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Detlev Riesner

Publications and source records attributed to Detlev Riesner.

18 recordsLinked to original sources
PubMed2007

Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis.

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

Amino Acid Sequence↗
PubMed2007

Interaction of the cellular prion protein with raft-like lipid membranes.

Conversion of the cellular isoform of the prion protein (PrP(C)) into the disease-associated isoform (PrP(Sc)) plays a key role in the development of prion diseases. Within its cellular pathway, PrP(C) undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrP(C) with model membranes, we purified posttranslationally modified PrP(C) from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrP(C) free in solution were analyzed by analytical ultracentrifugation. The interaction of PrP(C) with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrP(C) association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrP(C) released from the membrane into aqueous solution was estimated at between 10(-9) and 10(-7) M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrP(C) to PrP(Sc) are discussed.

Animals↗
PubMed2006

Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop.

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.

Plant Viruses↗
PubMed2006

Acid inactivation of prions: efficient at elevated temperature or high acid concentration.

Scrapie prion rods isolated from hamster and non-infectious aggregates of the corresponding recombinant protein rPrP(90-231) were incubated with hydrochloric acid. The amount of PrP and of infectivity that survived incubation in HCl at varying times, acid concentrations and temperatures was quantified by Western blot densitometry and bioassays, respectively. Prion rods and rPrP aggregates showed similar HCl hydrolysis kinetics of PrP, indicating structural homology. For 1 M HCl and 25 degrees C, the rate of PrP hydrolysis follows first-order kinetics at 0.014 h(-1); the rate of infectivity inactivation is 0.54 h(-1). Hydrolysis for 1 h at 25 degrees C was only slightly proportional to HCl concentration up to 5 M, but complete loss of infectivity and PrP reduction to <2 % was observed at 8 M HCl. The temperature dependence of unhydrolysed PrP, as well as infectivity at 1 M HCl for 1 h, showed a slight decrease up to 45 degrees C, but a sigmoidal decrease by several orders of magnitude at higher temperatures. The slow hydrolysis of PrP and inactivation of infectivity by acid treatment at room temperature are attributed to solvent inaccessibility of prion rods and rPrP aggregates, respectively. The more effective hydrolysis and inactivation at temperatures above 45 degrees C are interpreted as thermally induced disaggregation with an activation energy of 50-60 kJ mol(-1). Most importantly, infectivity was always inactivated faster or to a higher extent than PrP was hydrolysed at several incubation times, HCl concentrations and temperatures.

Animals↗
PubMed2006

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion.

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.

Animals↗
PubMed2005

Search for a prion-specific nucleic acid.

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.

Animals↗
PubMed2005

Assembly of natural and recombinant prion protein into fibrils.

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.

Amyloid↗
PubMed2005

The polysaccharide scaffold of PrP 27-30 is a common compound of natural prions and consists of alpha-linked polyglucose.

An inert polysaccharide scaffold identified as a 5-15% component of prion rods (PrP 27-30) is unambiguously distinguishable from the N-glycosyl groups and the GPI anchor of PrP, and consists predominantly of 1,4-linked glucose with some branching via 1,4,6-linked glucose. We show that this polysaccharide scaffold is a common secondary component of prions found in hamster full-length PrP(Sc), prion rods and in mouse ScN2a prions from cell culture. The preparation from prion rods was improved, resulting in a polysaccharide scaffold free of remaining infectivity. Furthermore, we determined the stereochemistry of the glycoside linkages as pre-dominantly if not entirely alpha-glycosidic. The origin of the polysaccharide, its interaction with PrP and its potential relation to glycogen and corpora amylacea are discussed.

Animals↗
PubMed2004

Prevention of Alzheimer's disease-associated Abeta aggregation by rationally designed nonpeptidic beta-sheet ligands.

A new concept is introduced for the rational design of beta-sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a beta-sheet efficiently block the solvent-exposed beta-sheet portions in Abeta-(1-40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of Abeta aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a beta-sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10(-5) m. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of Abeta-(1-40) in the absence and in the presence of the ligands. From the comparable decrease in Abeta monomer concentration, we conclude that these beta-sheet ligands do not prevent the initial oligomerization of monomeric Abeta but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble Abeta aggregates formed in the presence of both inhibitors from 3- to 15-mers.

Alzheimer Disease↗
PubMed2004

Synthetic mammalian prions.

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

Amyloid↗
PubMed2004

Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of viroid "thermomutants" to tomato and Brassica species.

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the "pathogenicity" domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid "thermomutants" progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid "thermomutants"; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in "hairpin II" domain (C-->A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50-80 days pi) when infection reached 70-90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.

Arabidopsis↗
PubMed2003

Structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by NMR and thermodynamic analysis.

The 69 nucleotide left-terminal domain (T(L)) of the potato spindle tuber RNA viroid (PSTVd) constitutes one of its five structural elements. Due to a twofold complementary sequence repeat, two possible conformations are proposed for the T(L) secondary structure; an elongated-rod and a bifurcated form. In the present study, two T(L) mutants were designed that remove the symmetry of the sequence repeats and ensure that either the bifurcated or the elongated-rod conformation is thermodynamically favored. Imino 1H and 15N resonances were assigned for both mutants and the native T(L) domain based on 1H-1H NOESY and heteronuclear 1H-15N HSQC high-resolution NMR spectra. The NMR secondary structure analysis of all constructs establishes unambiguously the elongated-rod form as the secondary structure of the native T(L) domain. Temperature-gradient gel electrophoresis and UV melting experiments corroborate these results. A combined secondary structure and sequence analysis of T(L) domains of other Pospiviroidae family members indicates that the elongated-rod form is thermodynamically favored for the vast majority of these viroids.

Base Sequence↗
PubMed2003

Biochemistry and structure of PrP(C) and PrP(Sc).

In a brief historical description, it is shown that the prion model was developed from the biochemical and biophysical properties of the scrapie infectious agent. The biochemical properties of the prion protein which is the major, if not only, component of the prion are outlined in detail. PrP is a host-encoded protein which exists as PrP(C) (cellular) in the non-infected host, and as PrP(Sc) (scrapie) as the major component of the scrapie infectious agent. An overview of the purification techniques is given. Although chemically identical, the biophysical features of PrP(Sc) are drastically different in respect to solubility, structure, and stability; furthermore, specific lipids and a polyglucose scaffold were found in prions, whereas for nucleic acids their absence could be proven. The structure of recombinant PrP in solution is known from spectroscopic studies and with high resolution from NMR analysis. Structural models of PrP(Sc) were derived recently from electron microscopic analysis of two-dimensional crystals. Conformational transitions of PrP in vitro were studied with different techniques in order to mimic the natural PrP(C) to PrP(Sc) conversion. Spontaneous transitions can be induced by solvent changes, but at present infectivity cannot be induced in vitro.

Amino Acid Sequence↗
PubMed2002

Detection and analysis of hairpin II, an essential metastable structural element in viroid replication intermediates.

In (-)-stranded replication intermediates of the potato spindle tuber viroid (PSTVd) a thermodynamically metastable structure containing a specific hairpin structure (HP II) has been proposed to be essential for viroid replication. In the present work a method was devised allowing the direct detection of the HP II structure in vitro and in vivo using a biophysical approach. An RNA oligonucleotide was constructed which specifically binds to the HP II loop region in transient (-)-strand intermediates. Analysis of the resulting oligonucleotide/HP II complexes on temperature-gradient gels enabled us to follow the formation of HP II during in vitro transcription by T7 RNA polymerase. Moreover, we were able to demonstrate the formation of HP II during viroid replication in potato (Solanum tuberosum) cells.

Base Sequence↗
PubMed2001

Heat stability of prion rods and recombinant prion protein in water, lipid and lipid-water mixtures.

Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP(90-231), were autoclaved in water, bovine lipid or lipid-water mixtures for 20 min at temperatures from 100 to 170 degrees C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or lipid-water as well as for rPrP(90-231) in lipid-water. The presence of lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in pure lipid gave different results - not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in pure lipid exceeded that in lipid-water mixtures. Degradation factors larger than 10(4) were reached at 170 degrees C in the presence of lipids and at 150 degrees C in the absence of lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 degrees C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.

Animals↗