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S Brunner

Publications and source records attributed to S Brunner.

At least 19 recordsLinked to original sources
PubMed2003

A large rearrangement involving genes and low-copy DNA interrupts the microcollinearity between rice and barley at the Rph7 locus.

Grass genomes differ greatly in chromosome number, ploidy level, and size. Despite these differences, very good conservation of the marker order (collinearity) was found at the genetic map level between the different grass genomes. Collinearity is particularly good between rice chromosome 1 and the group 3 chromosomes in the Triticeae. We have used this collinearity to saturate the leaf rust resistance locus Rph7 on chromosome 3HS in barley with ESTs originating from rice chromosome 1S. Chromosome walking allowed the establishment of a contig of 212 kb spanning the Rph7 resistance gene. Sequencing of the contig showed an average gene density of one gene/20 kb with islands of higher density. Comparison with the orthologous rice sequence revealed the complete conservation of five members of the HGA gene family whereas intergenic regions differ greatly in size and composition. In rice, the five genes are closely associated whereas in barley intergenic regions are >38-fold larger. The size difference is due mainly to the presence of six additional genes as well as noncoding low-copy sequences. Our data suggest that a major rearrangement occurred in this region since the Triticeae and rice lineage diverged.

Chromosome Mapping↗
PubMed2001

A phosphate transporter expressed in arbuscule-containing cells in potato.

Arbuscular mycorrhizas are the most common non-pathogenic symbioses in the roots of plants. It is generally assumed that this symbiosis facilitated the colonization of land by plants. In arbuscular mycorrhizas, fungal hyphae often extend between the root cells and tuft-like branched structures (arbuscules) form within the cell lumina that act as the functional interface for nutrient exchange. In the mutualistic arbuscular-mycorrhizal symbiosis the host plant derives mainly phosphorus from the fungus, which in turn benefits from plant-based glucose. The molecular basis of the establishment and functioning of the arbuscular-mycorrhizal symbiosis is largely not understood. Here we identify the phosphate transporter gene StPT3 in potato (Solanum tuberosum). Functionality of the encoded protein was confirmed by yeast complementation. RNA localization and reporter gene expression indicated expression of StPT3 in root sectors where mycorrhizal structures are formed. A sequence motif in the StPT3 promoter is similar to transposon-like elements, suggesting that the mutualistic symbiosis evolved by genetic rearrangements in the StPT3 promoter.

Cloning, Molecular↗
PubMed2001

Tumor targeting with surface-shielded ligand--polycation DNA complexes.

Incorporation of the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) into DNA/polyethylenimine (PEI) complexes was found to enhance gene transfer into tumor cell lines in a receptor-dependent manner. In systemic applications, the surface charge of DNA complexes dominated the in vivo characteristics of gene transfer. Administration of surface-shielded Tf-polycation/DNA complexes into the tail vein of A/J mice resulted in preferential gene delivery into distantly growing subcutaneous Neuro2a tumors. In contrast, application of positively charged DNA/PEI complexes directed gene transfer primarily to the lung. Two alternatives of masking the surface charge of complexes were accomplished. In the first case, shielding was obtained by covalently coating of DNA/Tf-PEI complexes with polyethylene glycol (PEG). Alternatively, incorporation of sufficient Tf protein into the DNA complexes resulted in charge shielding even without PEGylation. In the latter case lower-molecular weight polycations (25 kDa PEI for Tf-PEI complexes, or 32 kDa polylysine for AVET complexes) were used.

Animals↗
PubMed2001

Can the API (RAPID) Coryne system be used for identification of rapidly growing mycobacteria?

Fifty-six strains of rapidly growing mycobacteria (RGM) and 14 strains of aerobic actinomycetes as quality controls (QC) were tested in the API (RAPID) Coryne system version 2. Both groups yielded codes with low identification scores, considerable overlaps, and similar diagnoses. No species-specific codes were observed. Thus, the system would not be useful for the identification of RGM.

Actinomycetales↗
PubMed2001

Genetic characterization of a large, historically significant Utah kindred with facioscapulohumeral dystrophy.

In 1950, Tyler and Stephens reported a remarkable kindred affected with facioscapulohumeral dystrophy (FSHD), consisting of 1249 descendants of a man who emigrated to Utah in 1840. Members of this kindred are still seen in our clinic and, to our knowledge, no member had been tested for deletions at the FSHD1A locus on chromosome 4q35, the common chromosomal rearrangement associated with FSHD. We have identified 971 additional members of this kindred who either were not included in or unborn at the time of the report by Tyler and Stephens, and have identified 120 living members as affected by history or by examination. Members of this kindred contribute to a disease prevalence of nearly 1:15 000 in the Utah/southern Idaho region. We have demonstrated that affected members carry a disease-associated 20 kb deletion allele at the FSHD1A locus. This allele is the same size in multiple, distantly-related branches of the kindred, confirming the meiotic stability of the FSHD1A deletion. This large, genetically homogeneous population of patients represents a unique resource with which to study current questions about FSHD, including the possibilities of anticipation and parental transmission effects.

Chromosomes, Human, Pair 4↗
PubMed2001

DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression.

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor "mediated uptake is less effected by excessive PEGylation.

DNA↗
PubMed2001

[MR angiography of pelvic and leg arteries: initiation with time-resolved data acquisition of the lower legs].

58 patients suffering from peripheral arterial vascular disease were examined using contrast-enhanced MR angiography with the intention of optimizing the visualization of lower leg arteries. Different from the customary acquisition order, were first the arteries of the lower legs depicted with three time-resolved phases. Afterwards, the iliacal and femoral vessels were imaged by applying the floating-table technique in two steps. In all cases, the lower leg arteries were depicted without overlying veins. By injecting the contrast agent in two phases, imaging quality of the iliofemoral arteries was not significantly reduced.--In conclusion, we would recommend the hybrid technique of peripheral contrast-enhanced MRA with primarily starting the acquisition of the lower legs in cases of foot infections or ulcerations where the transit time is reduced bi- or unilaterally.

Adult↗
PubMed2000

The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.

The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.

Electron Transport↗
PubMed2000

Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis.

BACKGROUND: Autoantibodies are a hallmark of autoimmune hepatitis, but most are not disease specific. Autoantibodies to soluble liver antigen (SLA) and to liver and pancreas antigen (LP) have been described as disease specific, occurring in about 30% of all patients with autoimmune hepatitis, but no standardised assays are available. Methods We tested 2000 serum samples from patients with various liver diseases and controls for SLA autoantibodies by inhibition ELISA. Serum samples positive for SLA antibodies were used for immunoscreening of cDNA expression libraries. Identified clones were tested against a panel of serum samples positive for SLA and LP autoantibodies and control serum samples, and the epitope mapped by deletion mutants and exonuclease digestion. FINDINGS: SLA and LP autoantibodies were identical. Of 2000 serum samples screened, 35 were positive for SLA autoantibodies. These positive samples came from patients with autoimmune hepatitis; three from patients with an overlap syndrome (primary biliary cirrhosis and secondary autoimmune hepatitis). Expression cloning and absorption experiments identified a 422 aminoacid protein present in two splice variants as the sole target antigen. Aminoacids 371-409 were critical for immune recognition. INTERPRETATION: The identified cDNA encodes the primary target antigen of SLA/LP autoantibodies. The SLA/LP antigen has a previously unknown aminoacid sequence, and presumably codes for an unindentified enzyme, suggested to be UGA-suppressor tRNA-associated protein. SLA/LP autoantibodies are disease specific and recognise a dominant epitope, suggesting a specific antigen-driven immune response. Identification of the SLA/LP target antigen will allow establishment of a reliable, widely available diagnostic assay. Furthermore, its role in the pathogenesis of autoimmune hepatitis can now be studied.

Autoantibodies↗
PubMed2000

Automated ribotyping and pulsed-field gel electrophoresis reveal a cluster of group A streptococci in intravenous drug abusers.

BACKGROUND: In 1998, an unexpected increase of group A streptococci (GAS) in blood cultures was observed at our institution. MATERIALS AND METHODS: To determine whether they were from unrelated sporadic cases or attributable to a common source, all isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and by automated ribotyping. RESULTS: Two clusters were found. All isolates, except one, of cluster 2 had been isolated from intravenous drug abusers. CONCLUSION: All patients were hospitalized either in different hospitals or at different times in the same hospitals indicating that the putative common source is to be found in the drug community rather than in a particular hospital.

Adult↗
PubMed2000

Catalase-negative Actinomyces neuii subsp. neuii isolated from an infected mammary prosthesis.

In this case report a catalase-negative strain of Actinomyces neuii subsp. neuii is described as the possible causative agent of an infected mammary prosthesis. DNA hybridization studies and 16S rRNA analysis confirmed that the strain belongs to the species Actinomyces neuii subsp. neuii. Since this strain is the first A. neuii subsp. neuii strain reported to be catalase negative, the catalase reaction should no longer be considered a key reaction for the diagnosis of this species but must be interpreted in conjunction with other characteristics.

Actinomyces↗
PubMed2000

Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.

The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory. Gene Therapy (2000) 7, 401-407.

Adenoviridae↗
PubMed1999

Differential behaviour of lipid based and polycation based gene transfer systems in transfecting primary human fibroblasts: a potential role of polylysine in nuclear transport.

DNA delivery systems for gene therapy applications have to be able to trigger the uptake of plasmid DNA into the nucleus. We have tested two types of non-viral vector systems, lipofection (cationic lipid-based, using Lipofectamine) and polyfection (cationic polymer-based, using glycerol enhanced transferrinfection), for their ability to transfect confluent, contact inhibited primary human fibroblasts. While both systems worked well with growing fibroblasts, polyfection was superior with confluent cells. A slight reduction in cell associated plasmid DNA was observed with resting cells, but it was similar for both types of complexes. Lipofectamine showed a prevalence for transfecting cycling cells as judged by costaining transfected cells with cell cycle markers. No such bias was observed when glycerol enhanced transferrinfection was used. Microinjection of plasmid DNA/polylysine complexes into the cytoplasm of fibroblasts resulted in a higher percentage of expressing cells than injection of plasmid DNA, offering an explanation for the higher transfection levels obtained with transferrinfection in non-growing cells.

Biological Transport↗
PubMed1999

Development of cytochrome P450 2D6-specific LKM-autoantibodies following liver transplantation for Wilson's disease -- possible association with a steroid-resistant transplant rejection episode.

BACKGROUND/AIMS: Antibodies to cytochrome P450 2D6, also known as LKM1-autoantibodies, are characteristic for a subgroup of patients with autoimmune hepatitis, but can also occasionally be found in hepatitis C. We observed the occurrence of LKM1-autoantibodies 4 months after liver transplantation for Wilson's disease, in close association with a steroid-resistant rejection episode, in the absence of evidence for autoimmune hepatitis or hepatitis C. METHODS: Sera from several time points prior to and following transplantation were tested for LKM-reactivity by immunofluorescence, ELISA and Western blotting. Antigen specificity was confirmed by Western blotting analysis on different cytochrome P450 isoenzymes. The absence of viral hepatitis C and hepatitis G virus infection was confirmed by polymerase chain reaction. The serum of the organ donor was also tested. RESULTS: All the sera prior to transplantation and up to 4 months after transplantation were LKM-negative by all assay systems used. In the course of a steroid-resistant rejection episode at this time, the patient developed LKM antibodies at high titre (70% in inhibition ELISA) and has remained positive since (now more than 4 years). Reactivity was exclusively to the cytochrome isoenzyme 2D6. Hepatitis C infection never occurred, but hepatitis G was transiently present many years prior to transplantation. The donor serum was negative for all autoantibodies and for hepatitis C and G virus infection. DISCUSSION: We here describe a patient developing LKM1-autoantibodies without evidence of autoimmune or viral hepatitis. The close temporal association with a transplant rejection episode suggests immunological mechanisms of rejection together with hepatocellular injury as a pathogenetic mechanism.

Adult↗
PubMed1999

PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery.

We investigated the in vitro and in vivo properties of DNA/transferrin-polyethylenimine (800 kDa) complexes before and after covalent coupling of poly(ethylene glycol) (PEG). Upon incubation with plasma, the positively charged non-PEGylated DNA complexes form aggregates. Plasma proteins such as IgM, fibrinogen, fibronectin and complement C3 were found to bind to non-PEGylated DNA complexes. At DNA concentrations relevant for in vivo gene delivery a strong aggregation of erythrocytes was also observed. PEGylation of the complexes strongly reduces plasma protein binding and erythrocyte aggregation. Furthermore, PEGylated complex size was stabilized and had a reduced surface charge. Prolonged circulation in the blood of the PEGylated complexes was also observed when injected intravenously. In tumor bearing mice, application of non-PEGylated complexes through the tail vein resulted in reporter gene expression in tail and lung, but severe toxicity was observed in some mice. In contrast, PEGylated complexes mediated reporter gene transfer to the tumor without significant toxicity.

Animals↗
PubMed1999

Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function.

Amino Acid Sequence↗
PubMed1998

Functional analysis and cell-specific expression of a phosphate transporter from tomato.

For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter c DNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains. It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi. When expressed in a Pi-uptake-deficient yeast mutant, the L. esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 MicroM. The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5. The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H + - ATPase. Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs. In situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root. The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells. Under Pi-deprivation condition, mRNA levels are also detectable in young stelar tissue. This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface.

Amino Acid Sequence↗