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O Ullrich

Publications and source records attributed to O Ullrich.

18 recordsLinked to original sources
PubMed1997

Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis.

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm x 75 microns i.d.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25 degrees C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10(5)-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Intracellular metabolism of 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.

The intracellular metabolism of 4-hydroxynonenal (HNE), a secondary product of lipid peroxidation and mediator of inflammation, which was found in the joints of patients with rheumatoid arthritis, was investigated in primary cultures of rabbit synovial fibroblasts. A consumption rate of 27.3 nmol/min x 10(6) cells was measured for the cultivated fibroblasts. It could be shown, that 4-hydroxynonenal enters the synovial fibroblasts and is metabolized mainly oxidatively to 4-hydroxynonenoic acid, intermediates of the tricarboxylic acid cycle and water and by formation of the glutathione-HNE adduct. The share of protein-bound HNE was about up to 8% of the total added HNE after 10 min of incubation. All metabolites accumulates intracellularly within the incubation time except of 4-hydroxynonenal itself. An increase of 4-hydroxynonenoic acid could be detected also extracellularly during the intracellular metabolism of 4-hydroxynonenal. Therefore, an involvement of synovial fibroblasts in the secondary antioxidant defense system of the joints during conditions of higher HNE concentrations like rheumatoid arthritis is suggested.

Aldehydes↗
PubMed1996

GTPase activity of Rab5 acts as a timer for endocytic membrane fusion.

The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport. Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein. To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5. Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.

ADP-Ribosylation Factors↗
PubMed1996

Inhibition of poly(ADP-ribose) formation by 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.

The formation of poly(ADP-ribose) in primary cultures of rabbit synovial fibroblasts after treatment with active oxygen released by xanthine/xanthine oxidase is inhibited by addition of 1 and 10 microM 4-hydroxy-2,3-trans-nonenal (HNE). The endogenous formation of HNE by the xanthine/xanthine oxidase system is not responsible for the inhibitory effect of the aldehyde, owing to the low accumulation rate of the lipid peroxidation product in the system used. HNE is able to inhibit the isolated nuclear enzyme ADP-ribosyltransferase, as shown by an in vitro assay with an Ki of 4 mumol/litre. Therefore the molecular basis of HNE-mediated effects on cell proliferation, differentiation and transformation might be due to the inhibitory effect of poly(ADP-ribos)ylation.

Aldehydes↗
PubMed1996

Rab11 regulates recycling through the pericentriolar recycling endosome.

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

Animals↗
PubMed1996

The effect of the lipid peroxidation product 4-hydroxynonenal and of its metabolite 4-hydroxynonenoic acid on respiration of rat kidney cortex mitochondria.

In rat kidney cortex mitochondria, 4-hydroxynonenal inhibits state 3 respiration as well as uncoupled respiration at micromolar concentrations. The inhibition is more distinct for NAD-linked than for FAD-linked respiration. 4-Hydroxynonenal increases the state 4 respiration. It is assumed that 4-hydroxynonenal behaves like a decoupling agent. 4-Hydroxynonenal augments the inhibitory effect of 2,4-dinitrophenol observed at superoptimal concentrations. 4-Hydroxynonenal is metabolised by renal mitochondria, and 4-hydroxynonenoic acid is one of the metabolites generated. This metabolite is without effect on respiration at concentrations up to 50 microM. Therefore, the effect of 4-hydroxynonenal on respiration is not mediated by this fatty acid derivative formed during respiratory measurements.

Aldehydes↗
PubMed1995

Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion.

We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport. It is mainly cytosolic, but a fraction colocalizes with Rab5 to early endosomes. Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged endosomes. Overexpression of rabaptin-5 alone is sufficient to promote expansion of early endosomes. Rab5 recruits rabaptin-5 to purified early endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily. Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion. Rabaptin-5 is thus a Rab effector required for membrane docking and fusion.

Amino Acid Sequence↗
PubMed1995

Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells.

We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.

Animals↗
PubMed1994

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by mitochondria isolated from rat kidney cortex.

The cytosolic lipid peroxidation product 4-hydroxynonenal (HNE) is rapidly metabolized in mitochondria isolated from rat kidney cortex. About 80% of HNE was degraded within 3 min of incubation. Main products of HNE which were identified in mitochondria were the hydroxynonenoic acid, the 1,4-dihydroxynonene and the glutathione-HNE-conjugate. Furthermore, formation of metabolites of the tricarboxylic acid cycle from HNE is suggested. The quantitative share of HNE binding to proteins was high with about 8% of total HNE consumption after 3 min of incubation. Therefore, rapid degradation of HNE by mitochondria might be involved in an intracellular antioxidative defense system.

Aldehydes↗
PubMed1994

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis.

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.

Animals↗
PubMed1994

Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange.

The Rab GTPases function as specific regulators of membrane transport. The GTP/GDP cycle is believed to control shuttling of Rab proteins between the cytosol and organelle membranes. In vitro, Rab proteins are removed from membranes by a protein that inhibits GDP dissociation (rabGDI), which leads to formation of a cytosolic complex of Rab with the inhibitor protein. Here we use a purified Rab5-rabGDI complex in a permeabilized cell system to investigate how the cytosolic complexed form of Rab reassociates with the membrane. We find that exogenous Rab5 is correctly targeted and induces the formation of enlarged early endosomes, demonstrating that it is functionally active. Binding of Rab5 to the acceptor membrane is accompanied by release of the rabGDI protein into the cytosol. A transient GDP-Rab5 intermediate was detected which was subsequently converted into the GTP-bound form. Our results indicate that there is a multistep mechanism for the insertion of Rab5 into the membrane which is mediated by a guanine-nucleotide-exchange factor.

Animals↗
PubMed1994

Distinct structural elements of rab5 define its functional specificity.

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.

Amino Acid Sequence↗
PubMed1993

Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins.

Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic. These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles. Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane. In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins. Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins. However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif. Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways. Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI.

Amino Acid Sequence↗
PubMed1993

Elevated cAMP levels induce multilayering of MDCK cells without disrupting cell surface polarity.

The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seeded in high densities in media containing FCS a regular monolayer was formed. However, in serum-free medium supplemented with insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine, the development of a multilayer with intercellular lumina was observed. In hormone-depletion studies we identified PGE1 as the inducer of these multilayers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that an elevated intracellular cAMP level resulted in formation of the multilayer. Further analysis by electron microscopy and immunocytochemistry revealed a polarized organization of the multilayered cells. Junctional complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surrounding the intercellular lumina. Surprisingly, cells participating in the formation of both the free surface and the surface of the intercellular lumen, exhibited two distinct membranes with microvilli, each separated by junctional complexes. Immunolocalization of membrane marker proteins demonstrated that an apical 114 kDa membrane protein was localized to the free cell surfaces, the same membrane domains where extensive microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These results provided evidence that nontransformed epithelial MDCK cells form multilayers in response to elevated cAMP levels; however, they retain the potential of developing cell surface polarity.

Alprostadil↗
PubMed1991

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line.

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.

Amino Acid Sequence↗