Ricarda Jahnel

Publications (6)23.15 Total impact

• Article: Characterization of rat transient receptor potential vanilloid 1 receptors lacking the N-glycosylation site N604.

  Kerstin Wirkner, Huge Hognestad, Ricarda Jahnel, Ferdinand Hucho, Peter Illes
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  ABSTRACT: Whole-cell patch-clamp recordings were performed on HEK293 cells transiently transfected with the rat (r) wild-type transient receptor potential vanilloid 1 (TRPV1) (rTRPV1) receptor or with a mutant that lacks the potential N-glycosylation site at position N604 (rTRPV1-N604T). Replacement of Asn by Thr (N604T) depressed the maximum of the concentration-response curve for capsaicin and decreased the EC50 value of this agonist. Further, such a manipulation modified the sensitivity to the TRPV1 receptor-antagonist capsazepine and altered the dependence of the capsaicin effect on extracellular pH. Hence, glycosylation may affect the basic functional characteristics of the rTRPV1 receptor channel in accordance with the knowledge that N-glycosylation may regulate ligand binding or gating properties of ionotropic neurotransmitter receptors.
  Neuroreport 07/2005; 16(9):997-1001. · 1.66 Impact Factor
• Article: Analysis of the dorsal spinal cord synaptic architecture by combined proteome analysis and in situ hybridization.

  Mathias Dreger, Joanna Mika, Annette Bieller, Ricarda Jahnel, Clemens Gillen, Martin K H Schaefer, Eberhard Weihe, Ferdinand Hucho
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  ABSTRACT: The proteomic analysis of tissue samples is an analytical challenge, because identified gene products not only have to be assigned to subcellular structures, but also to cell subpopulations. We here report a strategy of combined subcellular proteomic profiling and in situ hybridization to assign proteins to subcellular sites in subsets of cells within the dorsal region of rat spinal cord. With a focus on synaptic membranes, which represent a complex membrane protein structure composed of multiple integral membrane proteins and networks of accessory structural proteins, we also compared different two-dimensional gel electrophoresis systems for the separation of the proteins. Using MALDI mass spectrometric protein identification based on peptide mass fingerprints, we identified in total 122 different gene products within the different synaptic membrane subfractions. The tissue structure of the dorsal region of the spinal cord is complex, and different layers of neurons can be distinguished neuroanatomically. Proteomic data combined with an in situ hybridization analysis for the detection of mRNA was used to assign selected gene products, namely the optical atrophy protein OPA-1, the presynaptic cytomatrix protein KIAA0378/CAST1, and the uncharacterized coiled-coil-helix-coiled-coil-helix domain containing protein 3 (hypothetical protein FLJ20420), to cell subsets of the dorsal area of the spinal cord. Most striking, KIAA0378/CAST1 mRNA was found only sparsely within the dorsal horn of the spinal cord, but highly abundant within the dorsal root ganglion. This finding, combined with the identification of KIAA0378/CAST1 within the synaptic membrane fraction of the spinal cord at the protein level, are consistent with the reported presynaptic localization of CAST, predominantly within the tissue we investigated primarily attributable to primary afferent sensory neurons. Our approach may be of use in broader studies to characterize the proteomes of neural tissue.
  Journal of Proteome Research 02/2005; 4(2):238-49. · 5.11 Impact Factor
• Article: Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

  Ricarda Jahnel, Olaf Bender, Lisa M Münter, Mathias Dreger, Clemens Gillen, Ferdinand Hucho
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  ABSTRACT: The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.
  European Journal of Biochemistry 12/2003; 270(21):4264-71. · 3.58 Impact Factor
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  Available from: Arnold Grünweller

  Article: Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA.

  Arnold Grünweller, Eliza Wyszko, Birgit Bieber, Ricarda Jahnel, Volker A Erdmann, Jens Kurreck
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  ABSTRACT: Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA-DNA-LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2'-O-methyl RNA-DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA-DNA-LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1-green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC50 approximately 70 nM). In contrast, the efficiency of a 2'-O-methyl-modified oligonucleotide (IC50 approximately 220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA-DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.
  Nucleic Acids Research 07/2003; 31(12):3185-93. · 8.03 Impact Factor
• Article: Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I.

  Jens Kurreck, Birgit Bieber, Ricarda Jahnel, Volker A Erdmann
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  ABSTRACT: The efficiencies of 32 antisense oligodeoxynucleotides, 35 DNA enzymes and 6 ribozymes to bind and cleave the full-length messenger RNA of the vanilloid receptor subtype I were analyzed. Systematic screening of the mRNA revealed that good accessibility of a putative cleavage site for antisense oligodeoxynucleotides is a necessary but not a sufficient prerequisite for efficient DNA enzymes. Comparison of DNA enzymes and ribozymes against the same target sites revealed: 1) DNA enzymes were more active with longer recognition arms (9 nucleotides on either side), whereas ribozymes revealed higher activities with shorter recognition arms (7 nucleotides on either side). 2) It does not only depend on the target site but also on the enzyme sequence, whether a DNA enzyme or a ribozyme is more active. 3) The most efficient DNA enzyme found in this study had an approximately 15-fold higher reaction rate, k(react), and a 100-fold higher k(react)/K(m) under single turnover conditions compared with the fastest ribozyme. DNA enzymes as well as ribozymes showed significant activity under multiple turnover conditions, the DNA enzymes again being more active. We therefore conclude that DNA enzymes are an inexpensive, very stable and active alternative to ribozymes for the specific cleavage of long RNA molecules.
  Journal of Biological Chemistry 04/2002; 277(9):7099-107. · 4.77 Impact Factor
• Article: Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line

  Ricarda Jahnel, Mathias Dreger, Clemens Gillen, Olaf Bender, Jens Kurreck, Ferdinand Hucho
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  ABSTRACT: The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma × rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers.Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.
  European Journal of Biochemistry. 10/2001; 268(21):5489 - 5496.