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W Möller

Publications and source records attributed to W Möller.

At least 19 recordsLinked to original sources
PubMed2006

Effects of altered gravity on the actin and microtubule cytoskeleton of human SH-SY5Y neuroblastoma cells.

Human SH-SY5Y neuroblastoma cells were used to study the effects of altered gravity on the actin and microtubule cytoskeleton dynamics. A cholinergic stimulation of the cells during a 6 min period of changing gravity (3 parabolas) resulted in an enhanced actin-driven protrusion of evoked lamellipodia. Likewise, the spontaneous protrusive activity of nonactivated cells was promoted during exposure to changing gravity (6 up to 31 parabolas). Ground-based experiments revealed a similar enhancement of the spontaneous and evoked lamellar protrusive activity when the cells were kept at 2 g hypergravity for at least 6 min. This gravity response was independent of the direction of the acceleration vector in respect to the cells. Exposure of the cells to "simulated weightlessness" (clinorotation) had no obvious influence on this type of lamellar actin cytoskeleton dynamics. A 20 min exposure of the cells to simulated weightlessness or to changing gravity (6 to 31 parabolas) - but not to 2 g (hypergravity, centrifugation) - resulted in an altered arrangement of microtubules indicated by bending, turning, and loop formation. A similar altered arrangement was shown by microtubules which had polymerized into lamellipodia after release from a taxol block at simulated weightlessness (clinorotation) or during changing gravity (5 parabolas). Our data suggest that in human SH-SY5Y neuroblastoma cells, microgravity affects the dynamics and spatial arrangement of microtubules but has no influence on the Rac-controlled lamellar actin cytoskeleton dynamics and cell spreading. The latter, however, seems to be promoted at hypergravity.

Acetylcholine↗
PubMed2006

Negligible clearance of ultrafine particles retained in healthy and affected human lungs.

Ambient particles are believed to be a specific health hazard, although the underlying mechanisms are not fully understood. There are data in the literature indicating fast and substantial systemic uptake of particles from the lung. The present authors have developed an improved method to produce ultrafine particles with more stable radiolabelling and defined particle size range. Fifteen subjects inhaled technetium 99m (99mTc)-labelled carbonaceous particles of 100 nm in size. Radioactivity over the lung was followed for 70 h. The clearance of these ultrafine particles from the lungs and specifically translocation to the circulation was tested. Lung retention for all subjects at 46 h was mean+/-sd 99+/-4.6%. Cumulative leaching of 99mTc activity from the particles was 2.6+/-0.96% at 70 h. The 24-h activity leaching in urine was 1.0+/-0.55%. No evidence of a quantitatively important translocation of 100-nm particles to the systemic circulation from the lungs was found. More research is needed to establish if the approximately 1% cleared activity originates from leached activity or insoluble translocated particles, and whether a few per cent of translocated particles is sufficient to cause harmful effects.

Administration, Inhalation↗
PubMed2006

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Animals↗
PubMed2004

In vitro anti-proliferation/cytotoxic activity of sixty natural products on the human SH-SY5Y neuroblastoma cells with specific reference to dibenzyl trisulphide.

Sixty natural products belonging to the following structural classes: artemisinins, coumarins, flavonoids, tannins, tetrahydroberberine alkaloids, tetracyclic triterpenes, tetranortriterpenoids and polysulphides were screened against the human SH-SY5Y neuroblastoma cell line revealing differences in their effects on cell morphology and in anti-proliferation/cytotoxic activity. Based on the data obtained, dibenzyl trisulphide is the most effective anti-proliferative/cytotoxic compound. In addition, we hereby propose the human SH-SY5Y cell line as a sensitive and uncomplicated in vitro test system for detecting compounds with potential anti-proliferation/cytotoxic activity.

Antineoplastic Agents↗
PubMed2003

In vitro anti-proliferation/cytotoxic activity of cantharidin (Spanish Fly) and related derivatives.

The anti-cancer therapeutic promise of cantharidin is limited because of its high mammalian toxicity. In order to find new anti-cancer lead compounds with reduced toxicity of the cantharidin prototype, the following seven derivatives were screened against the human SH-SY5Y neuroblastoma and MCF-7 breast cancer cells in vitro: 2,3-dimethyl-7-oxabicylo-[2.2.1]heptane-2,3-dicarboxylic anhydride (cantharidin) [1], 1-cyclohexen-1,2-dicarboxylic anhydride [2], cis-4-cyclohexen-1,2-dicarboxylic anhydride [3], cis-1, 2-cyclohexanedicarboxylic anhydride [4], exo-7-oxabicyclo[2.2.1]hept-5-ene-2-3 dicarboxylic anhydride [5], exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin) [6], and (S)-(-)-O-acetylmalic anhydride [7]. Cantharidin, was found to be the most effective anti-proliferative compound on both cell lines. However, on the human neuroblastoma cells cantharidin was of equal toxicity to compound [6]. Mode of action studies revealed that cantharidin inhibited growth factor-mediated activation of mitogen activated protein kinase (MAPkinase) and attenuated the de-phosphorylation of the extracellular regulated kinases 1 and 2 (erk1 and erk2).

Anhydrides↗
PubMed2002

[Pneumological diagnostics by means of models aerosols. IV. Clearance of aerosol particles from the pulmonary alveoli. Foundations and first clinical results].

The elimination of deposited particles (inorganic and organic particles, bacteria, viruses) from the periphery of the human lung (alveolar clearance) implies phagocytosis by alveolar macrophages, intracellular digestion, migration and translocation. Alveolar clearance of poorly soluble particles happens very slowly and primarily depends on the function of alveolar macrophages. In humans, the transport of particles to the bronchial tree is of secondary relevance, suggesting that the elimination of particles primarily depends on digestion (dissolution) processes within macrophages. The dissolved material is excreted via urine, if there is no further metabolization within the body. The pathophysiology of the alveolar clearance mechanisms in the human lung can be studied by a magnetic tracer technique (magnetopneumography). Ferromagnetic magnetite test-particles are deposited in the periphery of the lung by controlled inhalation. After magnetization and particle alignment in a strong external magnetic field pulse, the amount of retained particles can be detected by a sensitive magnetic field sensor (SQUID, superconducting quantum interference device). Long lasting cigarette smoking and chronic lung inflammations (sarcoidosis, interstitial lung fibrosis) induce a significant impairment of alveolar clearance capacity, while patients with chronic bronchitis show only a moderate impairment of alveolar clearance.

Aerosols↗
PubMed2001

Retention of the potential energy of multiply charged argon ions incident on copper.

The retained fraction of the potential energy of argon ions incident on copper has been measured using stationary calorimetry at charge states up to 9+ and kinetic energies ranging from 75 to 240 eV per ionic charge. An average fraction of 30% to 40% is found with little dependence on the charge number and on the kinetic energy. The retention of the total energy ranges from 60% to 75% and can mainly be accounted for by the retained fraction of the potential energy and the collisional energy lost by reflected ions and sputtered target atoms.

Journal Article↗
PubMed2000

Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs.

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.

Acrylamide↗
PubMed2000

Reduction of amphetamine hydroxylamine and other aliphatic hydroxylamines by benzamidoxime reductase and human liver microsomes.

For the reduction of N-hydroxylated derivatives of strongly basic functional groups, such as amidines, guanidines, and aminohydrazones, an oxygen-insensitive liver microsomal system, the benzamidoxime reductase, has been described. To reconstitute the complete activity of the benzamidoxime reductase, the system required cytochrome b(5), NADH-cytochrome b(5)-reductase, and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to homogeneity from pig liver. It was not known if this enzyme system was also capable of reducing aliphatic hydroxylamines. The N-hydroxylation of aliphatic amines is a well-known metabolic process. It was of interest to study the possibility of benzamidoxime reductase reducing N-hydroxylated metabolites of aliphatic amines back to the parent compound. Overall, N-hydroxylation and reduction would constitute a futile metabolic cycle. As examples of medicinally relevant compounds, the hydroxylamines of methamphetamine, amphetamine, and N-methylamine as model compounds were investigated. Formation of methamphetamine and amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines were easily reduced by benzamidoxime reductase to their parent amines with reduction rates of 220.6 nmol min(-1) (mg of protein)(-1) for methamphetamine, 5.25 nmol min(-1) (mg of protein)(-1) for amphetamine, and 153 nmol min(-1) (mg of protein)(-1) for N-methylhydroxylamine. Administration of synthetic hydroxylamines of amphetamine and methamphetamine to primary rat neuronal cultures produced frank cell toxicity. Compared with amphetamine or the oxime of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells. The benzamidoxime reductase is therefore involved in the detoxication of these reactive hydroxylamines.

Amphetamines↗
PubMed1999

Hypoxic upregulation of tyrosine hydroxylase gene expression is paralleled, but not induced, by increased generation of reactive oxygen species in PC12 cells.

Oxygen sensing was investigated in rat pheochromocytoma PC12 cells. They respond to hypoxia with an increased intracellular generation of reactive oxygen species (ROS), measured by oxidation of dihydrorhodamine 123. This increase is abolished by intracellular superoxide scavenging by Mn(III)-tetrakis(1-methyl-4-pyridyl)-porphyrin, and reduced or absent in the presence of the flavoprotein/complex I inhibitors, diphenyl-eneiodonium and rotenone. The same inhibitors, but neither intra- nor extracellular (superoxide dismutase) superoxide scavenging, abolish the hypoxia-induced increase in tyrosine hydroxylase (TH) gene expression. Thus, ROS production increases in PC12 cells during hypoxia, but this is not the cause of hypoxic TH mRNA upregulation that involves a flavoprotein.

Animals↗
PubMed1999

Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus.

Gas chromatography-mass spectrometry was used to measure the oxidative DNA damage in diabetic subjects and controls. Levels of multiple DNA base oxidation products, but not DNA base de-amination or chlorination products, were found to be elevated in white blood cell DNA from patients with type II diabetes as compared with age-matched controls. The chemical pattern of base damage is characteristic of that caused by an attack on DNA by hydroxyl radical. An increased formation of the highly reactive hydroxyl radical could account for many of the reports of oxidative stress in diabetic subjects. There was no evidence of an increased DNA damage by reactive nitrogen or chlorine species.

Base Pairing↗
PubMed1999

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

BACKGROUND: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation. RESULTS: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences. CONCLUSIONS: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.

Amino Acid Sequence↗
PubMed1999

Measurement of local viscoelasticity and forces in living cells by magnetic tweezers.

We measured the viscoelastic properties of the cytoplasm of J774 macrophages with a recently developed microrheometer. Ferromagnetic beads (1.3 microm in diameter) were used to determine the local viscoelastic moduli. Step-force pulses were applied to the magnetic beads and the displacement was observed by single particle tracking. By analyzing the creep response curves in terms of a triphasic mechanical equivalent circuit, we measured the shear elastic modulus, the effective viscosities, and the strain relaxation time. The values of the shear modulus vary by more than an order of magnitude within the cell population (range, 20-735 Pa; average, 343 Pa) and by a factor of 2 within single cells. The effective viscosity of the cytoplasm exhibits a relatively sharp distribution about an average of eta = 210 Pa s (+/- 143 Pa s). We measured the displacement field generated by the local forces by observing the induced motion of nonmagnetic beads. Even at distances of the order of 1 microm, no induced motion was seen, suggesting that the cytoplasm is composed of clusters of densely packed and cross-linked filaments separated by soft regions. In another series of experiments we analyzed the magnetophoretic motion of the ferromagnetic beads at a constant magnetic force. Measuring the bead velocity parallel and perpendicular to the applied force showed that local active forces on the beads varied from 50 to 900 pN.

Animals↗
PubMed1999

Treatment of diabetic polyneuropathy with the antioxidant thioctic acid (alpha-lipoic acid): a two year multicenter randomized double-blind placebo-controlled trial (ALADIN II). Alpha Lipoic Acid in Diabetic Neuropathy.

Short-term trials with the antioxidant thioctic acid (TA) appear to improve neuropathic symptoms in diabetic patients, but the long-term response remains to be established. Therefore, Type 1 and Type 2 diabetic patients with symptomatic polyneuropathy were randomly assigned to three treatment regimens: (1) 2 x 600(mg of TA (TA 1200), (2) 600)mg of TA plus placebo (PLA) (TA 600) or (3) placebo and placebo (PLA). A trometamol salt solution of TA of 1200 or 600 mg or PLA was intravenously administered once daily for five consecutive days before enrolling the patients in the oral treatment phase. The study was prospective, PLA-controlled, randomized, double-blind and conducted for two years. Severity of diabetic neuropathy was assessed by the Neuropathy Disability Score (NDS) and electrophysiological attributes of the sural (sensory nerve conduction velocity (SNCV), sensory nerve action potential (SNAP)) and the tibial (motor nerve conduction velocity (MNCV), motor nerve distal latency (MNDL)) nerve. Statistical analysis was performed after independent reviewers excluded all patients with highly variable data allowing a final analysis of 65 patients (TA 1200: n = 18, TA 600: n = 27; PLA: n = 20). At baseline no significant differences were noted between the groups regarding the demographic variables and peripheral nerve function parameters for these 65 patients. Statistically significant changes after 24 months between TA and PLA were observed (mean +/- SD) for sural SNCV: +3.8 +/- 4.2 m/s in TA 1200, +3.0+/-3.0m/s in TA 600, -0.1+/-4.8m/s in PLA (p < 0.05 for TA 1200 and TA 600 vs. PLA); sural SNAP: +0.6+/-2.5 microV in TA 1200, +0.3+/-1.4 microV in TA 600, -0.7 +/- 1.5 microV in PLA (p = 0.076 for TA 1200 vs. PLA and p < 0.05 for TA 600 vs. PLA), and in tibial MNCV: +/- 1.2 +/- 3.8 m/s in TA 1200, -0.3 +/- 5.2 m/s in TA 600, 1.5 +/- 2.9 m/s in PLA (p < 0.05 for TA 1200 vs. PLA). No significant differences between the groups after 24 months were noted regarding the tibial MNDL and the NDS. We conclude that in a subgroup of patients after exclusion of patients with excessive test variability throughout the trial, TA appeared to have a beneficial effect on several attributes of nerve conduction.

Action Potentials↗
PubMed1998

Expression, purification, and spectroscopic studies of the guanine nucleotide exchange domain of human elongation factor, EF-1beta.

Two guanine nucleotide exchange domains, corresponding to the C-terminal region of the human translational elongation factor EF-1beta (which consists of 225 amino acids), were produced by DNA recombinant overexpression techniques in Escherichia coli. We describe here a fast and efficient method for purifying these two protein fragments and for concentrating their solutions rapidly to a level as high as 25 mg/ml. This technique permitted the isolation of 20-30 mg of pure, native protein per liter of bacterial culture. Both fragments were able to form a complex with their natural substrate, elongation factor EF-1alpha, as detected by gel filtration experiments. The domain of 110 residues was slightly more active than the 91-amino-acid domain in guanine nucleotide exchange assays. Folding and stability of the two C-terminal domains were explored by circular dichroism (CD) and NMR spectroscopy. In spite of optimal conditions concerning NaCl concentration, temperature, and pH, during the NMR experiments both proteins showed signs of aggregation after approximately 7 days at 303 degreesK, a time period and temperature required for future heteronuclear NMR experiments. Also, the longer fragment suffered from proteolysis in the N-terminal region, suggestive of flexibility in that part of the structure. The secondary structure content for these two EF-1beta fragments was estimated, using data from both CD and NMR. The results of both methods agree very well and indicate for each fragment the presence of approximately 20% alpha-helix and approximately 50% beta-sheet. Elucidation of the three-dimensional structure of the exchange domain of EF-1beta by NMR spectroscopy appears therefore feasible.

Animals↗
PubMed1997

Termination of quiescence in crustacea. The role of transfer RNA aminoacylation in the brine shrimp Artemia.

In quiescent embryos of the brine shrimp Artemia, the level of aminoacylation of transfer RNAs is low. During resumption of development the charging level of transfer RNAs increases, concomitant with the activation of protein synthesis. The total level of charging rises dramatically from an average of 4% to 50% within a period of 24 h of development. The restriction of in vitro translation of the quiescent embryo extract can be partially released by the addition of charged aminoacyl-tRNA, which apparently starts the flow of ribosomes into polyribosome structures. Complete reactivation of translation by aminoacyl-tRNA occurs when mRNA from preformed mRNA-ribosome complexes, like the polyribosomes extracted from developing embryos or poly(U)-programmed ribosomes, are offered to quiescent embryo extracts. With respect to the mechanism of in vivo recharging of tRNAs, we observed that the level of several aminoacyl-tRNA synthetases increase during development. Methionyl-tRNA synthetase rises more than 10-fold. In the case of valyl-tRNA synthetase, the activation is lower and shown to be due to the de novo synthesis of its mRNA and the corresponding protein product as well. We conclude that protein synthesis and thereby the gradual animation of cryptobiotic Artemia embryos is determined to a large extent by the rate by which aminoacyl-tRNAs are replenished during development at both the initiation and elongation level.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Isolation and characterization of the protein components of the liver microsomal O2-insensitive NADH-benzamidoxime reductase.

Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochrome b5, its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83-92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b5 and NADH cytochrome b5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxic N-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason why N-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.

Amino Acid Sequence↗