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Biomedical subjects

B Schäfer

Publications and source records attributed to B Schäfer.

At least 19 recordsLinked to original sources
PubMed2006

Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media.

A process for equine influenza virus vaccine production using a microcarrier system (Cytodex 1) in a 2 L Wave bioreactor is described. Growth of Madin Darby canine kidney (MDCK) cells in serum containing GMEM medium (SC) is compared to growth in serum-free Ex-Cell MDCK medium (SF) without washing steps and medium exchange before infection. Cultivations with microcarrier concentrations of 2 and 4 g/L for both media are shown. Metabolic data from carbon and amino acid metabolism are discussed. Additionally, in roller bottle experiments the influence of multiplicity of infection (moi) and trypsin concentration on the HA value was investigated. Analysis of HA and TCID(50) at 37 degrees C showed a stable HA of maximum 2.6 log HA/100 microL for 2 weeks. Peak TCID(50) titers of 10(7.7) viruses/mL were achieved 20h post infection, but infectivity was below detection limit after 150 h. Cell attachment onto microcarriers under serum-free conditions was improved by Ca(2+) addition and by cell harvesting without trypsin using only an EDTA/PBS solution. For the wave cultivation maximum virus titers of 2.3-2.6 log HA units/100 microL were reached from infection with a moi of 0.05. However, in SF medium pH dropped to less than pH 6.8 which resulted in lower HA titers of 1.7 log HA units/100 microL. For the higher microcarrier concentration (4 g/L) medium exchange steps (500 mL) were needed for both media. Omission of the washing step and medium exchange before infection in SF medium clearly simplified the influenza production process; however, for higher virus yields a better pH control of the wave bioreactor would be required. Higher cell densities (2.8 x 10(6) cells/mL for 2 g/L microcarrier) and better attachment compared to stirred tank bioreactors showed, that the wave bioreactor is a good alternative to stirred tank processes for expanding production capacities in case of a pandemic.

Amino Acids↗
PubMed2004

The in vivo form of the murine class VI POU protein Emb is larger than that encoded by previously described transcripts.

The class VI POU domain family member known as Emb in the mouse (rat Brn5 or human mPOU/TCFbeta1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5' RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the -COOH terminal, and an -NH(2) terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene.

Alternative Splicing↗
PubMed2004

Genetic aspects of targeted insertion mutagenesis in yeasts.

Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

Gene Expression Regulation, Fungal↗
PubMed2004

A novel complex regulates cardiac actin gene expression through interaction of Emb, a class VI POU domain protein, MEF2D, and the histone transacetylase p300.

Expression of the mouse cardiac actin gene depends on a distal enhancer (-7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3' AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation.

Acetyltransferases↗
PubMed2003

Cloning and characterization of boar epididymal secretory proteins by homology to the human.

Northern blot analysis suggested that the boar epididymis produces closely related counterparts to human epididymal proteins HE1, HE3, HE4, HE5 and HE12. 'Full-length' cloning by nucleic acid and amino acid sequence similarity was achieved by RT-PCR methods in the case of the porcine counterparts of HE3 and HE4, while the homologues of HE5 and HE12, despite their cross-hybridization during Northern blot analysis, have not yet been cloned. The two novel porcine cDNAs were derived from moderately abundant epididymal mRNAs that were 75 and 83% identical to HE3 and HE4 cDNAs, respectively. To emphasize their relationship to the corresponding HEs, they were named Se3 and Se4 cDNAs. Their open reading frames predicted small secretory proteins with 55% (Se3) and 76% (Se4) conserved amino acids. Monospecific antipeptide antibodies to HE secretory proteins identified He3- and HE12-related proteins on Western blots of porcine epididymal fluid and semen. Both Northern and Western analyses indicated that the Se proteins were produced in a regionalized pattern and accumulated in the cauda fluid.

Amino Acid Sequence↗
PubMed2002

[Systematic development of a scale for determination of health-related quality of life in multiple trauma patients. The Polytrauma Outcome (POLO) Chart].

Even years after having sustained multiple injuries patients often suffer from its sequelae. These comprise restrictions in physical function, but also pain, social and psychological impairments. Although the Meran Consensus Conference in 1990 defined the contents of "quality of life" (QoL) measures in surgery, still no instrument is available for the valid assessment of all relevant QoL domains in multiple injured patients. This paper describes the systematic development of a modular instrument for the assessment of health related QoL. Within three phases (phase I: generation of items, phase II: item reduction, phase III: pre-testing in 70 multiple injured and control patients) a questionnaire of 57 items was developed, which measures all relevant trauma-related aspects of QoL after acute hospital care. In combination with the Glascow Outcome Scale (GOS), the EUROQOL and the SF-36, the newly developed instrument builds the Polytrauma Outcome Chart (POLO-Chart) which will also be used as "Part E" for outcome assessment within the "Trauma registry" of the German Society for Trauma Surgery. In phase IV, the POLO-Chart will finally be validated in five trauma centres (Celle, Essen, Hanover, Cologne und Munich).

Activities of Daily Living↗
PubMed2002

Quality of life in children with traumatic brain injury--basic issues, assessment, and recommendations.

An international Task Force was convened under the guidance of BMBF Conference so as to review the "State of the art" for measuring quality of life (QoL) in children who have suffered traumatic brain injury (TBI). After expert review of instruments and evaluation of two independent literature reviews this work group established "inclusion criteria" for the review of current tools that could contribute to the measurement of QoL in children with TBI. Six instruments were determined to meet all or most of the criteria required to be used in current clinical practice and research for children with TBI.

Age Factors↗
PubMed2001

Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein.

We attempted to devise a transcription system in which a particular DNA sequence of interest could be inducibly expressed under the control of a modified polymerase III (pol III) promoter. Its activation requires a mutated transcription factor not contained endogenously in human cells. We constructed such a promoter by fusing elements of the beta-lactamase gene of Escherichia coli, containing a modified TATA-box and a pol III terminator, to the initiation region of the human U6 gene. This construct functionally resembles a 5'-regulated pol III gene and its transcribed segment can be exchanged for an arbitrary sequence. Its transcription in vitro by pol III requires the same factors as the U6 gene with the major exception that the modified TATA-box of this construct only interacts with a TATA-binding protein (TBP) mutant (TBP-DR2) but not with TBP wild-type (TBPwt). Its transcription therefore requires TBP-DR2 exclusively instead of TBPWT: In order to render the system inducible, we fused the gene coding for TBP-DR2 to a tetracycline control element and stably transfected this new construct into HeLa cells. Induction of such a stable and viable clone with tetracycline resulted in the expression of functional TBP-DR2. This system may conceptually be used in the future to inducibly express an arbitrary DNA sequence in vivo under the control of the above mentioned promoter.

Base Sequence↗
PubMed2000

Domestication differentially affects cochlear nucleus subdivisions in the gerbil.

We analyzed the effects of domestication on the subdivisions of the cochlear nucleus in the gerbil (Meriones unguiculatus) by comparing their volumes and rostrocaudal extents in laboratory gerbils and in age-matched F1 offspring of gerbils caught in the wild. In addition, soma size was systematically analyzed in the anteroventral cochlear nucleus of both groups. Total cochlear nucleus volume and rostrocaudal extent were not significantly different between groups either for young (postnatal day 9) animals before the onset of hearing or for young 4-month-old animals. However, the dorsal cochlear nucleus was significantly larger and the anteroventral cochlear nucleus was significantly smaller in young adults of the wild strain. Thus the relative proportions of the cochlear nucleus subdivisions differed between the groups. In addition, soma size was significantly larger in the low-frequency portion of the anterovental cochlear nucleus in domesticated gerbils compared to wild gerbils. To our knowledge, this is the first reported instance of a well-defined brain structure (e.g., the antreovental cochlear nucleus) being larger in the domesticated than in the wild form.

Animals↗
PubMed2000

In vitro differentiation of endothelial cells from AC133-positive progenitor cells.

Recent findings support the hypothesis that the CD34(+)-cell population in bone marrow and peripheral blood contains hematopoietic and endothelial progenitor and stem cells. In this study, we report that human AC133(+) cells from granulocyte colony-stimulating factor-mobilized peripheral blood have the capacity to differentiate into endothelial cells (ECs). When cultured in the presence of vascular endothelial growth factor (VEGF) and the novel cytokine stem cell growth factor (SCGF), AC133(+) progenitors generate both adherent and proliferating nonadherent cells. Phenotypic analysis of the cells within the adherent population reveals that the majority display endothelial features, including the expression of KDR, Tie-2, Ulex europaeus agglutinin-1, and von Willebrand factor. Electron microscopic studies of these cells show structures compatible with Weibel-Palade bodies that are found exclusively in vascular endothelium. AC133-derived nonadherent cells give rise to both hematopoietic and endothelial colonies in semisolid medium. On transfer to fresh liquid culture with VEGF and SCGF, nonadherent cells again produce an adherent and a nonadherent population. In mice with severe combined immunodeficiency, AC133-derived cells form new blood vessels in vivo when injected subcutaneously together with A549 lung cancer cells. These data indicate that the AC133(+)-cell population consists of progenitor and stem cells not only with hematopoietic potential but also with the capacity to differentiate into ECs. Whether these hematopoietic and endothelial progenitors develop from a common precursor, the hemangioblast will be studied at the single-cell level.

Animals↗
PubMed2000

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.

Animals↗
PubMed1999

[Difference between gastric mucosal pCO2 and arterio-intramucosal pCO2 during orthotopic liver transplantation].

Tonometry is a clinically accepted method to monitor blood flow of the splanchnic region, which is of particular interest in orthotopic liver transplantation (OLT). We investigated the hemodynamic changes and the tonometrically registered perioperative course of the difference between gastric mucosal pCO2 (prCO2) and arterial mucosal CO2 (CO2 gap) in 23 patients undergoing OLT without veno-venous bypass. Gastric mucosal pH (pHi) was additionally calculated. Despite significant changes in systemic hemodynamics during the anhepatic stage and after reperfusion and a significant drop in pHi during anhepacy, the difference between prCO2 and CO2 was constant. These contrasting findings of tonometry, i.e. solely a drop in pHi is, in our opinion, a consequence of the poor metabolic capacity of the liver in the perioperative OLT period, which influenced the calculation of the pHi with the Henderson-Hasselbalch equation. We conclude that, due to methodical problems, calculated pHi is not a reliable indicator of splanchnic blood flow and oxygenation during OLT. We therefore suggest that the prCO2 and the CO2 gap be used to monitor the splanchnic region. These parameters, obtained perioperatively, do not indicate a further reduction in splanchnic oxygenation despite profound changes in systemic hemodynamics during OLT without veno-venous bypass.

Adult↗
PubMed1999

Study of single-molecule dynamics and reactions with classic light microscopy.

Single-molecule studies in the life sciences often deal with observation or spectroscopy. Studies of reactions are rare, and the light microscope has been used for such experiments only occasionally. In an experimental environment, for example, as is required for most nearfield scanning or electron microscopies, it is difficult to study single-molecule reactions of biological relevance. Therefore, we have developed techniques to study single-molecule reactions with classic (nonscanning) farfield light microscopy. The conversion of nicotinamide adenine dinucleotide (NAD+) and lactate to NADH (a reduced form of NAD+), pyruvate, and H+ catalyzed by a few LDH-1 enzyme molecules has been studied in substrate solutions with different viscosity using the NADH autofluorescence. It is even possible to monitor the progress of the reaction by phase-contrast microscopy via scattering or absorption by product molecules. As an example for a single-molecule reaction with a macromolecule as substrate, the handling and enzymatic cutting of fluorescently stained lambda-DNA is studied. In solutions containing 10 mM magnesium and 66 mM potassium ions at pH 7.9, an individual DNA molecule tends to collapse into a globular structure. When moved through an aqueous solution, it becomes stretched by viscosity drag. After stopping the motion, the molecule collapses and the dynamics of this process can be quantified. When a restriction enzyme is present, sequence-specific cutting can be directly observed in the light microscope. The theoretical restriction pattern, as predicted from the sequence of the molecule, can be generated directly under visual inspection.

Bacteriophage lambda↗
PubMed1999

A novel group-II intron in the cox1 gene of the fission yeast Schizosaccharomyces pombe is inserted in the same codon as the mobile group-II intron aI2 in the Saccharomyces cerevisiae cox1 homologue.

We describe herein a large group-II intron which is inserted in the mitochondrial cox1 gene of the Schizosaccharomyces pombe strain EF2. The intron RNA consists of 2492 nucleotides which can be folded into a secondary structure with all the expected sequence motifs of subgroup-IIA1 introns (Michel et al. 1989). Determination of the exact splice point revealed that the intron is inserted in the same codon, but 1 bp downstream, as the mobile intron aI2 in the Saccharomyces cerevisiae cox1 homologue. A total of nine nucleotide changes was observed around the insertion site of the intron in the cox1 gene of strain EF2 compared with the reference strain ade7-50h(-). Seven of these changes are clustered within the 51 bp upstream of the splice point. Only one sequence deviation was found in the downstream exon. The intron is capable of splicing despite the fact that both the EBS1/IBS1 and the EBS2/IBS2 sequence motifs, thought to be necessary for correct splicing, extend over 5 instead of 6 bp. The maturase, endonuclease and reverse transcriptase domains of the putative protein encoded by the newly described S. pombe group-II intron were not closer to those encoded by the other two, cobI and cox2I, S. pombe group-II introns than to the group-II intron-encoded proteins in Allomyces, Marchantia, Podospora and Saccharomyces.

Amino Acid Sequence↗
PubMed1998

Mosaic structure of the cox2 gene in the petite negative yeast Schizosaccharomyces pombe: a group II intron is inserted at the same location as the otherwise unrelated group II introns in the mitochondria of higher plants.

In contrast to homologous genes in other fungal mitochondrial genomes, the gene encoding subunit 2 of cytochrome oxidase (cox2) in several Schizosaccharomyces pombe strains contains a large group II intron. Its 2436 nucleotides can be folded into a typical group II intron secondary structure, possessing all the expected sequence motifs for subgroup IIA1 (Michel et al., 1989). This intron is remarkable for the following reasons: (i) Five nucleotide changes were observed compared with the continuous form of the cox2 gene in the reference strain 50 at the 3'-exon sequence, but not in the 5'-exon. (ii) One of these changes occurred at the splice point leading to a serine instead of a threonine residue in the deduced cox2 polypeptide. In all cases, the alterations resulted in the replacement of more frequently used codons by rare ones. (iii) Although the intron is able to undergo splicing, the sequence motifs thought to be necessary for interaction between the 5'-exon and the intron during the splicing process (the EBS1/IBS1 as well as the EBS2/IBS2 pairings) are unusual. (iv) The intron is inserted at the same location in the cox2 gene as the otherwise unrelated intron from higher plants.

Alleles↗
PubMed1998

Stroke from internal carotid artery occlusion during mumps infection.

During the terminal incubation period of severe mumps infection, a 40-year-old patient suffered from large infarction in the right middle cerebral artery territory. The proximal right internal carotid artery (ICA) was occluded on angiography. Computed tomography of the neck detected a hemorrhage located ventromedial to the right common carotid artery. Four months later the right ICA was partly recanalized. Carotid surgery revealed an atherosclerotic plaque and a vessel wall, which was fragile and less compact than usually. A strong inflammatory reaction to mumps infection may have contributed to the pathogenesis of the cervical hemorrhage and to acute thrombosis and occlusion of the ICA.

Adult↗
PubMed1998

Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH.

Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh(-)-mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol. The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus. Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis. PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.

Alcohol Dehydrogenase↗