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D Hülser

Publications and source records attributed to D Hülser.

12 recordsLinked to original sources
PubMed2004

Yeast two-hybrid systems confirm the membrane- association and oligomerization of BC1 but do not detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic geminivirus.

Most of the plant begomoviruses use two proteins to transport their DNA from cell to cell, BV1 to shuttle it between nucleus and cytoplasm and BC1 to facilitate movement across plasmodesmata. In order to analyse their interaction for Abutilon mosaic geminivirus (AbMV) in yeast ( Saccharomyces cerevisiae), BC1 and BV1 genes were cloned into various plasmid vectors suitable for two-hybrid analysis. BC1 was fused to the binding domain (GBD) and BV1 to the activation domain (GAD) of the GAL4 transcription factor to check for interactions in the nucleus. Additionally, BC1 as well as BV1 were integrated into pMyr or pSos vectors to analyze protein binding at the plasma membrane using the CytoTraptrade mark system. Using freeze-fracture immuno-labelling (FreeFI), singly-expressed GBD:BC1 was localized at the plasma membrane although it was fused to a nuclear localization signal provided by the construct. GAD:BV1 was found in the nucleus of transformed cells as expected. Upon co-transformation of both constructs, cells grew poorly and exhibited symptoms of autolysis without any detectable level of GBD:BC1 or GAD:BV1, as revealed by FreeFI. In conclusion, both fusion proteins did not meet in the same compartment and appeared to be harmful to yeast if constitutively co-expressed. When expressed from pSos vector, BC1 induced the CytoTrap detection signal in the absence of pMyr indicating that BC1 protein alone is able to target the effector protein to the inner face of the plasma membrane. A mutated form of BC1 (DeltaBC1) lacking the previously identified membrane-binding domain was no longer able to auto-induce the CytoTrap signal cascade. Using DeltaBC1, an N-terminal, or a C-terminal third of BC1 revealed a homo-oligomerization of the C-terminal region of BC1 in two-hybrid analysis, but no interaction of BC1 with BV1.

Biological Transport↗
PubMed2002

Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling.

The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors. Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation. Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria. Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane. The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed. In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.

Cell Membrane↗
PubMed1998

Characterization of the gap junction protein connexin37 in murine endothelium, respiratory epithelium, and after transfection in human HeLa cells.

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29-303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

Animals↗
PubMed1994

TNF receptors TR60 and TR80 can mediate apoptosis via induction of distinct signal pathways.

TNF membrane receptors are usually co-expressed in many tissues but their relative contribution to cellular TNF responses is for most situations unknown. In a TNF cytotoxicity model of KYM-1, a human rhabdomyosarcoma cell line, we recently demonstrated that each of the two TNFRs is on its own capable of inducing cell death. Here we show that both receptors are able to induce apoptosis, as revealed from a similar onset of DNA fragmentation and typical morphologic criteria. To obtain additional information about the signaling pathways involved in TR60- and TR80-induced programmed cell death, we have used a series of selective inhibitors of intracellular signaling molecules. The overall pattern emerging from these experiments provides strong evidence for distinct signal pathway usage of TR60 and TR80, indicating protein kinase(s)-mediated control of TR60 signaling and a tight linkage of TR80 to arachidonate metabolism. The subsequent establishment of KYM-1-derived cell lines that display TNFR selective resistance further supports a segregation of TR60 and TR80 signaling pathways for induction of apoptotic cell death. Moreover, these results demonstrate an independent control of the distinct signaling cascades used by TR60 and TR80. This allows a highly flexible regulation of a cellular TNF response in those cases in which both receptors contribute to overall TNF responsiveness.

Apoptosis↗
PubMed1991

Growth inhibition of oncogene-transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions.

We have studied the proliferation of rat 208F cells (a derivative of Rat-1 cells) transformed by activated c-Ha-ras, v-fgr, v-raf, v-fms, or v-src oncogenes during cocultivation with an excess of early passage rat embryonic fibroblasts or immortal 208F cells. The total number and size of foci formed by oncogene-transformed 208F cells were strongly reduced by cocultured normal fibroblasts. The extent of growth suppression of transformed foci appears to be dependent on the type of transforming oncogene and on the type of normal fibroblasts rather than on the extent of gap-junctional communication between transformed and normal cells. Total inhibition of fluorescent dye transfer between normal and transformed cells by the 3 beta-O-hemisuccinate of 18 alpha-glycyrrhetinic acid (18 alpha-carbenoxolone), an inhibitor of gap-junctional communication in human fibroblasts, did not prevent growth inhibition of transformed cells in the cocultivation assay. Since adjacent cells remained electrically coupled in the presence of this inhibitor it is possible that the strongly reduced metabolic cooperation, as indicated by the lack of fluorescent dye transfer, is sufficient for mediating the growth-inhibitory effect of normal fibroblasts. 208F cell-conditioned medium, however, also caused strong growth inhibition of transformed derivatives, suggesting that the effect is in part mediated by release of stable growth inhibitor(s) from 208F cells.

Animals↗
PubMed1989

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes.

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.

Animals↗
PubMed1983

Timing the early events during sea urchin fertilization.

To determine precisely the timing, duration, and sequences of the earliest events during sea urchin (Lytechinus variegatus) fertilization, the bioelectric recordings of microelectrode-impaled eggs were electronically superimposed, by video mixing, over the microscopic differential interference contrast image of the same egg at insemination. Videotape analysis, utilizing a slow-motion analyzer, demonstrates that the successful sperm triggers the bioelectric membrane potential reversal within 3.36 +/- 3.02 sec (0.72-9.76 sec range; sigma = 23 eggs) of sperm-egg attachment. This sperm, actively gyrating about its attachment site, is indistinguishable from the other, unsuccessful sperm until 12.66 +/- 2.72 sec (6.72-16.60 sec range; sigma = 15) later when the sperm tail ceases its beating and sperm incorporation ensues. The cortical granules begin to discharge, and the fertilization coat starts to elevate at the fusion site at 20.79 +/- 3.18 sec (13.62-26.08 sec range; sigma = 12) after the onset of the fertilization potential, i.e., an average of about 8 sec after the cessation of sperm-tail motility during incorporation. In most cases, the bioelectric responses starts within 7 sec of sperm adhesions; if the data are analyzed excluding the few slow cases, the fertilization potential is found to start 1.93 sec (+/- 1.28 sec) after sperm attachment. These results indicate that the first successful sperm triggers the fast block to polyspermy within 3.4 sec, perhaps as quickly as 1.9 sec, of sperm-egg adhesion, about 13 sec before the first morphological indication of fertilization, and about 21 sec before the characteristic elevation of the fertilization coat responsible for the late block to polyspermy.

Animals↗
PubMed1983

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions.

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.

Animals↗
PubMed1981

Increased radioresistance of cells in cultured multicell spheroids. I. Dependence on cellular interaction.

Monolayers of six different cell lines were investigated with respect to ionic coupling using micro-electrode techniques. In parallel, survival after Co-gamma-irradiation of monolayer- and spheroid cultures of these lines was compared. It was found that spheroids of coupled cell lines were more radioresistant than monolayers ("contact effect"). however, cell coupling did not enhance the survival of monolayers over single cells. This suggests that the contact effect is a "tissue phenomenon" requiring cellular interaction but is expressed only under conditions of three-dimensional growth.

Animals↗
PubMed1975

Cell communication by periodic cyclic-AMP pulses.

At the surface of aggregating cells of the slime mould, Dictyostelium discoideum, two different sites interacting with extracellular cAMP are detectable: binding sites and cycl-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 short-term response. The system is, consequently, adapted to the recognition of pulses rather than to steady-state concentrations of cAMP. The ce,lls 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. The cAMP recognition system is part of a molecular network involved in the generation of spatio-temporal patterns of cellular activities. This system controls the periodic formation of chemotactic signals and their propagation from cell to cell. The phosphodiesterase limits the duration of the cAMP pulses and thus sharply separates the periods of signalling; the binding sites at the cell surface are supposed to be the chemoreceptors. The control of cellular activities via cAMP receptors can be studied with biochemical techniques with cell suspensions in which spatial inhomogeneities are suppressed by intense stirring, whereas the temporal aspect of the spatiotemporal pattern is preserved. Under these conditions it can be shown that the extracellular cAMP concentration changes periodically, and that the phase of the cellular oscillator can be shifted by external pulses of cAMP. It can also be shown that small cAMP pulses induce a high output of cAMP, which demonstrates signal amplification, a function necessary for a cellular relay system.

3',5'-Cyclic-AMP Phosphodiesterases↗