Search PubMed⌕ Search

Biomedical subjects

S Hoffmann

Publications and source records attributed to S Hoffmann.

At least 55 records · Page 3Linked to original sources
PubMed2001

Accurate disulfide formation in Escherichia coli: overexpression and characterization of the first domain (HF6478) of the multiple Kazal-type inhibitor LEKTI.

The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chromatography. Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies. Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy. Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E. coli cytoplasm.

Carrier Proteins↗
PubMed2001

Binding of phage-displayed HIV-1 Tat to TAR RNA in the presence of cyclin T1.

The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle. Together with cellular cyclin T1 and an RNA element (transactivation response; TAR) located at the 5' end of all viral transcripts, it forms a ternary complex that ultimately enhances the expression of all viral genes. In this ternary complex, cyclin T1 interacts directly with Tat and TAR. The presence of cyclin T1 is essential for high TAR RNA affinity and specificity of Tat. To study protein-protein and protein-RNA interaction, we developed a phage display system that displays functional Tat on the surface of bacteriophage M13. The addition of recombinant cyclin T1 to the selections yielded a phage display system that mirrors all binding properties of the cyclin T1-Tat-TAR complex known from cell assays and biochemical studies. Phage-displayed Tat protein as well as the cyclin T1 are fully functional. The relative binding capabilities of wild-type- and mutant Tat-displaying phages show that the presence of cyclin T1 significantly reduces the importance of basic residues in the basic sequence region of Tat for its binding to TAR.

Amino Acid Sequence↗
PubMed2001

Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

Angiotensin II↗
PubMed2001

[Physiotherapy in osteoarthritis--a review of literature on conservative therapy of knee and hip osteoarthritis].

In this review, 18 studies have been evaluated about the effectiveness of exercise therapy in the physiotherapy in osteoarthritis of hip or knee. All studies emphasised the general profit of exercise therapy compared to alternative programs (such as "health education", etc.). The effect sizes were mostly indicated to be moderate to large. There were no study comparing two or more alternative exercise programs in regard to the size of effectiveness. Only two studies gave informations about the radiographic grade of the OA. Today the effectiveness is proved only for aerobic, dynamic strengthening exercises. There were no study about the effectiveness of physiotherapy in Hip-OA.

Humans↗
PubMed2001

A procedure for evaluation and documentation of susceptibility test methods using the susceptibility of Klebsiella pneumoniae to ciprofloxacin as a model.

A new procedure for the evaluation and documentation of susceptibility test methods is described. To illustrate the procedure, four basically different susceptibility test methods were examined in a routine laboratory. The test parameter detection of decreased susceptibility to ciprofloxacin (breakpoint MIC 0.25 mg/L) among 94 selected isolates of Klebsiella pneumoniae was used. In addition to comparison of frequency histograms and regression analysis, the accuracies of the susceptibility test methods were determined using the receiver operating characteristic procedure. For each of the methods, the sensitivity (SN), specificity (SP), positive predictive value (PV+) and negative predictive value (PV-) for detection of decreased susceptibility to ciprofloxacin were calculated and plotted against a range of ciprofloxacin inhibition zones determined by the various susceptibility test methods or MICs determined by the Etest (Etest MICs). The results illustrate the accuracy and the robustness of the methods, which can be used to expose the need for training and instruction of laboratory staff. It becomes possible to optimize and justify the choice of inhibition zone breakpoints or Etest MIC breakpoints according to the SN and SP of the method employed. Furthermore, the consequences of adjustments of these breakpoints on the PV+ and PV- can be analysed and related to different clinical and epidemiological situations. We believe that our approach can be used as a model for the evaluation and documentation of susceptibility test methods in general.

Anti-Infective Agents↗
PubMed2001

Scanning force microscopy observation of tumor cells treated with hematoporphyrin IX derivatives.

Cells from human head-neck squamous cell carcinoma (hypopharynx, UMSCC11, and UMSCC22) and lung tumor (alveolus, UMSCC7) were investigated by light and scanning force microscopy (SFM) observation. In the present study a less-invasive contact mode was used to scan living cells in air covered with a thin fluid layer. The investigations were done without any pretreatment of the specimens for such observations as chemical fixation, or staining. Untreated tumor cells, and those treated with the antitumor drug hematoporphyrin IX derivative (HpD) were studied without photosensitizing. Additionally, the temperature influence on cell proliferation was studied. Three-dimensional topographic images and their magnifications offer highly informative insights into the untreated cell surface. In the present study, the cell structure destruction and cell death could partly be visualized by observing the head-neck tumor cells incubated with HpD. Most of the drug-treated head-neck tumor cells died (only 2%-5% of survivors). However, HpD could not affect cells of the lung tumor cell type. They, well known as more resistant against oxidation processes, survived to a great extent. Also, no distortion of the membrane under drug treatment could be observed. The possibilities and limits in the use of SFM for studying the topography of cell surfaces are presented in many details.

Antineoplastic Agents↗
PubMed2001

Phosphatidylcholine molecular species in lung surfactant: composition in relation to respiratory rate and lung development.

Surfactant reduces surface tension at the air-liquid interface of lung alveoli. While dipalmitoylphosphatidylcholine (PC16:0/ 16:0) is its main component, proteins and other phospholipids contribute to the dynamic properties and homeostasis of alveolar surfactant. Among these components are significant amounts of palmitoylmyristoylphosphatidylcholine (PC16:0/ 14:0) and palmitoylpalmitoleoylphosphatidylcholine (PC16:0/ 16:1), whereas in surfactant from the rigid tubular bird lung, PC16:0/14:0 is absent and PC16:0/16:1 strongly diminished. We therefore hypothesized that the concentrations of PC16:0/14:0 and PC16:0/16:1 in surfactants correlate with differences in the respiratory physiology of mammalian species. In surfactants from newborn and adult mice, rats, and pigs, molar fractions of PC16:0/14:0 and PC16:0/16:1 correlated with respiratory rate. Labeling experiments with [methyl-(3)H]choline in mice and perfused rat lungs demonstrated identical alveolar proportions of total and newly synthesized PC16:0/14:0, PC16:0/16:1, and PC16:0/16:0, which were much higher than those of other phosphatidylcholine species. In surfactant from human term and preterm neonates, fractional concentrations not only of PC16:0/16:0 but also of PC16:0/14:0 and PC16:0/ 16:1 increased with maturation. Our data emphasize that PC16:0/14:0 and PC16:0/16:1 may be important surfactant components in alveolar lungs, and that their concentrations are adapted to respiratory physiology.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Fusion proteins from artificial and natural structural modules.

The purpose of preparing fusion proteins from designed and natural sequences is mainly twofold; it aims at the stabilization of structure and at the modification of biological activity. Fusion with beta-galactosidase, for example, can increase the intracellular stability and DDT-degrading activity of an artificial DDT-binding peptide, and fusions with a leucine zipper produce mono- and bifunctional single-chain variable domain antibody fragments or homodimeric and heterodimeric DNA-binding proteins like an artificial homodimeric HIV-1 enhancer-binding protein with increased binding specificity and repressor activity. Of importance are also short leader sequences that mediate the translocation of proteins across the cytoplasmic and the nuclear membrane. An interesting by-product of the leucine zipper-mediated dimerization of an HIV-1 enhancer-binding protein was the synthesis and the structural as well as functional characterization of a retro-leucine zipper.

Amino Acid Sequence↗
PubMed2001

The laboratory surveillance system of Chlamydia trachomatis and Neisseria gonorrhoeae infections in Denmark.

This article presents results of surveillance of C. trachomatis and N. gonorrhoeae infection in Denmark. In 1999 and 2000, there were 291 and 309 male cases of laboratory-confirmed gonorrhoeae, whereas the number of female cases was 43 for both years. The penicillin resistance was steady, with a prevalence of 40% in 1999, whereas the prevalence of fluoroquinolone resistance increased from 0% in 1990 to 27% in 1999. The number of laboratory-confirmed C. trachomatis infections remains around 13 000 cases per year. Men aged 20-29 years and women aged 15-24 are the most at risk.

Adolescent↗
PubMed2001

A survey of academic and industry professionals regarding the preferred skillset of graduates of medical informatics programs.

Identification of the skills needed by graduates of medical informatics masters degree programs is needed so that students will know what is desired in the workplace and curriculum designers can assure that courses cover relevant areas. We conducted a mail survey of representatives of the informatics job market to discover what they think is most important. A survey instrument was designed after analyses of job ads and curricula in the U.S. and interviews with representative employers. The survey was mailed to 1000 randomly selected members of AMIA and HIMMS plus EMR vendors. Respondents were asked to rank skills and groups of skills according to perceived utility. The results indicate higher rankings for organizational and interpersonal skills than for more technical credentials. Statistical analysis indicates the existence of relatively few underlying constructs to the skill list.

Data Collection↗
PubMed2000

VEGF release by retinal glia depends on both oxygen and glucose supply.

Isolated retinae or isolated Müller cells were cultured in vitro, and vascular endothelial growth factor (VEGF) was assayed as protein (by ELISA) and as mRNA (by semi-quantitative RT-PCR). In both types of cultures, hypoxia (5% O2) resulted in an upregulated VEGF release. While the unstimulated VEGF secretion was virtually independent of glucose (0.125 - 25 mM), elevated glucose concentrations (10 - 25 mM) blocked most of the stimulatory effect of hypoxia on VEGF mRNA synthesis (determined in Müller cell cultures) as well as on VEGF release (in both retina and Müller cell cultures). It is concluded that in retinal glial (Müller) cells, being responsible for retinal VEGF synthesis (and, thus, for undesirable neovascularization), the metabolic effects of hypoxia can be compensated by a surplus of glucose.

Animals↗
PubMed2000

Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II.

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.

Biological Transport↗
PubMed2000

Origin of dynamic heterogeneities in miscible polymer blends: A quasielastic neutron scattering study

In order to investigate the origin of the often invoked nanoheterogeneities in miscible polymer blends, we have performed quasielastic neutron scattering experiments on the component dynamics within the miscible polymer blend polyisoprene/polyvinyl ether including the pure components as a reference. We find that the apparent local heterogeneities observed by spectroscopic techniques originate from the chain specific crossover properties between entropy driven and local chain dynamics and are, thus, a purely dynamical phenomenon.

Journal Article↗
PubMed2000

Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga.

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH(2)-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.

Amino Acid Sequence↗
PubMed2000

Decreased aminoacylation of mutant tRNAs in MELAS but not in MERRF patients.

Mutations in human mitochondrial tRNA genes are associated with a number of multisystemic disorders. Using an assay that combines tRNA oxidation and circularization we have determined the relative amounts and states of aminoacylation of mutant and wild-type tRNAs in tissue samples from patients with MELAS syndrome (mito- chondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes) and MERRF syndrome (myoclonus epilepsy with ragged red fibers), respectively. In most, but not all, biopsies from MELAS patients carrying the A3243G substitution in the mitochondrial tRNA(Leu(UUR))gene, the mutant tRNA is under-represented among processed and/or aminoacylated tRNAs. In contrast, in biopsies from MERRF patients harboring the A8344G substitution in the tRNA(Lys)gene neither the relative abundance nor the aminoacylation of the mutated tRNA is affected. Thus, whereas the A3243G mutation may contribute to the pathogenesis of MELAS by reducing the amount of aminoacylated tRNA(Leu), the A8344G mutation does not affect tRNA(Lys)function in the same way.

Acylation↗
PubMed2000

R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil inhibit human retinal pigment epithelial cell contraction.

PURPOSE: To evaluate the effects of R-(+)-verapamil, S-(-)-verapamil, and the racemate on in vitro contraction of human retinal pigment epithelial cells. METHODS: RPE cells from human donor eyes were enzymatically dissociated and cultured on collagen gels. Transdifferentiated RPE cells (seventh and eighth passage) were used for experiments. The contraction assays were treated with different concentrations (10-1000 microM) of R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil. RESULTS: Verapamil inhibited the gel contraction of human RPE cells. R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil acted equally in a dose-dependent manner from 10-1000 microM. CONCLUSION: The results indicate the effectiveness of verapamil to inhibit the contraction of RPE cells. R-(+)-verapamil is recommended because it is associated with fewer systemic and local side effects.

Adolescent↗