for autoradiography at the electron microscope
(1966) Hülser, Dieter F.; Rajewsky, Manfred F.
Drei handelsübliche Kernspuremulsionen, Gevaert NUC 307, Ilford L4 und Kodak NTE, wurden wegen ihrer geringen Korngröße auf ihre Eignung zur elektronenmikroskopischen Autoradiographie untersucht. Korngrößenverteilungskurven wurden aufgenommen und ein geeigneter Entwickler ausgesucht. Zur Bestimmung der Empfindlichkeit dieser drei Emulsionen wurden Einkornschichten im Elektronenmikroskop mit Elektronen einer Energie von 5,7 keV, der mittleren beta-Energie des Tritiums, bestrahlt. Anschließend wurden die Emulsionen entwickelt, aber nicht fixiert. Mit dem Anteil der entwickelten AgBr-Körner kann dann über Trefferkurven die Empfindlichkeit der Emulsionen bestimmt werden. Man benötigt zur Bildung eines latenten Bildkeimes für die Ilford L4-Emulsion 1 - 1,4 Elektronen pro AgBr-Korn, für die Gevaert NUC 307-Emulsion 2 - 3 und für die Kodak NTE-Emulsion 4 - 5 Elektronen pro AgBr-Korn. Folgerungen für das Auflösevermögen bei radioaktiven Punkt- und Flächenquellen werden diskutiert. Fortschritte in der Mikroautoradiographie werden von der Entwicklung feinkörniger Emulsionen abhängen, deren Empfindlichkeit bei etwa einem Elektron pro AgBr-Korn liegen sollte.
Open Access
Characterization of gap junctions by electrophysiological and electronmicroscopical methods
(1990) Hülser, Dieter F.; Paschke, Dietmar; Brümmer, Franz; Eckert, Reiner
Gap junctions are ubiquitous in the animal kingdom from mesozoa to vertebrates. They must be discriminated from desmosomes which anchor cells together to form structural or functional units as well as from tight junctions which seal membranes of epithelial cells to each other so that the paracellular path becomes impermeable to molecules and a polarity of apical and basolateral surface is maintained.
Open Access
Closing and opening of gap junction pores between two- and threedimensionally cultured tumor cells
(1982) Hülser, Dieter F.; Brümmer, Franz
Intercellular signal transfer via gap junction pores in cultured multicell spheroids of BICR/M1R-K cells decreases with increasing spheroid age. In two days old spheroids the pores allow passage of Lucifer yellow molecules. Two days later, this fluorescent dye is retained in the injected cell even though the cells are still electrically coupled. Gap junction plaques of considerable size are still found in 9 days old spheroids, when the cells are completely uncoupled. The same cells growing as monolayer cultures do not exhibit such a gradual closing of their gap junction pores: Their coupling is established at first cell contact, probably by a gradual opening of the pores, which remain open even up to 9 days in culture.
Open Access
Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes
(1989) Traub, Otto; Look, Jutta; Dermietzel, Rolf; Brümmer, Franz; Hülser, Dieter F.; Willecke, Klaus
Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.
Open Access
Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes
(1984) Bräuner, Thomas; Hülser, Dieter F.; Strasser, Reto J.
The usefulness of a new voltage-sensitive fluorescent dye, the membrane permeant negatively charged oxonol dye diBA-C4-(3)−, was evaluated by measuring the membrane potentials of BICR/M1R-k and L cells with glass microelectrodes and simultaneously recording the fluorescence of the stained cells. The membrane potential of BICR/M1R-k cells was varied between −25 mV and −90 mV by changing the bicarbonate concentration in the medium or by voltage clamping. To avoid any interference by the inserted electrodes with the fluorescence measurement of the cytoplasm, the cells were fused by polyethyleneglycol to form giant cells (homokaryons). These homokaryons also allowed penetration by two glass microelectrodes without causing a serious leakage of the plasma membrane. The slow responding dye diBA-C4-(3)− had a fluorescence response of about 1% per mV. Mathematical analysis of the fluorescence changes after voltage clamping revealed a first-order reaction with a rate constant between 0.1 min−1 and 0.8 min−1, depending on the cell size which was determined by the number of nuclei per homokaryon. A model for the mechanism of the fluorescence changes is proposed.
Open Access
Contact cooperation in stimulated lymphocytes : 2, Electrophysiological investigations on intercellular communication
(1972) Hülser, Dieter F.; Peters, Johann H.
Intercellular communication-as defined by the flow of ions between cells through low-resistance junctions-exists in agglutinates of phytohemagglutinin-stimulated lymphocytes. It is shown that this communication starts within minutes after the addition of the stimulant, at the same time as other functions of the lymphocyte membrane are altered. In contrast, neither stimulation nor intercellular communication can be detected under conditions where agglutination was established by a lymphocyte-agglutinating anti-serum, indicating that communication may be closely correlated with the process of stimulation.