PubMed Health⌕ Search

Biomedical subjects

Rainer Fischer

Publications and source records attributed to Rainer Fischer.

At least 55 records · Page 3Linked to original sources
PubMed2004

A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides.

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8-10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.

Antioxidants↗
PubMed2004

Plant-based production of biopharmaceuticals.

Plants are now gaining widespread acceptance as a general platform for the large-scale production of recombinant proteins. The first plant-derived recombinant pharmaceutical proteins are reaching the final stages of clinical evaluation, and many more are in the development pipeline. Over the past two years, there have been some notable technological advances in this flourishing area of applied biotechnology, as shown by the continuing commercial development of novel plant-based expression platforms. There has also been significant success in tackling some of the limitations of plant bioreactors, such as low yields and inconsistent product quality, that have limited the approval of plant-derived pharmaceuticals.

Biological Factors↗
PubMed2004

Over-expression and production of plant allergens by molecular farming strategies.

Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future. To supply the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by molecular farming is an alternative to microbial expression systems. Especially as post-translational modifications of the allergens, e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen. An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in this paper.

Agrobacterium tumefaciens↗
PubMed2004

Chemolabile cellular microarrays for screening small molecules and peptides.

Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transfer of substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specific for peptides extended with the cell-penetrating peptide.

Amino Acid Sequence↗
PubMed2004

Plant cell cultures for the production of recombinant proteins.

The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems.

Biological Products↗
PubMed2004

Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencing.

Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3' end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp-specific siRNAs and of significant methylation within the gfp-coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5' regions.

Base Sequence↗
PubMed2004

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues.

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.

Journal Article↗
PubMed2004

Measurement of antibody-membrane interactions by surface plasmon resonance.

Due to problems of immobilizing functional tumor antigens in their natural conformation on surfaces for immunoassays, it is often difficult to evaluate the binding of antibodies derived from phage display libraries depleted and selected by panning on cell lines and living tumor cells. Performing cell membrane based ELISA methods does not reveal any up front kinetic binding information and depends on the performance of secondary antibodies and substrates. To overcome these limitations, we developed a new method to visualize direct antibody-cell membrane interactions by surface plasmon resonance using the Biacore 3000 and on-line signal subtraction on antigen-negative cell membrane vesicles. Conditions for the coating of cell membrane preparations to a carboxymethyl dextran hydrogel surface of a commercially available chip and the proof of concept for this application by the analysis of different formats of anti-CD30 and anti-carcinoembryonic antigen (CEA) antibodies interacting with coated membrane vesicles of CD30-positive/CEA-negative and CD30-negative and CEA-positive cell lines are described.

Antibodies↗
PubMed2003

Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells.

CD64, the high affinity receptor for IgG (FcgammaRI) is expressed on acute myeloid leukemia blast cells and has recently been described as a specific target for immunotherapy. To generate a recombinant immunotoxin, the anti-CD64 single chain fragment (scFv) m22 was cloned into the bacterial expression vector pBM1.1 and fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Genetically modified Escherichia coli BL21 Star (DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the 70-kDa His(10)-tagged m22(scFv)-ETA' was directed into the periplasmic space and purified by a combination of metal-ion affinity and molecular size-chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays, using CD64-positive AML cells. Binding specificity of m22(scFv)-ETA' was verified by competition with the parental anti-CD64 monoclonal antibody m22. The recombinant immunotoxin showed significant toxicity toward the CD64-positive cell lines HL-60 and U937 reaching 50% inhibition of cell proliferation at a concentration (IC(50)) of 11.6 ng/ml against HL-60 cells and 12.9 ng/ml against U937 cells. Approximately 41% of primary leukemia cells from a patient with CD64-positive AML were driven into early apoptosis by m22(scFv)-ETA' as measured by flow cytometric analysis. This is the first article documenting the specific cytotoxicity of a novel recombinant immunotoxin with major implications for immunotherapy of CD64-positive diseases.

ADP Ribose Transferases↗
PubMed2003

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein.

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5'-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome.

Amino Acid Sequence↗
PubMed2003

The glutathione synthetase of Schizosaccharomyces pombe is synthesized as a homodimer but retains full activity when present as a heterotetramer.

Glutathione synthetase was overexpressed as a histidine-tagged protein in Schizosaccharomyces pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56-kDa subunits and a heterotetrameric protein composed of two 32-kDa and two 24-kDa subfragments. Both forms are encoded by the GSH2 gene. The 56-Da protein corresponds to the complete GSH2 open reading frame, while the subfragments are produced following the cleavage of this larger protein by a metalloprotease. A stable homodimer was obtained by site-directed mutagenesis to remove the protease cleavage site, and this showed normal activity. A structural model of the fission yeast glutathione synthetase was produced, based on the x-ray coordinates of the human enzyme. According to this model the interacting domains of the proteolytic subfragments are strongly entangled. The subfragments were therefore coexpressed as independent proteins. These subfragments assembled correctly to yield functional heterotetramers with equivalent activity to the wild type enzyme. Furthermore, a permuted version of the protein was created. This also showed normal levels of glutathione synthetase activity. These data provide novel insight into the mechanisms of protein folding and the structure and evolution of the glutathione synthetase family.

Amino Acid Sequence↗
PubMed2003

'Molecular farming' of antibodies in plants.

'Molecular farming' is the production of valuable recombinant proteins in transgenic organisms on an agricultural scale. While plants have long been used as a source of medicinal compounds, molecular farming represents a novel source of molecular medicines, such as plasma proteins, enzymes, growth factors, vaccines and recombinant antibodies, whose medical benefits are understood at a molecular level. Until recently, the broad use of molecular medicines was limited because of the difficulty in producing these proteins outside animals or animal cell culture. The application of molecular biology and plant biotechnology in the 1990s showed that many molecular medicines or vaccines could be synthesised in plants and this technology is termed 'molecular farming'. It results in pharmaceuticals that are safer, easier to produce and less expensive than those produced in animals or microbial culture. An advantage of molecular farming lies in the ability to perform protein production on a massive scale using hectares of cultivated plants. These plants can then be harvested and transported using the agricultural infrastructure. Thus, molecular farming allows rapid progress from genetic engineering to crop production, and new cash crops producing recombinant proteins are already being commercially exploited. We speculate that as functional genomics teaches us more about the nature of disease, molecular farming will produce many of the protein therapeutics that can remedy it.

Animals↗
PubMed2003

Production of antibodies in plants and their use for global health.

Recombinant antibodies can be used to diagnose, treat and prevent disease by exploiting their specific antigen-binding activities. A large number of drugs currently in development are recombinant antibodies and most of these are produced in cultured rodent cells. Although such cells produce authentic functional products, they are expensive, difficult to scale-up and may contain human pathogens. Plants represent a cost-effective, convenient and safe alternative production system and are slowly gaining acceptance. Five plant-derived therapeutic recombinant antibodies (plantibodies) are undergoing clinical evaluation, three of which can be used as prophylactics.

Animals↗
PubMed2003

Production of therapeutic antibodies in plants.

Antibodies are versatile tools for the diagnosis and treatment of many diseases. Their use has increased dramatically with the advent of recombinant antibody (rAb) technology, allowing the production of immunological reagents with improved and novel properties. The main challenge now lies in achieving cost-effective production on a large scale. Over the past 15 years, the potential of plants for the production of pharmaceutical proteins has become well-established. Plants represent an inexpensive, efficient and safe alternative to traditional systems used for the commercial-scale synthesis of rAbs. This review describes the current status of antibody production in plants, focusing on their advantages compared with other expression systems and the remaining obstacles to widespread acceptance.

Animals↗