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W Möller

Publications and source records attributed to W Möller.

At least 37 records · Page 2Linked to original sources
PubMed1997

Probing mechanical properties of living cells by magnetopneumography.

Magnetopneumography (MPG) has been used to study long-term particle clearance from human lungs as well as cellular motility of pulmonary macrophages (PMs). This study describes an extension of the method enabling the measurement of mechanical properties of PM cells in vivo. Ferromagnetic microparticles are inhaled and then retained in the alveolar region of the lungs, where they are phagocytized within hours by PMs. The magnetic particles can be rotated in weak magnetic fields, and the response to this twisting shear (force) is detected as a macroscopic magnetic field producing a measure of cytoskeletal mechanics. Cytoplasmic viscosity is very high compared with that of water and is strongly non-Newtonian. Under rotational stresses from 0.4 to 6.4 Pa, it acts like a pseudoplastic fluid showing a characteristic shear rate dependence. The viscosity as well as the stiffness of the cytoskeleton increases with increasing shear stress as seems typical for living tissue and evidence for an intact cytoskeletal matrix. The particle recoil as measured by the amount of recoverable strain following a short twisting force describes a cytoplasmic elasticity that depends on both level and duration of stress. These investigations on the mechanical properties of living human cells are promising and should lead to better understanding of cellular dysfunction in disease as well as pathways for drug administration.

Adult↗
PubMed1996

Smoking impairs alveolar macrophage activation after inert dust exposure.

Magnetopneumography was applied to investigate intracellular phagosome motion in alveolar macrophage cells of healthy subjects (non-smokers and smokers). Ingested magnetic microparticles are inhaled and phagocytized by alveolar macrophages within hours. Thereby the particles are transferred into phagolysosomes. After magnetization the particles produce a macroscopic magnetic field of the lungs. Cellular motility causes a decay of the field (relaxation) by stochastic disorientation of the dipole particles (phagolysosomes) in the cells. Our studies have shown that the deposition of magnetite test particles induces a non-specific activation of the macrophage cells with a faster relaxation. This activation vanishes within the first day after particle deposition. This macrophage activation due to dust exposure was not present in smokers. It follows that cigarette smoking either causes a damage of the cellular defense or causes an adaptation of the macrophage cells to the permanent cigarette smoke inhalation.

Administration, Inhalation↗
PubMed1996

Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1 beta gamma delta in the endoplasmic reticulum.

The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1 alpha, EF-1 beta, EF-1 gamma and EF-1 delta which induce efficient transfer of aminoacyl-tRNA to the ribosome. In this process EF-1 alpha.GTP acts as the carrier of the aminoacyl-tRNA on its way to the ribosome. After release of aminoacyl-tRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1 alpha.GDP form is recycled to EF-1 alpha.GTP by EF-1 beta gamma delta. In eukaryotic cells the concentration of EF-1 alpha exceeds that of the complex beta gamma delta by a factor of 5-10. In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed. In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1 beta gamma delta, was found to co-localize with the endoplasmic reticulum (ER), displaying a distinct fine-structure in its staining pattern. The guanine nucleotide-binding subunit of EF-1, EF-1 alpha, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.

Amino Acid Sequence↗
PubMed1996

Stimulation of integrin receptors using a magnetic drag force device induces an intracellular free calcium response.

Mechanical loading of cells is of fundamental relevance in physiological processes and induces several functional responses in cells. Integrins, a family of adhesion receptors, which are responsible for the interaction with the extracellular matrix, may play a role in transmission of mechanical signals into cells. The osteogenic cell line U-2 OS expresses different integrin subunits which are uniformly distributed over the cell surface. We applied defined physical forces on individual integrin receptor subunits using paramagnetic microbeads coated with anti-integrin antibodies. Application of an inhomogeneous magnetic field consequently leads to a mechanical stress on the receptor. Intracellular Ca2+ increased when the alpha 2 or the beta 1 integrin subunits were stressed, whereas mechanical loading of the transferrin receptor had a significantly lower effect. This result indicates that forces specifically exerted to individual integrin receptors induce signal transduction pathways.

Calcium↗
PubMed1995

Valyl-tRNA synthetase from Artemia. Purification and association with elongation factor 1.

Two components of the protein biosynthetic machinery, valyl-transfer RNA synthetase (VRS) and elongation factor 1 (EF-1), have been isolated as a complex from several mammalian tissues. However, yeast VRS, which lacks an amino-terminal extension, does not associated with EF-1. We purified VRS from the brine shrimp Artemia and investigated its interaction with EF-1. Western blotting of crude Artemia extracts revealed the presence of two forms of VRS, differing in size and capacity to associate with EF-1. About 80% of the total VRS corresponds to a polypeptide of 130 kDa which behaves as a monomer upon gel filtration. Only the larger form of 140 kDa coelutes, cosediments and co-immunoprecipitates with the EF-1 alpha 2 beta gamma delta complex. The ratio of the two forms of VRS remains constant throughout early development. The possible origin and mode of expression of the two forms of VRS present in Artemia are discussed.

Amino Acid Sequence↗
PubMed1994

A mutant insulin receptor induces formation of a Shc-growth factor receptor bound protein 2 (Grb2) complex and p21ras-GTP without detectable interaction of insulin receptor substrate 1 (IRS1) with Grb2. Evidence for IRS1-independent p21ras-GTP formation.

The activation of p21ras by receptor tyrosine kinases involves the translocation of the growth factor receptor bound protein 2-mammalian son of sevenless protein (Grb2-SOS) complex to the plasma membrane where p21ras is localized. Insulin receptors induce p21ras-GTP formation by two possible mechanisms: tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent association with Grb2, or Shc phosphorylation and its subsequent association with Grb2. We investigated the contribution of the major tyrosine autophosphorylation sites Tyr1158, Tyr1162, and Tyr1163 of the insulin receptor to IRS1.Grb2 and Shc.Grb2 association and the formation of p21ras-GTP. Chinese hamster ovary-derived cell lines were used overexpressing mutant insulin receptors in which the major tyrosine autophosphorylation sites were stepwise replaced by phenylalanines. In cell lines expressing wild type or mutant Y1158F,Y1162,Y1163 (FYY) receptors, insulin stimulated tyrosine phosphorylation of IRS1 and Shc and the formation of IRS1.Grb2 and Shc.Grb2 protein complexes, together with an increase in p21ras-GTP. Cell lines expressing mutant Y1158,Y1162F,Y1163F (YFF) receptors showed insulin-induced tyrosine phosphorylation of Shc, Shc.Grb2 complex formation, and p21ras-GTP formation, whereas tyrosine phosphorylation of IRS1 was strongly decreased and formation of IRS1.Grb2 complexes was undetectable. The activity of FYY and YFF receptors to mediate p21ras-GTP formation correlated with their activity to induce Shc phosphorylation and Shc.Grb2 association. The mutant insulin receptors Y1158F,Y1162F,Y1163 and Y1158F,Y1162F,Y1163F were inactive in inducing any of these responses. We conclude that phosphorylation of Tyr1158 and Tyr1162 of the insulin receptor is linked to distinct post-receptor processes and that YFF receptors generate p21ras-GTP via the Shc.Grb2 pathway rather than one involving IRS1.Grb2 interaction.

Adaptor Proteins, Signal Transducing↗
PubMed1994

The subunit structure of elongation factor 1 from Artemia. Why two alpha-chains in this complex?

Elongation factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the elongation phase of protein biosynthesis. Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex. We describe here the complete subunit structure of elongation factor 1, and discuss its change during development of Artemia. Elongation factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1. Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha 2 beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached. Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the elongation step of prokaryotic protein synthesis.

Amino Acid Sequence↗
PubMed1994

The leucine-zipper in elongation factor EF-1 delta, a guanine-nucleotide exchange protein, is conserved in Artemia and Xenopus.

Elongation factor 1, a complex involved in protein biosynthesis, contains two guanine-nucleotide-exchange proteins EF-1 beta and EF-1 delta. The sequence of EF-1 delta of Artemia was determined with the purified protein. When compared to EF-1 delta from Xenopus, a high degree of identify (80%) was found in the C-terminal domains of the proteins, which contain the guanine-nucleotide-exchange activity. The N-terminal domains share only 23% of the amino acids at identical positions, and therefore they were further analysed for less obvious types of homology. To this end, a published approach for sequence analysis, which can detect peculiar amino acid patterns in proteins was applied. In this way, a weak albeit unmistakable similarity between the two EF-1 delta proteins was demonstrated in the region of the leucine-zippers, apart from the leucine repeat itself. Apparently, they display a common structural pattern in their N-terminal domains, which so far has been observed mainly in transcription factors.

Amino Acid Sequence↗
PubMed1994

Activation of overexpressed receptors for insulin and epidermal growth factor interferes in mitogenic signaling without affecting the activation of p21ras.

Activated receptors with a tyrosine kinase activity induce a variety of responses like changes in the differentiation and mitogenic status of cells. These responses are mediated in part by p21ras. Some of these activated receptors induce in certain cell types a pronounced, but transient, increase in Ras-GTP. We have stimulated cells with insulin, epidermal growth factor (EGF), and fetal calf serum (FCS), and the mitogenic response, as reflected by stimulation of [3H]thymidine incorporation, was compared with the magnitude of the transient increase in Ras-GTP levels. Cell lines were used that expressed both physiological and elevated numbers of p21ras and receptors for insulin and EGF, respectively. In all the examined cell lines 9% FCS did not induce a marked increase in Ras-GTP despite its high mitogenic potency. Pronounced increases in Ras-GTP levels were observed in insulin-stimulated CHO cells which overexpress insulin receptors whereas in the parental CHO cells only a small increase is seen. Insulin (1 microM) and FCS (9%) stimulate [3H]thymidine incorporation in parental CHO cells to a similar high level whereas in insulin receptor overexpressing CHO cells the maximum of insulin-stimulated [3H]thymidine incorporation is only 55% of the level reached by 9% FCS. In those cells the maximum is already reached at low (1 nM) insulin concentrations. Remarkably, at higher insulin concentrations stimulation of [3H]thymidine incorporation starts to decrease strongly despite the fact that the magnitude of the transient increase in Ras-GTP and subsequent MAPkinase activation increases. Similarly, when EGF receptors are overexpressed in Rat-1 cells, the mitogenic response is also decreased at higher EGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A mutation in the insulin receptor that impairs proreceptor processing but not insulin binding.

Here we report the identification of a new mutation in the alpha-chain of the insulin receptor, changing Trp412 into Ser using DNA from consanguineous parents who gave birth to a child with leprechaunism. The mutant receptor was expressed stably in CHO and transiently in COS-1 cells. It was found that the Ser412 mutant is not cleaved into alpha- and beta-subunits and remains as a 210-kDa proreceptor at an intracellular site. This property of the mutant receptor is in line with the observed decreased insulin binding to the parental fibroblasts. Cross-linking experiments show that the Ser412 proreceptor is able to bind insulin with an affinity comparable to that of the wild-type alpha-chain. Despite its capacity to bind insulin, the mutant receptor is not autophosphorylated. We postulate that the patient was homozygous for the Trp412-->Ser mutation and that the mutation was responsible for the leprechaun phenotype. This is the first description of a transport-defective receptor with the mutation outside the tetrabasic processing site and a functional insulin binding domain. The ability of the Ser412 mutant to bind insulin in cross-linking experiments suggests that the impaired transport of the proreceptor to the cell surface is the primary cause for the binding defect to intact cells.

Animals↗
PubMed1994

In vivo and in vitro studies of the cellular defense system of the human lung.

Magnetic microparticles were used to investigate the defence system of the human lungs against foreign material. About 0.5 mg of spherical monodisperse magnetite particles were deposited in the alveolar region of the human lung by voluntary inhalation. After primary magnetization a remanent magnetic field (RMF) of the lung can be measured that allows estimation of the amount of dust retained in the lung. The decay of this RMF, called relaxation, results from a misalignment of the dipole particles due to the activity of pulmonary macrophages. This macrophage activity was characterized by a cell energy Ez. With a secondary magnetization the lung can be remagnetized by rotation of the dipole particles. This allows estimation of the intracellular viscosity and the motility of the alveolar macrophages in vivo. The macrophage cell-line J774 was used to verify the dynamic processes of the magnetic particles within the cells in vitro. In vitro and in vivo relaxation curves of polydisperse and of spherical monodisperse magnetite particles are presented. Thermal relaxation of mono-disperse and polydisperse particles within a viscous standard could be verified with the Brownian rotary diffusion model. Relaxation with monodisperse particles was double exponential in vivo as well as in vitro, suggesting that 2 different viscous compartments of the cytoplasm should be considered. Relaxation in the macrophage cell-line J774 was particle-size-dependent.

Administration, Inhalation↗
PubMed1994

Chemical dehydration for rapid paraffin embedding.

We describe chemical dehydration with 2,2-dimethoxypropane (DMP) for rapid paraffin embedding using a mixture of DMP and mineral oil followed by mineral oil as clearing intermediates. This method is useful for classical histological techniques as well as for histochemistry and immunocytochemistry.

Desiccation↗
PubMed1993

The human leucine zipper-containing guanine-nucleotide exchange protein elongation factor-1 delta.

Copy-DNA clones containing the complete coding region of the human elongation factor-1 delta (EF-1 delta) mRNA have been isolated and characterized. We present the deduced amino acid sequence and observe in it a leucine zipper motif seen recently in EF-1 delta from Artemia and Xenopus laevis. The human EF-1 delta sequence shows a strong conservation in its C-terminal domain. The homology between the N-terminal domains of EF-1 delta proteins is low and almost exclusively limited to the leucine zipper motif.

Amino Acid Sequence↗
PubMed1993

Epidermal-growth-factor receptors generate Ras.GTP more efficiently than insulin receptors.

Activation of the Ras proto-oncogene contributes in general to mitogenic activation of cells. We show here that epidermal growth factor (EGF) stimulates Ras.GTP formation very efficiently in a variety of cell lines expressing endogenous EGF receptors only. Maximal activation of the receptor converts up to 65% of cellular p21ras from the GDP form into the active GTP-bound state. This efficient activation occurs also in cultured primary human fibroblasts. Maximal insulin-induced Ras.GTP formation is less but in cells overexpressing the insulin receptor a similar high response of Ras.GTP formation is observed after insulin stimulation. Not only the efficiency but also the kinetics by which the EGF and insulin receptors stimulate Ras.GTP formation are quite distinct. In the Rat-1-derived cell line, H13IR2000, overexpressing both p21Ha-ras and the insulin receptor, the activated insulin receptor generates approximately 1 mol Ras.GTP/mol activated insulin receptor. The activated EGF receptor amplifies the signal, resulting in the activation of approximately 40 mol p21ras/mol receptor. Moreover, EGF-stimulated generation of Ras.GTP is transient with a maximum after 2 min of hormone stimulation and diminishes to near basal levels within 1 h whereas the insulin-induced Ras.GTP levels are maximal at 5-10 min and decline only slowly to half-maximal in 1 h. Desensitization of the EGF pathway by prolonged EGF stimulation, prevents subsequent stimulation of Ras.GTP formation by newly added EGF but not by insulin. Vice versa, in cells preincubated with insulin for 1 h, EGF stimulates Ras.GTP formation to near maximal values. These observations indicate that desensitization by prolonged hormone incubation does not involve inactivation of common signaling intermediates but rather components, specific for each pathway, like the particular receptors. The rapid down regulation of EGF receptors compared to insulin receptors corroborate this possibility. The observed high potency of EGF receptors to generate Ras.GTP may explain the, in general, stronger mitogenic activity of EGF compared to insulin.

3T3 Cells↗
PubMed1993

Behaviour of magnetic micro-particles in the human lung.

Magnetic micro-particles were used to investigate the defence system of the human lungs against foreign material. After primary magnetisation a remanent magnetic field (RMF) of the lung can be measured that allows estimation of the amount of dust retained in the lung. After calibration of the system with a lung phantom the magnetic contamination retained in the lungs of dental technicians and welders was estimated at mean values of 22 and 500 mg respectively. In normal controls only 0.3 mg was found. About 0.5 mg of spherical monodisperse magnetite particles was deposited in the alveolar region of the lung by voluntary inhalation. The decay of the RMF, called relaxation, results from a misalignment of the dipole particles due to the activity of pulmonary macrophages. This macrophage activity is characterised by a cellular energy Ez. With a secondary magnetisation the lung can be remagnetised by rotation of the dipole particles. This allows an estimation of the intracellular viscoelasticity and the motility of the alveolar macrophages in vivo. Secondary magnetisation and relaxation curves of spherical monodisperse magnetic particles are presented. Intracellular viscosity was estimated to be n approximately equal to 100 Pa.s at shear rates near 0.01 s-1, the rigidity modulus being v approximately equal to 4-8 Pa. Macrophage activity was described by a cellular energy EZ approximately 5 x 10(-18) J. Additionally, non-magnetic aerosol exposure resulted in a faster relaxation, which was interpreted to be due to activation of the macrophages. The magnetite particles were cleared with a half-time of approximately 110 days.

Administration, Inhalation↗
PubMed1992

Elongation factor-1 messenger-RNA levels in cultured cells are high compared to tissue and are not drastically affected further by oncogenic transformation.

Copy-DNA clones covering the complete coding sequence of human Elongation Factor-1 gamma mRNA have been isolated and characterized. The expression of Elongation Factor-1 in a variety of cell lines and a number of tissues shows a large increase in Elongation Factor-1 mRNA going from tissue to cultured cells (20-fold). Messenger-RNA levels for Elongation Factor-1 alpha, -1 beta and -1 gamma increase in parallel suggesting coordinate regulation of the expression of these genes. Oncogenic transformation in vitro does not strongly affect Elongation Factor-1 mRNA levels.

Amino Acid Sequence↗
PubMed1992

Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.

In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced.

Amino Acid Sequence↗