Biomedical subjects

M Böttger

Publications and source records attributed to M Böttger.

At least 19 recordsLinked to original sources

PubMed1998

Acid nuclear extracts as mediators of gene transfer and expression.

In an attempt to demonstrate transfection-active DNA packaging proteins in the cell nucleus, we prepared acid nuclear extracts with perchloric acid and subsequent protein fractions by stepwise acetone precipitation. The original extract and these fractions containing different compositions of nuclear proteins were used as DNA packaging agents. After the formation of complexes between these protein fractions and reporter genes, the addition of these complexes to the cells resulted in high transfection rates. Gel electrophoresis shows that the most active fractions contain histone H1 and HMG17. HMG1 exhibits a smaller activity. This result was confirmed by positive transfection experiments with commercial histone H1. Our results show that the transfection activity of acid nuclear protein fractions and histone H1 is dependent on the presence of calcium.

3T3 Cells↗

PubMed1997

Oxidoreductases in plant plasma membranes.

Electron transporting oxidoreductases at biological membranes mediate several physiological processes. While such activities are well known and widely accepted as physiologically significant for other biological membranes, oxidoreductase activities found at the plasma membrane of plants are still being neglected. The ubiquity of the oxidoreductases in the plasma membrane suggests that the activity observed is of major importance in fact up to now no plant without redox activity at the plasmalemma is known. Involvement in proton pumping, membrane energization, ion channel regulation, iron reduction, nutrient uptake, signal transduction, and growth regulation has been proposed. However, positive proof for one of the numerous theories about the physiological function of the system is still missing. Evidence for an involvement in signalling and regulation of growth and transport activities at the plasma membrane is strong, but the high activity of the system displayed in some experiments also suggests function in defense against pathogens.

Biological Transport↗

PubMed1997

High-mobility-group proteins and cancer--an emerging link.

In the last few years, considerable interest has been generated in the role of high-mobility-group (HMG) proteins, and HMG box proteins generally, in cancer development and therapy. These proteins were discovered in the early 1970s (Goodwin et al. 1973) as a group of nonhistone proteins. Some members of the HMG protein family (i) constitute a class of important architectural proteins involved in transcriptional regulation of genes, (ii) are frequently expressed in transformed cells at levels that correlate with the degree of neoplastic cell transformation, (iii) participate in gene rearrangements, which are linked to the emergence of benign solid tumors, (iv) confer the ability to recognize DNA-cisplatin adducts selectively, and (v) provide a new delivery system for efficient gene transfer. It should be considered that some HMG proteins, acting as architectural proteins that bring many of the transcription factors into precise three-dimensional shapes, may have a similar critical role in neoplastic transformation to that of some transcription factors themselves.

Cell Line, Transformed↗

PubMed1997

H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer.

In this article we describe the chromatographic separation of acid nuclear protein fractions which have previously been shown to be active in DNA transfection experiments. By combining anionic and cationic ion exchangers, we were able to separate and identify some of the active proteins. In addition to HMG1, already known for its transfection activity, we have identified histone H1 and HMG17 as further transfection-active proteins. The highest transfection activity was associated with H1 and another nonidentified protein showing a somewhat higher electrophoretic mobility than H1. We have also found that the presence of CaCl2 in a low concentration in the cell culture medium is an important requirement for transfection.

Animals↗

PubMed1997

Fast isolation of RNA to detect expression of tumor markers.

The expression status of several tumor-related proteins is of great interest in clinical examination and research. As a completion to conventional antibody staining, RT-PCR is often used today. Reliable isolation of RNA from a low number of cells is very often a critical stage of such an examination. We demonstrate here a simple and fast method to isolate RNA from only 10,000 cells and applied it to the detection of CEA, c-ERB-B2, and mdr-1 as often studied models for tumor markers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

The effect of boron on plasma membrane electron transport and associated proton secretion by cultured carrot cells.

Plasma membrane electron transport reactions and associated proton secretion were studied in boron-deficient carrot cells. It was found that the hormone-sensitive plasma membrane NADH oxidase was inhibited by boron deficiency and that under such conditions activity could be restored by exogenous boric acid with or without 2,4-dichlorophenoxy acetic acid. Gramicidin, a channel-forming protonophore, further stimulated NADH oxidase by carrot cells. Proton secretion, associated with plasma membrane H(+)-ATPase, was also affected by boron deficiency, but not as severely as ferricyanide-generated proton secretion, reflecting plasma membrane electron transport. The addition of 1 mM boric acid and 1 microM 2,4-dichlorophenoxy acetic acid to carrot cells fully restored the H+ secretion in presence of ferricyanide. The effect of boron deficiency in cultured carrot cells can, therefore, be directly associated with cell growth through its effect on the plasma membrane NADH oxidase and H+ secretion. Ferricyanide provides a probe which activates transmembrane electron transport that is only coupled to proton release when boron is present.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1992

Inhibitors of the plasma membrane redox system of Zea mays L. roots. The vitamin K antagonists dicumarol and warfarin.

The action of the 4-hydroxycoumarins dicumarol and warfarin, antagonists of probable vitamin K type components of the plasma membrane electron-transport system, on plasma membrane redox activity of intact maize roots was compared. Both effectors inhibited electron transfer to extracellular hexacyanoferrate III. While the effect of the strongly lipophilic dicumarol on the electron-transport system was irreversible by rinsing, the inhibition caused by the hydrophilic warfarin could be reverted completely by exchange of the incubation medium. We take these results as possible evidence for the integration of dicumarol into the plasma membrane. The action of warfarin may be confined to enzymic sites freely accessible from the aqueous apoplasmic solution.

Cell Membrane↗

PubMed1992

Effect of D2O on maize plasmalemma ATPase and electron transport coupled proton pumping.

Heavy water (D2O) has been used as a putative inhibitor of the plasma membrane H(+)-ATPase and the plasma membrane redox system. Concentrations above 50% D2O inhibited H+ secretion and the plasma membrane redox system of Zea mays L. roots. Inhibition of H+ secretion by vanadate was reduced in presence of D2O. The plasma membrane of roots was transiently depolarized after the addition of heavy water in concentrations above 5%. The repolarization of the plasma membrane that takes place while the H+ secretion is still reduced by heavy water indicates that, despite the overall inhibiting effect of D2O, the plant is still able to regulate the membrane potential.

Cell Membrane↗

PubMed1990

Electron donation to the plasma membrane redox system of cultured carrot cells stimulates proton release.

Membrane-permeable electron donors, duroquinol, diphenylcarbazide, pyrocatechol and tert-octylcatechol, promoted both reduction of an impermeant electron acceptor and proton transport with cultured carrot cells. These cells were preloaded with electron donors for 15, 30, 45 and 60 min. Aliquots of cells were removed at various times, washed free of excess electron donors and assayed for their effect on transplasma membrane redox with impermeable hexacyanoferrate (HCF III) as the electron acceptor and for simultaneous H+ excretion in the presence of hexacyanoferrate. All four electron donors stimulated HCF III reduction and associated H+ excretion. Below a rate of hexacyanoferrate reduction of 6 mumol/g dry wt. per min, the ratios of H+/e- were between 0.3 and 1 with low concentrations (0.1 mM) of the added electron donors. When hexacyanoferrate reduction exceeded 6 mumol/g dry wt. per min, proton release began to cascade to give ratios of 1 to 3, suggesting activation of an H(+)-ATPase or a proton transporter. This behavior by cultured carrot cells indicates that a certain threshold of proton concentration in a limited membrane domain must be reached in order for the proton channel to be opened.

Biological Transport, Active↗

PubMed1990

Transfection by DNA-nuclear protein HMG1 complexes: raising of efficiency and role of DNA topology.

We have developed a novel and efficient transfection method based on the introduction of foreign DNA into mammalian cells in form of complexes of vector DNA with the nuclear protein HMG1. In this study, it is shown that a stabilization of the complexes against dilution dissociation by addition of soluble CaCl2 or by excessive HMG1 enhances the transfection efficiency. Furthermore, there are no differences in the transfection abilities between the 3 topological DNA forms, viz., supercoiled, open relaxed and linear DNA, if delivered to cells as HMG1-DNA complexes. It is further shown that transfection-inactive complexes of the core histones with foreign DNA can be activated in transfection by the addition of HMG1.

Animals↗

PubMed1988

Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection.

The aim of this study was the search for a method of vector packaging using natural chromatin constituents. The interaction of the chromosomal non-histone protein HMG1 with a vector plasmid (pLTEneo) was studied by sedimentation analysis and electron microscopy at physiological salt concentration. At high protein input the complexes exist in a condensed, monodisperse form sedimenting with 80 S irrespective of the supercoiled or relaxed conformation of DNA. Saturation binding is already observed at much lower input ratios. Dilution of 80 S complexes results in decondensation of the complexes. In the decondensed complex form, HMG1 binds in a bead-like manner to specific DNA regions. Condensation by HMG1 is sufficient to introduce the vector into mammalian cells without the need for unphysiological additives. The transfection rates were similar to or even higher than those obtained by the calcium phosphate coprecipitation technique.

Animals↗

PubMed1985

Heterogeneity, molecular weight and stability of an oncogenic papovavirus of the Syrian hamster.

Sedimentation, isopycnic density gradient and molecular weight studies of an oncogenic papovavirus of the Syrian hamster have been performed. The sedimentation coefficient of 223 S, the buoyant density of 1.340 g/ml, and the molecular weight of 27.5 X 10(6) are in close correspondence with those of SV 40 (simian virus 40) and polyoma viruses. Deviating from these members of the papovavirus group, a sedimentation heterogeneity resulting in the presence of dimers and higher virion oligomers up to pentamers besides heterogeneous material of viral origin was demonstrated by sedimentation velocity. Also, a microheterogeneity of the buoyant density of the virus was demonstrable which is discussed to reflect this virion association. The stability of hamster papovavirus was examined after exposure to alkaline pH and the virus destabilizing agents dithiothreitol and ethyleneglycol-bis-N,N'-tetraacetic acid. In neutral conditions a nucleoprotein complex of 170 S was demonstrated. Further disintegration of this complex with increasing pH resulted in its conversion to nucleoprotein complexes of 125 S and 90 S in analogy to SV 40 and polyoma viruses.

Animals↗

PubMed1985

Physicochemical properties of salt-soluble, unsheared chromatin. Hydrodynamic model calculations supporting a double-fibrillar structure.

Hydrodynamic model calculations of a special, salt-soluble chromatin fraction were performed on the basis of former experimental data. Using the models of an extended helix and of a cylinder fitted to this helix we conclude that in 0.1 M (NH4)2SO4 this chromatin has the conformation of a nucleosome double-fiber helix, i.e. a structure in which two helices share a common axis. Besides this helical structure, a stretched, linear double-fibrillar form can be derived from sedimentation data on the basis of a simple cylinder model. From the kinetic behaviour of experimental sedimentation coefficients which decrease shortly after chromatin extraction a conformational transition of the helical to the fibrillar form is postulated. At 0.2 M (NH4)2SO4 where histone H1 is released, these double-structures are supposed to dissociate into two single molecules with similar dimensions.

Chemical Phenomena↗

PubMed1984

Interaction of histone H1 with superhelical DNA. Conformational studies and influence of ionic strength.

The interaction of histone H1 with superhelical SV40 DNA at low ionic strength (approximately 0.02 M NaCl) results in the formation of DNP double-fibers and bundle- and cablelike twisted side-by-side associates of several of these double-fibers. On the basis of simple cylindrical or ellipsoidal models the sedimentation properties of these structures can be calculated in accordance with the experiment allowing a direct assignment of electron microscopical and hydrodynamic results. Sedimentation measurements in dependence on the ionic strength indicate a redistribution of H1 resulting in the formation of associates at 0.04 M NaCl and of aggregates at higher salt concentration. Double-fibers are present up to physiological salt concentrations.

DNA, Superhelical↗

PubMed1983

In vitro transformation and mutation of Chinese hamster cells by different SV40 nucleoprotein complexes.

SV40 minichromosomes (MCH) either isolated from SV40 infected CV-I monkey cells (native MCH) or reconstituted in vitro from viral DNA and the H1 depleted calf thymus histone fraction could transform and mutate Chinese hamster (CH) cells in vitro. Whereas reconstituted MCH transformed and mutated CH cells with about the same efficiency as purified SV40 DNA, approximately 10-200-fold increase in the transforming activity had been demonstrated for native MCH. All transformed cell colonies and a major part of the isolated mutant cell clones recovered after inoculation of CH cells with SV40 MHC expressed the SV40 T antigen. Addition of H1 to both purified SV40 DNA and reconstituted MHC drastically diminished the transforming capacities of both agents. Possible reason(s) for the inhibition effect of H1 histone is discussed.

Animals↗

PubMed1982

Physicochemical properties of salt-soluble, unsheared chromatin. Molecular weight studies.

A chromatin fraction, which can reproducibly be extracted from rat liver nuclei at moderate salt concentration (0.1 M (NH4)2SO4, 0.1 M Tris-HCl, 2 mM MnCl2, pH 7.9), was analyzed with regard to changes of its molecular weight in the range of (NH4)2SO4 concentrations between 0.1 M and 0.4 M. With the transition from 0.1 M to 0.2 M (NH4)2SO4 histone H1 is released and the molecular weight obtained from both sedimentation-viscosity and light scattering is reduced by approximately one-half. A spatial expansion of the resulting half-molecules is observed with further increasing salt concentration. On the basis of these results a double-fibrillar structure of this chromatin fraction is proposed.

Animals↗

PubMed1982

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes.

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native 'minichromosomes'. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.

Animals↗

PubMed1982

Curative effect of alpha-difluoromethylornithine on fatal Trypanosoma congolense infection in mice.

The curative effect of alpha-difluoromethylornithine (DFMO) on fatal T. congolense infection in mice was investigated using various concentrations administered orally in drinking water. Permanent cure was achieved in mice receiving 4% for 3 days or 2% for 5 days. Mice receiving less became parasitemic again but then, without any further treatment, resisted death due to trypanosomes for an average of 34.5 days, while untreated controls were dead within 8 days after trypanosomes were first seen in their blood.

Animals↗