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Biomedical subjects

W Ansorge

Publications and source records attributed to W Ansorge.

At least 91 records · Page 5Linked to original sources
PubMed1994

Nonradioactive, solid-phase DNase I footprints analyzed on an A.L.F. DNA Sequencer.

Solid-phase DNase I footprinting provides a powerful tool for analyzing the sequence-specific interactions of DNA binding proteins. Classically this type of assay requires radioactively labeled DNA molecules. Substitution of the isotope by fluorescein labeling of the DNA fragments enables the analysis of footprint patterns on a standard automated laser fluorescent (A.L.F.) DNA Sequencer. The combination of solid-phase footprinting technology and fluorescence-based nonradioactive detection of fragments has unique advantages over established footprinting technologies.

DNA-Binding Proteins↗
PubMed1994

Improved fluorescent cycle sequencing protocol allows reading nearly 1000 bases.

Recently available thermostable DNA polymerases result in enhanced resolution and accuracy compared to thermal enzymes used previously in fluorescent cycle sequencing. These new enzymes produce less variations in peak intensities, enhance gel resolution and are less sensitive to unspecific termination caused by either DNA structure or impurities in the DNA preparation. Optimization of nucleotide ratios and the usage of high concentrations of detergents in the sequencing reaction result in sequence readings up to 1000 bases and improve overall reliability of the sequencing protocol; this works successfully in about 90% of cases.

DNA-Directed DNA Polymerase↗
PubMed1993

The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells.

Three members of the Src family of protein tyrosine kinases Src, Fyn, and Yes associate with the activated platelet-derived growth factor (PDGF) receptor in vivo. This interaction requires the Src homology 2 (SH2) domain of the Src family member and causes activation of the intrinsic activity of the Src family kinases. We microinjected cells with DNA encoding catalytically inactive forms of the Src and Fyn proteins and examined their effects on PDGF-mediated signaling in vivo. Kinase-inactive Src and Fyn inhibited PDGF-stimulated entry of cells into S phase, whereas kinase-active forms of the proteins had no inhibitory effects. An intact SH2 domain was required for inhibition. Furthermore, when kinase-inactive Fyn was comicroinjected with a plasmid expressing activated Ras, the cells could enter S phase, indicating that the expression of kinase-inactive Fyn did not damage cell viability. Injection of an antibody specific for Src, Fyn, and Yes also reduced signal transduction through the PDGF receptor but only when injected within 8 hr of PDGF stimulation. Together these results indicate that the ubiquitously expressed Src family members are required for PDGF-induced mitogenic signaling.

3T3 Cells↗
PubMed1993

Cell biological studies with monoclonal and polyclonal antibodies against human casein kinase II subunit beta demonstrate participation of the kinase in mitogenic signaling.

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active (alpha and/or alpha') and two regulatory (beta) subunits. It has been suspected that, among numerous other cellular functions, CKII might play a role in the control of mitogenic signaling. To test for such a role and its mechanism in intact cells, monoclonal antibodies (mAbs) were generated against CKII beta using a recombinant protein containing amino acids 20-200 of human CKII beta. The CKII beta-specific mAb with the highest reactivity, mAb IVG6 (classified as IgG1 with kappa light chains), was purified to homogeneity. It recognized a CKII beta epitope comprising the amino acids 140-156, a basic and highly conserved region. In addition, polyclonal antibodies (pAbs) were raised and made monospecific by affinity purification. pAbs-mediated quantitative immunofluorescence microscopy of human IMR-90 fibroblasts and/or Western blots of cell fractions revealed (i) CKII beta was present in exponentially growing cells at a 2-3-fold higher level than in quiescent cells, (ii) CKII beta was localized predominantly in the nucleus of cells (3-15-fold cytoplasmic level depending on cellular state and assay used), and (iii) the nuclear/cytoplasmic ratio of CKII beta was higher by a factor of 2 in exponentially growing cells. Consequently, mitogenic stimulation of quiescent cells by fetal calf serum doubled the nuclear/cytoplasmic ratio of CKII beta. The increase occurred within the 1st h of stimulation. The translocation of CKII beta into the nucleus was inhibited when mAb IVG6 was injected into the cytoplasm at the time of mitogenic stimulation. This microinjection also significantly inhibited the cell proliferation. The data imply that cytoplasmic CKII participates in the transmission of mitogenic signals by translocation into the nucleus.

Antibodies, Monoclonal↗
PubMed1993

System for quantitation of gene expression in single cells by computerized microimaging: application to c-fos expression after microinjection of anti-casein kinase II antibody.

A system which allows sensitive and fast automated analysis of weakly labeled fluorescent specimens is described. It is tested in the analysis of c-fos expression stimulated by fetal calf serum and calibrated by quantitation of defined solutions injected into cells with the automated microinjection system. Low light level imaging technology combined with quantitative image processing methods and computer control of the hardware allows fully automated analysis of fluorescent molecules in single living or fixed cells. Reliable methods for subtraction of fluorescent background and automated identification of objects of interest in double-stained cells are described. The accuracy of quantitation is considerably improved by normalizing the fluorescence intensities of respective fluorophores in the same object by the method of ratio imaging. The error rate in determining the relative protein content in single cells is less than 15%. The method is applied to microinjection studies with a monoclonal antibody against casein kinase II subunit beta. Microinjection of this antibody into synchronized cells specifically inhibits c-fos expression stimulated by fetal calf serum. In combination with the computer-automated capillary microinjection system, the technique will become a useful tool in experiments requiring quantitative single cell analysis.

Antibodies↗
PubMed1993

Import of firefly luciferase into mammalian peroxisomes in vivo requires nucleoside triphosphates.

The insect enzyme firefly luciferase (FL), which is known to be imported into mammalian peroxisomes as well, was introduced into the cytoplasm of chinese hamster ovary cells and human skin fibroblasts by microinjection. This model system was used to study the nucleoside triphosphate dependence of peroxisomal protein import by immunofluorescence staining of FL following depletion of cellular ATP. In energized cells a punctate staining pattern of the enzyme is observed between 30 min and 1 week after microinjection suggesting an organellar localization of FL. Evidence for its peroxisomal localization was gained by comparison of the FL staining pattern with that of catalase, a peroxisomal marker. Differential permeabilization of cells with digitonin prior to immunofluorescence staining demonstrated the intraperoxisomal localization of microinjected FL and excluded the possibility that FL is merely adhering at the cytosolic face of peroxisomes without being imported. Depletion of cellular ATP by the metabolic inhibitors 2-deoxyglucose and NaN3 completely prevented import of FL into peroxisomes whereas upon reenergizing the cells FL import was restored. The import steps that may be responsible for the observed energy dependence are discussed.

Adenosine Triphosphate↗
PubMed1993

Membranes and polymer structures--biocompatibility aspects with respect to production limits.

Plasmapheresis can be performed by centrifugation and by use of membrane technology. With the latter technique we receive a plasma which is absolutely free from platelets. This is why membranes are gaining market shares in this particular field of medical application. Today plasmapheresis membranes are mostly fabricated from synthetic polymers, such as polypropylene (e.g. PLASMAPHAN), polysulfone, polyacrylonitrile, polymethylmethacrylate, polyvinylalcohol and others, the only exception being cellulose acetate. Parameters determining the biocompatibility of plasmapheresis membranes are generation of complement C3a or C5a, hemolysis and possible thrombus formation. These parameters depend on various properties of the membrane polymer: e.g. the nature of the molecular end/side-groups, the distribution of electrical charges on the polymer surface and the different chemical structures and conformation of the polymer. In addition, membrane properties like pore distribution and geometry or the flow characteristics of a particular device-design may trigger cell activation or influence biocompatibility through the adsorption of various plasmacomponents. Most of the polymers which are used today for manufacturing plasmapheresis membranes have not been developed for this purpose. They were originally selected to be used as textile fibers. Further, no present membrane polymer has been specifically developed to achieve high biocompatibility. The membrane profile was designed in such a way that pheresis properties were met rather than optimizing biochemical blood/polymer interactions. One reason for this decision may be that the market volume of plasmapheresis technology is too small in order to justify specific and high-cost developments of polymers for this purpose. Polymer selection to achieve excellent biocompatibility profiles is determined by polymer-availability, costs, membrane-forming processes and environmental aspects related to possible pollution during the manufacturing process. The production of PLASMAPHAN by the unique Accurel-process combines several of these parameters. The main membrane production processes and especially the Accurel-process are described here. The influence of polymer-surface properties, membrane structure and module-design on the biocompatibility of plasmapheresis treatments are discussed and explained by appropriate examples.

Biocompatible Materials↗
PubMed1993

Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts.

In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.

Amino Acid Sequence↗
PubMed1993

cDNA-derived molecular characteristics and antibodies to a new centrosome-associated and G2/M phase-prevalent protein.

Differential screening of a murine RNA-based cDNA library with cell cycle phase-specific transcripts released a cDNA clone (lambda CCD41) to a mRNA (1.349 kb) which, according to the mode of its detection, increases as expected during the cell cycle. The molecular characteristics of the protein (27 x 10(3) M(r)) encoded by this mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein. Immunofluorescence studies performed with PtK2 cells revealed that the amount of the antigen specified by the CCD41 sequence increases during the cell cycle out of proportion with the DNA content. In G1 phase cells, the antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes also detectable in perinuclear vesicles that increase in number and size, reaching a maximum in G2 phase cells. The centrosomal location of the CCD41 antigen was investigated in relation to another centrosomal antigen, centrosomin A. Since the latter antigen is detected by a monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome and to add new information about the general architecture of the organelle and its changes during the cell cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the centrosome, the CCD41-encoded protein appears to be localized as a compact structure inside the centrosome. Its epitopes are exposed throughout the cell cycle except during a brief period immediately after the formation of the daughter centrosome.

Amino Acid Sequence↗
PubMed1993

Automated low-redundancy large-scale DNA sequencing by primer walking.

Low-redundancy automated DNA sequencing by primer walking is described. T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 bases (in the unedited raw data). The low error rate allows efficient sequencing with low (2-3 times) redundancy. Plasmid subclones covering 20 kb of a cosmid insert were sequenced with an overall redundancy of 2.7 in the course of the European community Saccharomyces cerevisiae genome sequencing project. Neighboring plasmid subclones were linked by direct cosmid sequencing. Sets of ten walking primers are synthesized on the EMBL multiple segmental DNA synthesizer at low costs and used for sequencing with greater than 95% efficiency. The accuracy of the directed approach is improved by simultaneous walking on both strands by designing two primers in opposite directions in the same starting region. One primer is used to confirm sequence data on the opposite strand, and the other primer to obtain new sequence data.

Autoanalysis↗
PubMed1992

Human casein kinase II subunit alpha: sequence of a processed (pseudo)gene and its localization on chromosome 11.

A human 4.3 kb genomic DNA fragment, containing the information of a processed (pseudo)gene of casein kinase II subunit alpha (CKII alpha) was isolated and sequenced. The genomic CKII alpha sequence is 99% homologous to the CKII alpha cDNA, carries several nucleotide exchanges, a poly(A) stretch at its 3' end and is flanked at both ends by a 16 bp repeat. It has a promoter-like region including two TATA boxes and a CAAT box. Although translation of transcripts would be terminated by a stop codon after two third of the coding region, the resulting protein would still contain the catalytic domains. However, so far Northern blots with a 3' specific probe were negative. The 4.3 kb genomic fragment containing the processed CKII alpha (pseudo)gene was mapped by in situ hybridization to chromosome 11p15.

Base Sequence↗
PubMed1992

Cyclin A is required at two points in the human cell cycle.

Cyclins play a fundamental role in regulating cell cycle events in all eukaryotic cells. The human cyclin A gene was identified as the site of integration of hepatitis B virus in a hepatocarcinoma cell line; in addition, cyclin A is associated with the E2F transcription factor in a complex which is dissociated by the E1A oncogene product. Such findings suggest that cyclin A is a target for oncogenic signals. We have now found that DNA synthesis and entry into mitosis are inhibited in human cells microinjected with anti-cyclin A antibodies at distinct times. Cyclin A binds both cdk2 and cdc2, giving two distinct cyclin A kinase activities, one appearing in S phase, the other in G2. These results suggest that cyclin A defines novel control points of the human cell cycle.

Adenovirus Early Proteins↗
PubMed1991

Structure of the gene encoding human casein kinase II subunit beta.

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with numerous key functions in cell metabolism and growth. The human CKII has a tetrameric structure; two catalytic subunits (alpha and alpha') form the holoenzyme together with two presumably regulatory subunits (beta). The gene encoding CKII subunit beta was isolated from human genomic DNA and analyzed for its primary structure using exclusively nonradioactive procedures. The gene was found to span 4.2 kilobase pairs and to be composed of seven exons. Exon sizes range from 76 (exon 5) to 329 base pairs (bp) (exon 1), intron sizes from 145 (intron V) to 965 bp (intron II). All exon-intron junctional sequences conform to the canonical GT-AG rule. Primer extension analysis determined three transcription initiation sites, at 951, 919, and (minor) 840 bp upstream of the translation start site. The translation start is located early in the second exon; exon 1 is untranslated. The 3'-cleavage/polyadenylation signal sequence (AA-TAAA) is in the last exon at position 4173 bp relative to the first transcription initiation site. The coding sequence for CKII beta comprises 648 nucleotides identical to the published CKII beta-cDNA sequence (Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233). The upstream promoter region of the CKII beta gene contains multiple potential gene regulatory sequence elements, noticeable DNA structures, and the characteristics of a housekeeping gene (more than one transcription initiation site, lack of a TATA-box, presence of a CpG island, occurrence of multiple GC boxes and of nonstandard positioned CCAAT boxes). The CKII beta gene promoter shares common features with that of mammalian protein kinases and is closely related to the regulatory subunit gene promoter of cAMP-dependent protein kinase.

Amino Acid Sequence↗