Biomedical subjects

Walter Keller

Publications and source records attributed to Walter Keller.

41 records · Page 3Linked to original sources

PubMed2002

Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.

Allergens↗

PubMed2002

RNA editing by adenosine deaminases generates RNA and protein diversity.

RNA editing is defined as a post-transcriptional change of a gene-encoded sequence at the RNA level, excluding alterations due to processes such as pre-mRNA splicing and 3'-end formation. RNA editing is found in many organisms and can occur either by the insertion or deletion of nucleotides or by the substitution of bases by modification. The nucleoside inosine (I) was first detected in cytoplasmic tRNA and was later found in messenger RNA precursors (pre-mRNAs) and in viral transcripts. It is formed by hydrolytic deamination of a genomically encoded adenosine (A) at C6 of the base and this reaction is catalysed by a family of related enzymes. ADARs (for adenosine deaminases acting on RNA) catalyse A to I conversion either promiscuously or site-specifically in pre-mRNAs, viral RNAs and synthetic double-stranded RNAs (dsRNAs), whereas ADATs (for adenosine deaminases acting on tRNA) are involved in inosine formation in tRNAs. ADAT1 generates I at position 37 (3' of the anticodon) in eukaryotic tRNA(Ala). ADAT2 and ADAT3 function as a heterodimer which catalyses inosine formation at the wobble position (position 34) in eukaryotic tRNAs. Here, we review the state of knowledge on ADARs and ADATs and their RNA substrates, with an emphasis on the developments over the past few years that have increased the understanding of the mechanism of action of these enzymes and of the functional consequences of the widespread modification they catalyse.

Adenosine↗

PubMed2002

A role for SSU72 in balancing RNA polymerase II transcription elongation and termination.

Interactions of pre-mRNA 3'end factors and the CTD of RNA polymerase II (RNAP II) are required for transcription termination and 3'end processing. Here, we demonstrate that Ssu72p is stably associated with yeast cleavage and polyadenylation factor CPF and provide evidence that it bridges the CPF subunits Pta1p and Ydh1p/Cft2p, the general transcription factor TFIIB, and RNAP II via Rpb2p. Analyses of ssu72-2 mutant cells in the absence and presence of the nuclear exosome component Rrp6p revealed defects in RNAP II transcription elongation and termination. 6-azauracil, that reduces transcription elongation rates, suppressed the ssu72-2 growth defect at 33 degrees C. The sum of our analyses suggests a negative influence of Ssu72p on RNAP II during transcription that affects the commitment to either elongation or termination.

Blotting, Northern↗

PubMed2002

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.

Amino Acid Sequence↗

PubMed2002

Functional analysis of yeast snoRNA and snRNA 3'-end formation mediated by uncoupling of cleavage and polyadenylation.

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3'-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3' portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Base Sequence↗