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Biomedical subjects

S König

Publications and source records attributed to S König.

At least 37 records · Page 2Linked to original sources
PubMed2001

Transfection of dendritic cells (DCs) with the CIITA gene: increase in immunostimulatory activity of DCs.

Dendritic cells (DCs) are the major antigen-presenting cells. They are able to present tumor antigens to immunologic effector cells. MHC class II molecules on DC surfaces play an important role in priming effector cells against tumor cells and their antigens. The transactivator CIITA (MHC class II transactivator) is a non-DNA-binding transactivator, which regulates the expression of MHC class II, HLA-DM, and invariant chain and behaves as a master controller of constitutive and inducible MHC class II gene activation. Here, we transfected DCs with the CIITA gene using a novel transfection technique. The vector system consisted of a plasmid bound to an adenovirus via poly-L-lysine, which is covalently bound to a UV-irradiated adenovirus. After transfection, expression of MHC class II on DCs increased from 27% to 75% on day 2 after transfection. Transfected DCs were co-cultured with immunologic effector cells. Cytotoxicity of effector cells against tumor cells increased after co-culture with transfected DCs to 63% compared to 15% with effector cells co-cultured with irrelevantly transfected DCs (P=.037). This effect was dependent on the timing and period of co-culture. In conclusion, transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs. We can further conclude that DCs could be efficiently transfected with the CIITA gene. Transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs and may have a major impact on immunotherapeutic protocols for patients with cancer.

Adenoviridae↗
PubMed2001

Incidence of epilepsies and epileptic syndromes in children and adolescents: a population-based prospective study in Germany.

PURPOSE: To estimate the incidence rate of epilepsies and epileptic syndromes in German children and adolescents aged 1 month to <15 years, and to provide data on their classification. METHODS: A population-based prospective study was performed between July 1, 1999, and June 30, 2000. All children aged 1 month to <15 years with a newly diagnosed epilepsy or epileptic syndrome were recorded by private pediatricians, EEG laboratories, and the two University Children's Hospitals in the neighboring cities of Heidelberg and Mannheim. The diagnoses were classified according to the International Classification of Epilepsies and Epileptic Syndromes of the International League Against Epilepsy (ILAE). RESULTS: The total age-adjusted annual incidence rate was 60/100,000 (95% confidence interval, 42-84), with the highest incidence in the first year of life (146/100,000). Focal epilepsies or epileptic syndromes (58%; incidence rate, 35/100,000) were more common than were generalized ones (39%; incidence rate, 24/100,000), and 3% (incidence rate, 2/100,000) of the epilepsies or epileptic syndromes were undetermined. The rate of idiopathic (47%; incidence rate, 29/100,000) and symptomatic or cryptogenic epilepsies (50%; incidence rate, 30/100,000) was equal. No significant difference in incidence between boys and girls was found. CONCLUSIONS: Incidence rates for epilepsy in German children aged 1 month to <15 years are about equal to those of other countries in Europe and North America. In accordance with studies from the United States and from many European countries, incidence was highest in the first year of life, and no difference in the incidence between girls and boys was found. In Germany as throughout Europe, idiopathic generalized epileptic syndromes are more often diagnosed than in the United States.

Adolescent↗
PubMed2001

Evidence for perforin-like activity associated with earthworm leukocytes.

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Journal Article↗
PubMed2001

Sonographic monitoring of midline shift in space-occupying stroke: an early outcome predictor.

BACKGROUND AND PURPOSE: Transcranial color-coded duplex sonography (TCCS) allows bedside imaging of intracranial hemodynamics and parenchymal structures. It provides reliable information regarding midline shift (MLS) in space-occupying hemispheric stroke. We studied the value of MLS measurement to predict fatal outcome at different time points after stroke onset. METHODS: Forty-two patients with acute, severe hemispheric stroke were enrolled. Cranial computed tomography (CCT) and extracranial duplex sonography were performed on admission. TCCS was carried out 8+/-3, 16+/-3, 24+/-3, 32+/-3, and 40+/-3 hours after stroke onset. Lesion size was determined from follow-up CCT. RESULTS: Twelve patients died as the result of cerebral herniation (group 1); 28 survived (group 2). Two patients received decompressive hemicraniectomy and were therefore excluded from further evaluation. MLS was significantly higher in group 1 as early as 16 hours after onset of stroke. Specificity and positive predictive values for death caused by cerebral herniation of MLS >/=2.5, 3.5, 4.0, and 5.0 mm after 16, 24, 32, and 40 hours were 1.0. CONCLUSIONS: TCCS helps to estimate outcome as early as 16 hours after stroke onset and thus facilitates identification of patients who are unlikely to survive without decompressive craniectomy. Because of its noninvasive character and bedside suitability, sonographic monitoring of MLS might be a useful tool in management of critically ill patients who cannot undergo repeated CCT scans.

Aged↗
PubMed2000

Capillary electrophoresis and off-line capillary electrophoresis-electrospray ionization quadrupole time-of-flight tandem mass spectrometry of carbohydrates.

The use of off-line high-performance capillary electrophoresis in connection with nanospray electrospray ionization quadrupole time-of-flight tandem mass spectrometry for identification of complex carbohydrates of biological origin is presented. The method was applied to the identification of O-glycosylated amino acids and -glycopeptides from the urine of patients suffering from a hereditary disease - N-acetylhexosaminidase deficiency. Structural elements typical for O-glycosylation of proteins, like expression of core 1 and 2 type O-glycans with different numbers of N-acetyllactosaminyl repeats and different degrees of sialylation, can be directly detected.

Carbohydrate Sequence↗
PubMed2000

Moderation of the electroosmotic flow in capillary electrophoresis by chemical modification of the capillary surface with tentacle-like oligourethanes.

The surface chemistry of the inner wall of fused-silica capillaries is one important means to control the magnitude as well as the direction of the electroosmotic flow and the adsorption activity. A method was developed to change the surface characteristics of fused-silica capillaries by binding tentacle-like oligourethane groups onto the inner surface. The electroosmotic flow at a buffer pH of 6-9 was reduced by 15 to 40% compared to that in a bare fused-silica tubing, dependent on the type of coating. Sample adsorption is diminished at the same time resulting in a separation of proteins with higher resolution and good migration time precision. At a pH below 4.5 the electroosmotic flow is reversed into the anodic direction, which offers further possibilities for the separation of positively charged analytes as demonstrated for the separation of aromatic and biogenic amines.

Electrophoresis, Capillary↗
PubMed2000

Protection against FIV challenge infection by genetic vaccination using minimalistic DNA constructs for FIV env gene and feline IL-12 expression.

OBJECTIVE: To evaluate the efficacy of a genetic vaccination protocol based on minimalistic, immunogenic defined gene expression (MIDGE) vectors coding for domains of the feline immunodeficiency virus (FIV) env gene and feline IL-12. METHODS: Three groups of four cats each were immunized three times within 6 weeks by the ballistic transfer of gold particles coated with MIDGE vectors. Group 1 received non-coated gold beads, groups 2 and 3 MIDGE vectors expressing FIV surface plus part of the transmembrane protein. In addition, group 3 received feline IL-12 DNA. All cats were challenged by intraperitoneal injection of 25 TCID50 of infectious FIV Z2. The following criteria were monitored: clinical signs, antibodies to transmembrane protein, antibodies to whole FIV, haematological parameters and kinetics of CD4 and CD8 cells, FIV proviral load (determined by quantitative polymerase chain reaction; PCR) and cytotoxic T lymphocyte (CTL) activity (in selected cats). RESULTS: None of the cats developed a detectable antibody response during immunizations. Four weeks after challenge exposure, all cats in group 1 (control) and group 2 (FIV surface-transmembrane protein) had seroconverted and showed a high proviral load until week 19 (end of experiment). In contrast, only one of four cats in group 3 (surface-transmembrane protein and IL-12) showed antibodies; it was provirus positive at reduced virus load. Short-lived CTL activity was found in two cats in group 3. CONCLUSION: Genetic vaccination using a MIDGE-based construct for the expression of the surface-transmembrane protein domain of FIV env and feline IL-12 DNA led to protection against homologous virus challenge in three out of four vaccinated cats.

Animals↗
PubMed2000

Peptidylglycine-alpha-hydroxylating monooxygenase generates two hydroxylated products from its mechanism-based suicide substrate, 4-phenyl-3-butenoic acid.

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14.17. 3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.

Catalytic Domain↗
PubMed2000

Angiotensin II activates nuclear transcription factor kappaB through AT(1) and AT(2) in vascular smooth muscle cells: molecular mechanisms.

Nuclear factor-kappaB (NF-kappaB) regulates many genes involved in vascular physiopathology. We have previously observed in vivo NF-kappaB activation in injured vessels that diminished by angiotensin-converting enzyme inhibition. In the present work, we investigated the effect of angiotensin II (Ang II) on NF-kappaB activity in rat vascular smooth muscle cells, evaluating the molecular mechanisms and the specific receptor subtype involved. Ang II increased NF-kappaB DNA binding (5-fold, 10(-)(9) mol/L at 1 hour; electrophoretic mobility shift assay), nuclear translocation of p50/p65 subunits, and cytosolic inhibitor kappaBalpha (IkappaBalpha) degradation. Ang II elicited NF-kappaB-mediated transcription (transfection of a reporter gene) and expression of NF-kappaB-related genes (monocyte chemoattractant protein-1 and angiotensinogen). AT(1) (DUP753) and AT(2) (PD123319 and CGP42112) receptor antagonists inhibited Ang II-induced NF-kappaB DNA binding in a dose-dependent manner ( approximately 85% for each one; 10(-)(5) mol/L at 1 hour). The AT(2) agonist p-aminophenylalanine(6)-Ang II augmented NF-kappaB binding (4.6-fold, 10(-)(9) mol/L at 1 hour), p65 nuclear levels, and transcription of an NF-kappaB reporter gene. AT(1) antagonist markedly inhibited NF-kappaB-mediated transcription and gene expression. Some differences between AT(1)/AT(2) intracellular signals were found. Antioxidants and ceramide inhibitors, but not protein kinase C inhibitors, diminished NF-kappaB activation elicited by both Ang II and the AT(2) agonist, while tyrosine kinase inhibitors only decreased Ang II-induced NF-kappaB activity. Our results demonstrate that Ang II activates NF-kappaB via AT(1) and AT(2), although NF-kappaB-mediated transcription occurred mainly through AT(1). Both receptors share some signaling pathways (oxygen radicals and ceramide); however, tyrosine kinases only participate in AT(1)/NF-kappaB responses. These data provide novel insights into Ang II actions, suggesting a potential implication of the AT(2) in the pathobiology of vascular cells.

Angiotensin II↗
PubMed2000

Crystal versus solution structures of thiamine diphosphate-dependent enzymes.

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.

Artifacts↗
PubMed2000

Measurement of dipolar couplings in a transducin peptide fragment weakly bound to oriented photo-activated rhodopsin.

Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with 15N at Leu5 and Gly9, results in residual dipolar contributions to the 1J(NH) splittings for these two residues. Both residues show 1J(NH) splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu5 and Gly9 make angles of 48+/-4 degrees and 40+/-8 degrees, respectively, with the disk normal. These 'transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.

Amino Acid Sequence↗
PubMed2000

Genetic variation of European beech (Fagus sylvatica L.) along an altitudinal transect at Mount Vogelsberg in Hesse, Germany.

Allelic and genotypic variation at 13 different enzyme loci of autochthonous European beech (Fagus sylvatica L.) was investigated in six 110-160-year-old stands growing at elevations between 150 and 660 m above sea level on the western slope of mount Vogelsberg in central Germany. The highest elevated population showed the highest number of effective alleles (Ne), the highest total heterozygosity (He) and the highest population differentiation deltaT. Also, the genotype SKD-A2A3 of shikimate dehydrogenase was significantly more frequent at the two highest elevated stands (P = 11%) than at the three lowest elevated stands (P = 1%). Further differences in genotype frequencies between 11 of 15 stand pairs were elevation independent.

Alcohol Oxidoreductases↗
PubMed2000

The structural basis of substrate activation in yeast pyruvate decarboxylase. A crystallographic and kinetic study.

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.

Catalytic Domain↗
PubMed1999

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.

Amino Acid Sequence↗
PubMed1999

Comparison of safety and efficacy of carvedilol and metoprolol in stable angina pectoris.

In a double-blind, randomized, 3-month multicenter study, the safety and tolerability and the antianginal and anti-ischemic efficacy of carvedilol 25 to 50 mg twice daily were assessed in comparison with metoprolol 50 to 100 mg twice daily in younger and elderly patients with stable angina. After a 7-day placebo run-in at the end of which a symptom-limited bicycle ergometric exercise was performed, 368 patients were randomly allocated to the parallel treatment groups. After 4 weeks of therapy with a low dose, a further exercise test was performed and patients were titrated in single-blind fashion to the higher dose if the increase in total exercise time was < 1 minute, and there was no safety concern. After a further 8 weeks of treatment a third exercise test was performed. Carvedilol low dose/high dose was shown to be at least as safe and well tolerated as metoprolol low dose/high dose both in younger and elderly patients. There were no hitherto unknown adverse events and no marked change in the types of events after increase of the doses. Early adverse events after treatment initiation or uptitration were equal with both medications, indicating no particular risk associated with carvedilol's vasodilatory action. No rebound phenomena were observed. Both drugs showed good antianginal and anti-ischemic efficacy, with marked increases on uptitration including patients > or = 65 years of age. However, in the doses selected, which appeared equipotent with respect to beta blockade, carvedilol's improvement of time to 1-mm ST-segment depression was statistically significantly greater than that of metoprolol. This could be due to its additional vasodilatory or antioxidative actions. Based on the safety and efficacy data of the present study, use of the higher of the 2 recommended doses of carvedilol and metoprolol appears justified in younger and elderly patients without adequate therapeutic control at lower doses.

Adrenergic alpha-Antagonists↗
PubMed1999

[Significance of the height and width of the alveolar ridge in implantology in the edentulous maxilla. Analysis of 95 cadaver jaws and 24 consecutive patients].

Consideration of alveolar profiles and clinical experience demonstrate that the transversal dimension has been neglected in dental implantology so far. For a comprehensive evaluation of the impact of alveolar bone height and width, 95 edentulous bony maxillae with standardized, measured, and classified cross-sections were analyzed. With four types of implants (minimum length, 10 mm), 1076 insertions were simulated at 269 cross-sections and evaluated with regard to type of implant, position of cross-section, and class of atrophy. Similar evaluation was carried out in the clinical part of the study on 24 consecutive patients with edentulous maxillae. Implant insertion could only be simulated in 35% of the cadaver cross-sections, but had been expected in an additional 4.5% based on their sufficient bone height; length reductions were necessary in another 6%. These results depended largely on the class of atrophy. Anterior cross-sections offered better conditions than posterior ones. In contrast, implant insertion was impossible in all 24 patients. Height was primarily inadequate in 22 patients, and in two patients with sufficient bone height inadequate transversal dimensions were only recognised intraoperatively. These results allow a quantification of the impact of vertical and transversal maxillary alveolar bone dimensions. This impact primarily depends on bone height, but even with sufficient height, reductions of implant length often become necessary. Both for the cadaver maxillae (12% of the cross-sections with expected implant insertion) and for the patients (8%), alveolar profiles remain in which height measurement alone leads to incorrect assessment and may even result in the interruption of precisely planned surgical procedures. The complexity and expense of implant-borne rehabilitation and the consequences resulting from incorrect preoperative planning therefore generally justify extended cross-sectional diagnostic measuring.

Alveolar Process↗
PubMed1999

[Follow-up studies of 3-dimensional osteoplastic reconstruction of the extremely atrophied maxilla combined with implants].

In the severely resorbed maxilla, a 10-year success rate of only 49-74% of implants in combination with autogenous bone grafts has been reported. We developed a modified technique of antral inlay grafting and lateral and vertical onlay grafting of the severely resorbed maxilla for inserting implants to retain dentures. The clinical and radiologic results are presented. In 21 patients with severely resorbed edentulous maxillae, a total of 20 bilateral and one unilateral antral inlay graftings and lateral and vertical onlay graftings were performed after a prosthodontic setup. We opened the maxillary sinus by removing a bony window from the anterolateral wall and, after elevation of the sinus lining, grafted the sinus floor with corticocancellous iliac crest bone grafts. The maxilla was also augmented in the lateral and vertical direction. The bone grafts were fixed by osteosynthesis. After a median of 5 months, a total of 134 implants (Brånemark) were placed and later loaded by prosthodontic rehabilitation. Computed tomography (CT) scans were taken before the grafting procedure, immediately after grafting, after 4 months, and every year thereafter. A total of 94.8% of the implants were successful at the time of the abutment operation. After loading, two additional failures were seen in an average follow-up period of 2.5 years. Most patients were provided with implant-borne dentures. CT scans showed an average initial gain of vertical bone height of 3.7-17.7 mm. One year after grafting, a loss of 1.3 mm or 7% occurred. In the following 2 years, no major atrophy was observed. Statistical analysis showed no correlation between sex, bone height before augmentation, augmented bone height, and resorption of the grafted bone. We observed undisturbed healing and obtained large vertical bone heights, a high success rate, minimal resorption, and fully satisfactory prosthodontic rehabilitation; we can thus recommend our modified technique of reconstruction of the severely resorbed maxilla for routine use.

Alveolar Bone Loss↗