Biomedical subjects

M Büttner

Publications and source records attributed to M Büttner.

At least 19 recordsLinked to original sources

PubMed2006

Prion protein expression in bovine podocytes and extraglomerular mesangial cells.

The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

Animals↗

PubMed2005

The normal cellular prion protein (PrPc) is strongly expressed in bovine endocrine pancreas.

Expression of the cellular prion protein (PrP(c)) has been shown to be crucial for the development of transmissible spongiform encephalopathies and for the accumulation of the disease-associated conformer (PrP(sc)) in the brain and other tissues. One of the emerging hypotheses is that the conversion phenomenon could take place at the site where the infectious agent meets PrP(c). In this work we have studied whether PrP(c), a protein found predominantly in neurons, could also exist in pancreatic endocrine cells since neuroectoderm-derived cells and pancreatic islet cells share a large number of similarities. For this purpose we have examined the expression of PrP(c) in a series of fetal and postnatal bovine pancreatic tissue by immunohistochemistry and RT-PCR. Using immunostained serial sections and specific antibodies against bovine PrP(c), insulin, glucagon, somatostatin, chromogranin A and chromogranin B we found that PrP(c) is highly expressed in all endocrine cells of fetal and adult pancreatic islets with a particular strong expression in A-cells. Moreover it became evident that the PrP(c) gene-neighbour chromogranin B as well as chromogranin A are coexpressed together with PrP(c). The selective expression of PrP(c) in the bovine endocrine pancreas is of particular importance regarding possible iatrogenic transmission routes and demonstrates also that bovine pancreatic islet cells could represent an interesting model to study the control of PrP-gene expression.

Animals↗

PubMed2005

Gold cluster formation on a fullerene surface.

The growth of Au clusters on a fullerene thin film was investigated by in situ photoelectron spectroscopy in the ultraviolet (UPS) and x-ray (XPS) regime. Due to its highly corrugated surface fullerene films provide a wide range of bonding sites which could be exploited as molecular templates and serve to create a cluster superstructure. To gain insight into the fullerene-Au interaction two types of experiments were performed: (i) the deposition of Au on a fullerene surface, and (ii) the deposition of fullerenes on a Au surface. In both experiments an island growth mode is observed. The deposition of submonolayer amounts of C60 onto a gold film showed that the main interaction of the two species is due to chemisorption of the first C60 monolayer. In addition a constant band bending in the fullerene film is detected, but the UPS valence-band spectra show that there is no charge transfer from the Au to the C60 lowest unoccupied molecular orbital. In the reverse experiment, the cluster growth of Au on the corrugated C60 surface, the analysis of the Au core level does not reveal a specific bonding or nucleation site for Au atoms and clusters. This is in contrast to observations with Si clusters, which prefer to reside in the troughs between the fullerene molecules. The Au clusters grow continually from a size of about 55 atoms for the early stages of growth up to 150 atoms for the deposition of a nominal coverage of 1.5 nm. These data are derived from an analysis of the d-band splitting and the Au 4f core-level shift due to delayed photohole relaxation. The thermal stability of the Au-clusters-covered fullerene film was investigated by annealing in situ up to temperatures of 650 degrees C. For temperatures up to 450 degrees C a continuous growth of the clusters is detected, which is accompanied by a slight drop in Au concentration in the range of XPS for annealing temperatures higher than 350 degrees C. This may be due to a ripening of the clusters. The presence of Au apparently delays fullerene sublimation. The film shows a very good thermal stability and even after annealing at 650 degrees C there is still a fullerene film detectable in the photoelectron spectroscopy spectra.

Journal Article↗

PubMed2005

Some physico-chemical parameters that influence proteinase K resistance and the infectivity of PrPSc after high pressure treatment.

Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60 degrees C for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.

Animals↗

PubMed2005

Alloy formation of supported gold nanoparticles at their transition from clusters to solids: does size matter?

Gold nanoclusters of a size approaching the molecular limit (<3 nm) were prepared on Si substrates in order to study alloy formation on the nanometer scale. For this purpose, indium atoms are deposited on top of the gold particles at room temperature and the formation of AuIn(2) is studied by x-ray photoelectron spectroscopy in situ. It is observed that the alloy formation takes place independent of whether the particles electronically are in an insulating molecular or in a metallic state. Most important, however, closed packed full-shell clusters containing 55 Au atoms are found to exhibit an outstanding stability against alloying despite a large negative heat of formation of the bulk Au-In system. Thus, Au(55) clusters may play a significant role in the design of nanoscaled devices where chemical inertness is of crucial importance.

Journal Article↗

PubMed2005

Expression of a plant cell wall invertase in roots of Arabidopsis leads to early flowering and an increase in whole plant biomass.

In order to enhance sink strength, we expressed a heterologous plant cell wall invertase (CrCIN1) under the control of a root-specific promoter (ppyk10) in Arabidopsis thaliana. Slightly elevated apoplastic invertase activity resulted in apparent phenotypic changes. Transgenic plants developed more secondary roots and subsequently, possibly because of a higher capacity to acquire nutrients, a higher shoot and whole plant biomass. Furthermore, an early flowering phenotype was detected. The data presented here demonstrate that it is possible to modulate carbohydrate metabolism by ectopic expression of cell wall invertases and thereby influence sink organ size and whole plant development.

Arabidopsis↗

PubMed2003

Relatedness and heterogeneity at the near-terminal end of the genome of a parapoxvirus bovis 1 strain (B177) compared with parapoxvirus ovis (Orf virus).

The present study provides for the first time an extended investigation of individual genes located at the near-terminal right end of the genome of parapoxvirus bovis 1, Bovine papular stomatitis virus (BPSV) strain B177 and Orf virus (ORFV). Comparison of the respective DNA sequences of ORFV strain D1701 (9.9 kbp) and BPSV B177 (7.7 kbp) revealed a very similar organization of closely related genes transcribed in a rightward orientation. The most salient findings of this study were: (i) the absence of the ORFV-specific vascular endothelial growth factor (VEGF-E) gene in the BPSV isolate; (ii) the presence of an interleukin-10 (IL-10) orthologue; and (iii) the detection of three new genes encoding ankyrin-repeat-containing polypeptides. These results not only contribute to potential improvements of future molecular differentiation between the parapoxvirus species, but also shed new light on different pathobiologies among parapoxviruses.

Amino Acid Sequence↗

PubMed2002

Parapoxviruses: from the lesion to the viral genome.

Viruses of the genus parapoxvirus from the family poxviridae cause widespread but localized diseases of small and large ruminants. The economically most important disease is contagious pustular dermatitis or contagious ecthyma among sheep and goats, often simply called orf. The parapoxviruses (PPV) can be transmitted to man leading to localized lesions that are named pseudocowpox or milkers' node as being mostly restricted to the hands and fingers. In cattle two forms of PPV manifestation are commonly observed, the bovine papular stomatitis in young calves and the occurrence of lesions at the udder of cows. We here report about the recent efforts in molecular characterization of orf viruses and the state of the art about the generation of orf virus recombinants. In addition the current knowledge on immune responses against orf viruses and some new data on the behaviour of orf virus recombinants under non-permissive conditions are reported.

Animals↗

PubMed2002

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure.

The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41.1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

Amino Acid Motifs↗

PubMed2002

Orf virus encodes a functional dUTPase gene.

The present study is the first report on the functional activity of a parapoxvirus-encoded dUTPase. The dUTPase gene of the attenuated orf virus (ORFV), strain D1701, was expressed as a bacterial thioredoxin fusion protein. In vitro assays showed that ORFV dUTPase was highly specific for dUTP as substrate. The enzyme was active over a broad pH range (pH 6.0-9.0), with maximal enzymatic activity at pH 7.0 in the presence of Mg(2+) cations. Kinetic studies of the recombinant ORFV dUTPase revealed an apparent K(m) of 4.0 microM, which is more similar to that of the mammalian or African swine fever virus enzyme than to the K(m) of vaccinia virus dUTPase. Enzyme activity was also found with purified ORFV particles, indicating its virion association.

Deoxyuracil Nucleotides↗

PubMed2001

Genotypes of pestivirus RNA detected in live virus vaccines for human use.

Live virus vaccines for human use, 29 monovalent vaccines against measles, mumps, rubella or polio, eight polyvalent vaccines against measles-mumps-rubella and one bacterial polyvalent vaccine against Streptococcus pneumoniae, were tested by reverse transcriptase-nested PCR for the presence of petivirus or pestivirus RNA. Twenty-four samples were selected from European manufacturers, ten were from U.S.A. and four from Japan. Five (13.1%) out of 38 tested samples were positive for pestivirus RNA. Three vaccines (rubella and two measles) were from Europe and two (mumps and rubella) from Japan. The 5'-untranslated genomic region of the contaminant pestivirus RNA were amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary structures, characteristic to genotypes, revealed that the cDNA sequences belonged to bovine viral diarrhea virus (BVDV). A cDNA sequence, detected from one measles sample, belonged to BVDV-1b genotype. Pestiviral cDNA detected from the Japanese mumps and rubella vaccine samples, belonged to the BVDV genotypes 1a and 1c, respectively. Analysis on two cDNA sequences detected from measles and rubella vaccine samples from Europe showed their appurtenance to a new genotype, BVDV-1d. These findings indicate that contamination by animal pestivirus may occur in biological products for human use.

5' Untranslated Regions↗

PubMed2000

Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes.

Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome.

Animals↗

PubMed2000

Monosaccharide transporters in plants: structure, function and physiology.

Monosaccharide transport across the plant plasma membrane plays an important role both in lower and higher plants. Algae can switch between phototrophic and heterotrophic growth and utilize organic compounds, such as monosaccharides as additional or sole carbon sources. Higher plants represent complex mosaics of phototrophic and heterotrophic cells and tissues and depend on the activity of numerous transporters for the correct partitioning of assimilated carbon between their different organs. The cloning of monosaccharide transporter genes and cDNAs identified closely related integral membrane proteins with 12 transmembrane helices exhibiting significant homology to monosaccharide transporters from yeast, bacteria and mammals. Structural analyses performed with several members of this transporter superfamily identified protein domains or even specific amino acid residues putatively involved in substrate binding and specificity. Expression of plant monosaccharide transporter cDNAs in yeast cells and frog oocytes allowed the characterization of substrate specificities and kinetic parameters. Immunohistochemical studies, in situ hybridization analyses and studies performed with transgenic plants expressing reporter genes under the control of promoters from specific monosaccharide transporter genes allowed the localization of the transport proteins or revealed the sites of gene expression. Higher plants possess large families of monosaccharide transporter genes and each of the encoded proteins seems to have a specific function often confined to a limited number of cells and regulated both developmentally and by environmental stimuli.

Animals↗

PubMed2000

T helper 1-type cytokine transcription in peripheral blood mononuclear cells of pseudorabies virus (Suid herpesvirus 1)-primed swine indicates efficient immunization.

The induction of porcine cytokines, which are believed to be important for the regulation of T helper (Th)1- and Th2-specific immune responses of pigs, was analysed after in vitro restimulation with a herpesvirus, Suid herpes 1 (pseudorabies virus [PRV]), in peripheral blood mononuclear cells (PBMC). To this end, quantitative, competitive reverse transcription-polymerase chain reaction (RT-qcPCR) was established using constructed heterologous DNA MIMICS, which contain cytokine- or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primer-binding sites. This is a simple method that allows reliable determination of the differing regulation of cytokine mRNAs specific for porcine interleukin (IL)-2, -4 and -10, interferon gamma (IFN-gamma) and the housekeeping gene, GAPDH, as an endogenous control. PBMC derived from naive (innate response) and PRV-primed (memory response) outbred swine were analysed comparatively. The results demonstrated that restimulation with PRV significantly enhanced the transcription of Th1-type cytokines (IL-2 and IFN-gamma) but not of Th2-type cytokines (IL-4 and IL-10). This virus-specific cytokine response was only found with PBMC from swine protected against lethal PRV challenge infection, but not with naive PBMC or with PBMC from pigs immunized with plasmid DNA encoding PRV glycoprotein gC. Notably, PBMC derived from immune and naive pigs constitutively produced relatively high amounts of IL-10-specific mRNA, exceeding that of GAPDH mRNA, independently of the addition of viral antigen or the mitogen concanavalin A (Con A). The results of this work should help to provide a better understanding of the effector cell/cytokine network response to infection with, or vaccination against, PRV. Additionally, the simple, reliable and sensitive RT-qcPCR, when used to determine the porcine cytokine pattern, might be of prognostic value for the induction of protective immunity.

Animals↗

PubMed2000

A gelatin sponge implantation model in swine for the determination of local immune reactions.

For in vivo determination of innate and memory immune reactions we have implanted sterile gelatin sponges subcutaneously in swine for the monitoring of the following immunological parameters: 1. Analysis of local cell population phenotypes after vascularization of the gelatin sponges using flow cytometry. 2. Comparative analysis of sponge-infiltrating cells after loading with viral antigen in primed as well as naive animals. 3. Performance of reverse transcription quantitative competitive PCR (RT-qcPCR) for the detection of porcine cytokine mRNA indicative for IFN-gamma, IL-2, IL-4, IL-8 and IL-10. The in vitro analysis, e.g. by re-exposure to viral antigens, allows the determination of cytokine reaction patterns of sponge derived cells, draining lymph node cells as well as PBMC of the same individual. Studies of innate reactions and modulation of cellular infiltration in transplanted gelatin sponges are possible. Functional assays, e.g. cell-mediated cytotoxicity, antigen specific cell proliferation, using sponge-derived cells will provide additional information about the suitability of the model for example in vaccine potency tests.

Animals↗

PubMed1999

Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication.

The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage lambda. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.

Adenosine Diphosphate↗

PubMed1999

Parapoxviruses: potential alternative vectors for directing the immune response in permissive and non-permissive hosts.

Parapoxvirus (PPV) represents a genus of the poxviridae, and particularly PPV ovis (Orf virus, OV) seems to offer several potential advantages for the use of vector vaccine. Therefore, we started to investigate the genome of the highly attenuated OV strain D1701, which was only poorly characterised until now. Due to recombination of non-homologous sequences, part of the right hand end of the D1701 genome was duplicated and translocated to the opposite end of the genome. As a consequence gene deletion had occurred and the inverted terminal repeat region is increased. Results are described to identify viral genes, which are non-essential for virus replication and potentially influence viral pathogenesis, virulence, and host immunity. In more detail, we analysed the expression and functional activity of the OV-specific vascular endothelial growth factor (VEGF) gene homologue. Finally the construction and production of a D1701 mutant lacking the VEGF gene homologue is reported.

Animals↗

PubMed1999

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos.

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.

Animals↗