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Biomedical subjects

W Ansorge

Publications and source records attributed to W Ansorge.

At least 127 records · Page 7Linked to original sources
PubMed1989

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Ammonium Chloride↗
PubMed1989

Cellulosic versus synthetic membranes: a reasonable comparison?

Of the two main classes of dialysis membranes, cellulosic and synthetic, the former represents the standard membrane used in dialysis therapy. The disputed properties of cellulosic membranes related to biocompatibility have inspired a number of authors to compare these two membrane classes from a variety of perspectives. However, such a strict categorization as "synthetic" or "cellulosic" is of doubtful value from the point of view of polymer chemistry. Here, biocompatibility and performance properties of these two membrane classes are compared with the aim of investigating the validity of this categorization. The biocompatibility parameters studied are complement and leukocyte activation together with activation of the coagulation cascade. Analysis of a variety of both cellulosic and synthetic membranes with different degrees of biocompatibility showed that biocompatibility can be achieved by both classes of membranes and is, therefore, not a particular property of one class only. Furthermore, performance and beta 2-microglobulin removal properties of the two classes of membranes do not particularly favor one of these classes. Therefore, differences between cellulosic and synthetic membranes are not manifested in parameters like biocompatibility, hydraulic permeability, and overall performance.

Biocompatible Materials↗
PubMed1988

Automated Sanger dideoxy sequencing reaction protocol.

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.

Autoanalysis↗
PubMed1988

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.

Base Sequence↗
PubMed1988

Non-radioactive automated sequencing of oligonucleotides by chemical degradation.

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.

Automation↗
PubMed1988

Single cell assay with an automated capillary microinjection system.

An automated capillary microinjection system with computer-controlled positioning of the cells and of the capillary, and its applications and advantages, is described. The system is easy in handling and manipulation. About 1500 injections are possible in 1 h, with high reproducibility. In cytoplasmic and nuclear injections more than 90 and 85% of the cells are successfully injected. Using FITC-dextran at a concentration of 0.5% as a fluorescently labeled coinjection marker, 99% of the cells can be retrieved in culture medium even 48 h after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of an mRNA inhibiting DNA synthesis in cells. Functional screening by cell injections of cDNA libraries and of size-fractionated mRNA molecules can be performed efficiently with the automated microinjection system.

Autoradiography↗
PubMed1988

Performance of an automated system for capillary microinjection into living cells.

An automated capillary microinjection system for nuclear and cytoplasmic injections into cells is described. The system has been tested with samples of DNA, RNA and proteins. Movements of the capillary with precise cell positioning and time of injections are controlled by a computer. This first automated microinjection system allows injection of more than 1500 cells per hour with a minimum of practical training, volumes injected are more reproducible and cells are less damaged when compared with the standard manual injection technique. Retrieval of the injected cells is accurate to within 1% without complicated and laborious produced orientation marks on the cell support. The number of successfully injected cells is easily determined with great accuracy and the error of the statistical evaluation of the results is reduced to a minimum. Standardized procedures for pulling, handling and storage of the injection capillaries were developed.

Animals↗
PubMed1988

Automatic microinjection system facilitates detection of growth inhibitory mRNA.

Naturally quiescent human lymphocytes, consisting predominantly of T cells, contain mRNA(s) that can inhibit DNA synthesis when injected into either human diploid fibroblasts (IMR-90) or transformed recipient cells (HeLa). By using an automated capillary microinjection system and a fluorescent coinjection marker (fluorescein isothiocyanate-dextran), individually injected cells can be retrieved and analyzed for DNA synthesis. mRNA isolated from resting T cells is able to block the cells from entering the S phase. The block is reversible and leads to a delay in DNA synthesis. The inhibitory effect is not observed if the injected mRNA is isolated from growth-activated T cells. The disappearance of the inhibition coincides with the approach of the G1/S boundary in both the donor T cells and the recipient human fibroblasts. The mRNA of resting T cells was size-fractionated and the peak inhibitory activity was recovered in a fraction approximately equal to 1.5 kilobases long.

Cell Division↗
PubMed1988

The 89,000-Mr murine cytomegalovirus immediate-early protein stimulates c-fos expression and cellular DNA synthesis.

The immediate-early (IE) genes of murine cytomegalovirus (MCMV) are expressed in the absence of prior viral protein synthesis and regulate the transcription of MCMV early genes. The effect of MCMV IE genes on growth induction was studied. Different plasmids containing MCMV IE genes were microinjected into arrested NIH 3T3 mouse fibroblasts. Plasmids containing the ieI gene coding for the 89,000-Mr major IE protein pp89 were found to stimulate the expression of the c-fos protooncogene. Synthesis of pp89 and its transport to the nucleus appeared to be required for c-fos expression. DNA synthesis occurred in cells that were injected with MCMV IE genes and in neighboring cells that were not injected. The results suggest that the phosphoprotein pp89 stimulates cells to enter the cell cycle.

Animals↗
PubMed1988

Two different mRNAs are transcribed from a single genomic locus encoding the chicken erythrocyte anion transport proteins (band 3).

The chicken erythrocyte anion transport protein (band 3 of the erythrocyte cytoskeleton) is a central component taking part in two widely divergent functions of erythroid cells; it is a primary determinant of cytoskeletal architecture and responsible for electroneutral Cl-/HCO3- exchange across the plasma membrane. To analyze interesting aspects of the developmental regulation of this gene, we have cloned the cDNA and genomic counterparts of the erythroid-specific anion transport protein. We show that a single genetic locus for band 3 encodes two different erythroid cell-specific mRNAs, with different translational initiation sites, which predict polypeptides of sizes very close to those observed in vivo. In vitro translation and immune precipitation of synthetic mRNA derived from one putative fully encoding cDNA clone demonstrate that this clone gives rise to a protein which is identical in size and antigenicity to bona fide chicken erythroid band 3.

Amino Acid Sequence↗
PubMed1987

A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.

A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.

Autoanalysis↗
PubMed1987

Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.

Autoanalysis↗
PubMed1987

Ethylene oxide in dialyzer rinsing fluid: effect of rinsing technique, dialyzer storage time, and potting compound.

Ethylene oxide (ETO) is recognized as one of the main causes of dialyzer-associated hypersensitivity reactions. We studied the amount of ETO in the rinsing fluid of ETO-sterilized hollow-fiber dialyzers as a function of rinsing technique, dialyzer storage time, and the amount of potting compound (known to be an ETO reservoir) in the dialyzer. The results suggested that the initial 500 ml of rinsing fluid removes much of the residual ETO in the dialyzer. Ethylene oxide extraction was enhanced substantially by rinsing at 37 degrees C versus 5 degrees C. However, considerable amounts of ETO remained in the dialyzer after an initial 500 ml rinse, some of which could be removed by rinsing with an additional 1,500 ml. High concentrations of ETO were measured in fluid that had been recirculated through the dialyzer for 10 min or longer and in fluid that had been allowed to remain in the dialyzer for 10 min under zero-flow conditions. The amount of ETO in the rinsing fluid decreased markedly as the dialyzer storage time was increased from 4 to 8 weeks and in dialyzers in which a portion of the potting compound had been replaced with a polycarbonate ring. Our results suggest that the dose of ETO administered to the patient at the outset of dialysis can be minimized by rinsing the dialyzer with 2 L of fluid at 37 degrees C and by avoiding administration of rinsing fluid that has been allowed to remain in contact with the dialyzer for more than several minutes. Use of a long storage interval and use of dialyzers containing reduced amounts of potting material will also reduce the ETO load.

Ethylene Oxide↗
PubMed1986

A non-radioactive automated method for DNA sequence determination.

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.

Autoanalysis↗
PubMed1985

Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate.

A silver stain technique using treatment with potassium permanganate for visualisation of proteins and DNA separated by gel electrophoresis was developed and applied both to the very thin (thickness 0.1-0.2 mm) and to the usual 1-2 mm thick gels. The technique is reproducible, only stable chemicals are used and it is shortened to about 1 h even for the 1 mm gel (30 min for the 0.2 mm gel). It is applicable to gels thicker than 2 mm with somewhat longer washes. Amounts as low as 2 X 10(-10) g of protein per band have been detected. Protein bands of different colours are obtained. The technique has been used with success in continuous, discontinuous and 2D-gel systems. Bands have been detected which were not observed when the other silver staining techniques were used.

DNA↗
PubMed1984

Field gradients improve resolution on DNA sequencing gels.

A simple field gradient technique which leads to sharpening of bands and to an increase in the number of resolvable bases per gel is described. About 300 bases per sample application could be resolved on a 53-cm-long field gradient 6% gel, compared with about 200 on a standard gel. Gels of increasing cross-sectional area, producing the field gradients, are prepared by varying the thickness of spacers along the gel. Compared with the ionic strength gradient method, the field gradient method described here uses the standard sliding technique for preparation of very thin gels and does not require any special skills or preparation of more than one gel solution. Other possible gradient techniques are discussed.

Base Sequence↗
PubMed1984

System for DNA sequencing with resolution of up to 600 base pairs.

A system capable of resolving about 500 bases is of interest for sequencing of longer DNA molecules. Studies on further optimization of resolution on DNA sequencing gels were carried out. The effect of physico-chemical properties of gels and buffers on resolution were tested, e.g. ionic strength and pH of buffers, different buffer systems, acrylamide concentration, crosslinker concentration, type of crosslinker, temperature of polymerization, denaturing conditions, gel length and thickness. Tested were as well different running conditions like electric field, gel temperature, dimension of sample slots. Gels 0.1-0.2 mm thick and up to 1.2 m long were cast and tested routinely. Gel lengths of 60-70 cm (for sequencing up to 350-400 bases) to about 100 cm (above 400 bases) are practicable. Little is gained in resolution by increasing the gel length from 1 to 1.2 m. Resolution was improved using 0.1 mm thick gels, at a higher pH value of 8.6-8.8, and molarity increased to 0.2 M. The sequencing pattern in the region of higher bases could be better resolved on a twice-magnified picture of that region on the autoradiogram. With the long gels (70-120 cm), it is advantageous to obtain the sequence overlap by running in parallel gels of different concentrations, without re-application of samples, all loaded at the same time. Buffer chamber for running of two of three gels and thermostating plates up to 1.2 m long were designed. In this way four to six thermostated gels can be run from a power supply with two inputs. Three 1 m long gels (concentrations: 4%, 6%, 12-16%) are loaded with several samples of DNA to be sequenced and run in parallel without re-application of the samples. With good samples, the sequence overlap from the gels could be counted up to 500 base pairs, with exceptionally good samples closer to 600 bases. At present this number seems to be near the limit of the resolving power of the polyacrylamide gels.

Base Sequence↗