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Biomedical subjects

K Düring

Publications and source records attributed to K Düring.

9 recordsLinked to original sources
PubMed1999

The non-enzymatic microbicidal activity of lysozymes.

T4 lysozyme was thought to destroy bacteria by its muramidase activity. However, we demonstrate here that amphipathic helix stretches in the C-terminus of T4 lysozyme mediate its bactericidal and fungistatic activities. In heat-denatured T4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. Small synthetic peptides corresponding to amphipathic C-terminal domains of T4 lysozyme show a microbicidal activity. Its membrane disturbing activity was directly demonstrated for bacterial, fungal and plant cells but not in a hemolysis assay. Comparable results were obtained with hen egg white lysozyme. This opens up many new opportunities for optimization of lysozymes as antimicrobial agents in various applications by protein engineering.

Animals↗
PubMed1998

A plant transformation vector with a minimal T-DNA II. Irregular integration patterns of the T-DNA in the plant genome.

A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed. Frequent rearrangements of T4 lysozyme transgenes were detected. A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed. In transgenic potato plants, reduced variability in gene expression due to position effects was detected. When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed. However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained.

Blotting, Southern↗
PubMed1994

A plant transformation vector with a minimal T-DNA.

Plant transformation, via Agrobacterium tumefaciens, is usually performed with binary vectors. Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification. These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization. The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.

Agrobacterium tumefaciens↗
PubMed1993

Non-radioactive detection methods for nucleic acids separated by electrophoresis.

The different non-radioactive labelling and detection methods currently commercially available are compared and evaluated in this review. Minor factors such as electrophoresis and blotting techniques as well as choice of membrane and their impact on results are discussed. Two major labelling moieties, biotin and digoxigenin, and the various labelling methods are discussed in detail. A comparison of my own results and those from the literature favours application of the digoxigenin group as a routine label. Nevertheless, in several cases biotin will also lead to good results and may also serve as a second label. The most important factor within the non-radioactive systems is the detection of the targeted label. Colorimetric and chemiluminescent techniques are compared in terms of sensitivity, flexibility and applicability. Colorimetric detection can produce suitable results, but in most cases the major advantages of chemiluminescent techniques involving alkaline phosphatase and AMPPD or CSPD will make chemiluminescent detection the method of choice. A survey is given on applicability of the basic techniques to several important assay methods involving electrophoresis of nucleic acids. Finally, some examples of application of non-radioactive nucleic acid labelling and detection techniques in plant molecular biology and biomedicine are cited from the literature.

Animals↗
PubMed1993

A tightly regulated system for overproduction of bacteriophage T4 lysozyme in Escherichia coli.

Bacteriophage T4 lysozyme has been purified using the Ni-chelate affinity chromatography technique from overexpressing Escherichia coli cells by fusion to an N-terminal 6x His tail. Regulation of the lysozyme gene expression has been found to be critical during growth phase of the bacteria by comparing different plasmid constructions. Whereas a tac-promoter fusion construct alone did not lead to efficient production of T4 lysozyme because of early cell lysis, an improved repressor sequence and co-overproduction of the tac repressor resulted in high-level synthesis of the foreign protein after IPTG induction. Purification of the fusion protein from autolyzed crude cell extracts is possible in a simple one-step procedure.

Amino Acid Sequence↗
PubMed1991

Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology.

Nonradioactive detection methods for DNA, RNA, and protein analysis have been the subject of research for several years. In this paper the application of the digoxigenin nucleic acid labeling system, in combination with the new alkaline phosphatase substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl -1,2-dioxetane, to the special requirements of the analysis of transgenic plants is described. Earlier detection systems lacked the required ultrasensitive limits of detection necessary because of the large genomes found in plant cells. Routine detection of single-copy genes from transgenic plant species requires the detection of bands of picograms of specific DNA, which is easily achieved by employing the AMPPD substrate. Optimal conditions of genomic Southern analysis have been successfully adapted for Northern blotting techniques. Detection of foreign proteins in transgenic plants has proven difficult because of the very small amounts of detectable specific protein. Until now, utilization of biotinylated antibodies in combination with a streptavidin-alkaline phosphatase conjugate has been the most sensitive procedure. By introducing the AMPPD substrate, a further significant enhancement of sensitivity leading to detectable signals in the picogram range can be obtained.

Adamantane↗
PubMed1990

Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum.

Immunoglobulin light and heavy chains are synthesized in mammalian cells as precursors containing a signal peptide. Processing and assembling result in formation of active antibodies. Chimeric genes have been made containing the coding sequence of the barley alpha-amylase signal peptide which has been fused to cDNAs coding for either the mature light or the mature heavy chain of a monoclonal antibody. A plasmid was constructed linking both chimeric genes under the control of plant active promoters in an expression cassette. This DNA fragment was stably integrated into the genome of Nicotiana tabacum by Agrobacterium tumefaciens mediated gene transfer. Synthesis of light and heavy chains and assembly to antibodies was detected in transgenic tobacco tissue using specific secondary antibodies. By electron microscopic immunogold labeling, the presence of assembled antibody could be detected within the endoplasmic reticulum. Affinity chromatography indicated biological activity of the assembled immunoglobulin produced in plant cells. Unexpectedly, a significant amount of assembled antibodies was found within chloroplasts.

Amino Acid Sequence↗