Biomedical subjects

Petra Weber

Publications and source records attributed to Petra Weber.

11 recordsLinked to original sources

PubMed2006

Cell-free formation of misfolded prion protein with authentic prion infectivity.

Prion propagation has been modeled in vitro; however, the low infectious titer of PrP(Sc) thus generated has cast doubt on the "protein-only" hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP(Sc) and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP(Sc) provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.

Animals↗

PubMed2006

Microfluorometry of cell membrane dynamics.

Membranes of living cells are characterized by a combination of conventional and total internal reflection fluorescence microscopy (TIRFM) using the membrane marker laurdan. In the first case, all cellular membranes are assessed simultaneously, whereas in the second case, the plasma membrane is excited selectively by the evanescent electromagnetic field of a laser beam. A spectral shift depending on the phase of membrane lipids is used to characterize membrane stiffness, which decreases with temperature and increases with the amount of cholesterol. Spectral properties are evaluated and displayed as microscopic images.

2-Naphthylamine↗

PubMed2006

[The impact of the proportion of qualified nursing personnel on the incidence of pressure ulcers in nursing homes].

In Germany a minimal quota of 50% professionally qualified nursing personnel is statutory for the care of nursing home inhabitants. The influence of this quota on the quality outcome of nursing care is controversially discussed. The study investigated the question, whether there is an association between the proportion of qualified personnel and the incidence of pressure ulcers in nursing homes. A historical cohort study was performed. Incidence data were taken from a routine survey on pressure ulcers. Nursing homes participating in this survey were asked for information about their proportion of qualified personnel and some additional factors that could possibly influence the incidence of bedsores. Included nursing home inhabitants (n = 2813) were divided into three groups according to whether they have been cared for with a low (< 50%), medium (50-60%) or high (> or = 60%) proportion of qualified personnel. Multivariate logistic regression models were calculated. Comparing medium to low proportion of qualified personnel, an odds ratio of 1.5 (p = 0.455), comparing high to low proportion, an odds ratio of 0.8 (p = 0.703) and comparing high to medium proportion, an odds ratio of 0.54 (p = 0,09) was calculated. The study question can not be answered by the contradictory results.

Aged↗

PubMed2006

Photo-induced crosslinking of prion protein oligomers and prions.

Prion diseases are caused by a unique type of infectious agent, which is thought to consist of a misfolded beta-sheeted form of the alpha-helical cellular prion protein (PrPC). This misfolded isoform (PrPSc) tends to form insoluble amyloid-like aggregates, impeding classical structural analysis by X-ray crystallography or NMR. Intermolecular crosslinking may provide a means of stabilizing notoriously elusive oligomers for further analysis and may be used for analyzing aggregate architecture by characterising intermolecular contact sites. Using a photo-induced crosslinking method (PICUP), aggregates of recombinant PrP (rPrP) and PrPSc were linked at interacting surfaces via amino acid side chains. The degree of crosslinking within PrP aggregates was adjustable using varying light intensities and could efficiently be monitored by fluorescence correlation spectroscopy. Specific intermolecular crosslinking of PrPSc molecules was achieved even in crude brain homogenate. Functional studies showed that stabilized aggregates of rPrP did not loose their capacity to induce further protein aggregation and crosslinking of PrPSc did not alter significantly the level of infectivity, indicating that photo-induced covalent linkage of PrPSc does not destruct surfaces important for prion propagation.

Amyloid↗

PubMed2005

Automated PrPres amplification using indirect sonication.

Prions, which mainly consist of the scrapie isoform of the prion protein (PrP(Sc)), induce the misfolding of the physiological prion protein (PrP(C)). The Protein Misfolding Cyclic Amplification (PMCA), a process consisting of sonication and incubation, is one of the few methods thought to model autocatalytic prion replication and generation of proteinase K (PK)-resistant PrP (PrPres) in vitro. Here we show for the first time that the amplification may be achieved through direct as well as indirect sonication (water bath sonication using sealed sample containers), allowing the PMCA method to be automated. The automated method may serve as a valuable tool in high throughput screening for the diagnosis or compound identification for treatment of prion disease. The in vitro amplification process is weakly facilitated by divalent cations such as Mn, Zn and Ni, but not Cu, however, the presence of metal ions decreases the stability of PrPres against proteinase K digestion.

Animals↗

PubMed2005

Breakage of PrP aggregates is essential for efficient autocatalytic propagation of misfolded prion protein.

The conversion of cellular prion protein (PrP(C)) to the disease-associated misfolded isoform (PrP(Sc)) is an essential process for prion replication. This structural conversion can be modelled in protein misfolding cyclic amplification (PMCA) reactions in which PrP(Sc) is inoculated into healthy hamster brain homogenate, followed by cycles of incubation and sonication. In serial transmission PMCA experiments it has recently been shown that the protease-resistant PrP obtained in vitro (PrPres) is generated by an autocatalytic mechanism. Here, serial transmission PMCA experiments were compared with serial transmission reactions lacking the sonication steps. We achieved approximately 200,000-fold PrPres amplification by PMCA. In contrast, although initial amplification was comparable to PMCA reactions, PrPres levels quickly dropped below detection limit when samples were not subjected to ultrasound. These results indicate that aggregate breakage is essential for efficient autocatalytic amplification of misfolded prion protein and suggest an important role of aggregate breakage in prion propagation.

Amyloidosis↗

PubMed2005

[Quality in nursing care--action research in "Hamburger Behindertenhilfe"/Hamburg to disabled care].

A two year action research program with the title "Quality in Nursing Care" was carried out by a large institute for disabled care in Hamburg. Presently, the importance of quality in nursing as a profession is seen to be secondary to that of pedagogy in the area of disabled care. A major explanation for this could be the fact that 150 years ago the treatment and care of disabled people lay predominately in the field of theology and only later in that of medicine. In the second half of the 20th century, in the 60ies and 70ies to be precise, starting with an investigative commission "Psychiatryenquete "pedagogical science began to focus on the education of disabled people and their integration within society. At this point in time, seen from the pedagogical point of view, the care of disabled people in theory and practice freed itself from being a medicine concentrated profession and from the dominance of nursing care. However in today's society due to the increasing number of disabled people requiring expert nursing care there is a necessity to examine care related needs of assistance. With the research project "Quality in Nursing Care" an institute for disabled care has looked at different nursing care interventions and methods that our society may require for the changing situation we are presently being confronted with. An analysis of the requirements, the content and evaluation of the three chosen project related interventions, (the individual consulting of inhabitants with regard to the broadening of the competence skills, the development of expert nursing competence through further and continuous education of multipliers, and the development of codes and guidelines for activities) will be introduced. Beginning with the basic and primary principle of the action research, the researchers have developed, with the participating members of the institutions, the specific aims of the work undertaken and the interventions studied and executed. The most important discovery made during the project work undertaken is that the assessment of the extensively nursing linked problems experienced by the inhabitants in disabled care at this point in time is either simply not receiving enough attention or not being taken sufficiently into consideration by the predominately pedagogically trained staff. Taking this into account it is quite clear that professional nursing support in the long term has an important role to play in a successful disabled people orientated care concept.

Attitude to Health↗

PubMed2005

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Animals↗

PubMed2004

Autocatalytic self-propagation of misfolded prion protein.

Prions are thought to replicate in an autocatalytic process that converts cellular prion protein (PrP(C)) to the disease-associated misfolded PrP isoform (PrP(Sc)). Our study scrutinizes this hypothesis by in vitro protein misfolding cyclic amplification (PMCA). In serial transmission PMCA experiments, PrP(Sc) was inoculated into healthy hamster brain homogenate containing PrP(C). Misfolded PrP was amplified by rounds of sonication and incubation and reinoculated into fresh brain homogenate every 10 PMCA rounds. The amplification depended on PrP(C) substrate and could be inhibited by recombinant hamster PrP. In serial dilution experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed the structural conversion of PrP(C) as efficiently as PrP(Sc) from brain of scrapie (263K)-infected hamsters, yielding an approximately 300-fold total amplification of PrPres after 100 rounds, which confirms an autocatalytic PrP-misfolding cascade as postulated by the prion hypothesis. PrPres formation was not paralleled by replication of biological infectivity, which appears to require factors additional to PrP-misfolding autocatalysis.

Animals↗

PubMed2004

Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode.

Lifetime images of autofluorescence of cultivated endothelial cells were recorded using a novel picosecond laser diode in the near ultraviolet range (375 nm). In contrast to existing picosecond light sources this wavelength permits efficient excitation of the free and protein bound coenzyme NADH with fluorescence lifetimes of 0.4-0.5 ns and 2.0-2.5 ns, respectively. The effective fluorescence lifetime tau(eff) (depending on both lifetimes) was homogenously distributed over the cells with some shortening in the perinuclear region, possibly close to mitochondria. A slight decrease of tau(eff) was observed after inhibition of the mitochondrial respiratory chain, whereas a slight increase was observed after inhibition of the glycolytic pathway, thus indicating variations of the ratio of free and protein bound NADH. Although present applications are still limited by their low pulse energy (< or = 5 pJ), uv picosecond laser diodes have a large potential in high resolution fluorescence microscopy and fluorescence lifetime endoscopy.

Adenosine Triphosphate↗

PubMed2002

Localization of ecdysone receptor protein during colour pattern formation in wings of the butterfly Precis coenia (Lepidoptera: Nymphalidae) and co-expression with Distal-less protein.

Butterfly wing colour patterns are determined during late larval and early pupal development, a part of metamorphosis controlled by ecdysteroid hormones via their nuclear hormone receptors. We have sequenced a fragment of the common regions of the ecdysone receptor (EcR) from the butterflies Precis coenia and Bicyclus anynanaand found high identities (83.5% to 100%) to EcR from a moth, Manduca sexta. In P. coenia, we sequenced a putative EcR-B1 isoform with 80.4% identity with the A/B-region of the M. sexta-EcR-B1. Consequently, we used antibodies generated against MsEcR-B1 to localise EcR protein during wing development of P. coenia. Nuclear staining of EcR was observed in different cell types during the course of colour pattern formation. Major observations are as follows: EcR is expressed in cell nuclei corresponding to wing lacunae and prospective veins. EcR is expressed early in pupal wing development in "focal" cells which are thought to release determining signals in a process leading to eyespot formation. Scale forming cells differentiate first and show EcR signal in the eyespot foci and most of the wing sheet, but not in areas corresponding to prospective eyespots. In the eyespots, 20-24 h after pupation, EcR expression seems to play a role in formation of scale rows preceding a later expression (28 h) in scale-forming cells. The results demonstrate that EcR is locally expressed in correlation to all major events of wing development and colour pattern formation. In particular, EcR expression patterns in prospective eyespots show that these are special pattern elements which are specified in concert with other factors of colour pattern formation such as the transcription factor Distal-less. In eyespot foci, Distal-less is expressed simultaneously with EcR, but clearly precedes EcR expression in eyespot scale-forming cells. This indicates a potential interaction between "short-range" signalling systems and "long-range" hormonal systems.

Amino Acid Sequence↗