Biomedical subjects
Publications and source records attributed to S König.
The extensor mechanism of the hand consists of different tendons, originating from extrinsic and intrinsic muscles of the hand and forearm. Because of their course near to skin and bone separated only by a thin layer of paratenon, they are very sensitive to injuries and adhesions. Treatment protocols have to be adapted according to the different zones. This paper explains the basic principles of rehabilitation in extensor tendon injuries.
The aim of this project was to establish a method for the purification of total-RNA from fixed rat-retina. Two different established methods were used for RNA purification, and successful isolation was verified with RT-PCR for amplification of beta-actin (two different product-lengths) and subsequent gel-electrophoresis. Total-RNA was successfully isolated from fixed rat-retina. The house keeping gene, beta-actin could be detected after fixing the retina either with 1% formalin or with 4% paraformaldehyde (PFA). Hexamer-primer based RT-PCR gave better results than the oligo-d(T)-primer based RT-PCR method. Both the 698 and 225 bp beta-actin-fragments could be successfully amplified, where amplification of the latter was more efficient. This approach shows that tissue fixation prior to RNA-isolation facilitates the rapid isolation of undamaged RNAs in tissues such as the retina, which are known to yield low levels of RNA and are vulnerable to RNases.
The rigid tris- and bis(catecholamide) ligands H(6)A, H(4)B and H(4)C form tetrahedral clusters of the type M(4)L(4) and M(4)L(6) through self-assembly reactions with tri- and tetravalent metal ions such as Ga(III), Fe(III), Ti(IV) and Sn(IV). General design principles for the synthesis of such clusters are presented with an emphasis on geometric requirements and kinetic and thermodynamic considerations. The solution and solid-state characterization of these complexes is presented, and their dynamic solution behavior is described. The tris-catecholamide H(6)A forms M(4)L(4) tetrahedra with Ga(III), Ti(IV), and Sn(IV); (Et(3)N)(8)[Ti(4)A(4)] crystallizes in R3(-)c (No. 167), with a = 22.6143(5) A, c = 106.038(2) A. The cluster is a racemic mixture of homoconfigurational tetrahedra (all Delta or all Lambda at the metal centers within a given cluster). Though the synthetic procedure for synthesis of the cluster is markedly metal-dependent, extensive electrospray mass spectrometry investigations show that the M(4)A(4) (M = Ga(III), Ti(IV), and Sn(IV)) clusters are remarkably stable once formed. Two approaches are presented for the formation of M(4)L(6) tetrahedral clusters. Of the bis(catecholamide) ligands, H(4)B forms an M(4)L(6) tetrahedron (M = Ga(III)) based on an "edge-on" design, while H(4)C forms an M(4)L(6) tetrahedron (M = Ga(III), Fe(III)) based on a "face-on" strategy. K(5)[Et(4)N](7)[Fe(4)C(6)] crystallizes in I43(-)d (No. 220) with a = 43.706(8) A. This M(4)L(6) tetrahedral cluster is also a racemic mixture of homoconfigurational tetrahedra and has a cavity large enough to encapsulate a molecule of Et(4)N(+). This host-guest interaction is maintained in solution as revealed by NMR investigations of the Ga(III) complex.
The authors analyzed the association of L-carnitine treatment with hepatic survival in 92 patients with severe, symptomatic, valproate-induced hepatotoxicity. Forty-eight percent of the 42 patients treated with L-carnitine survived, but only 10% of the 50 patients treated solely with aggressive supportive care survived (p < 0.001). Early intervention with IV rather than enteral L-carnitine was associated with the greatest hepatic survival. Specifically, all 10 patients who were diagnosed in <5 days and treated with IV L-carnitine survived. Most patients had features of chronic illness and most children appeared to be malnourished.
The cleavage of the donor substrate d-xylulose 5-phosphate by wild-type and H263A mutant yeast transketolase was studied using enzyme kinetics and circular dichroism spectroscopy. The enzymes are able to catalyze the cleavage of donor substrates, the first half-reaction, even in the absence of any acceptor substrate yielding d-glyceraldehyde 3-phosphate as measured in the coupled optical test according to Kochetov (Kochetov, G. A. (1982) Methods Enzymol. 90, 209-223) and compared with the H263A variant. Overall, the H263A mutant enzyme is less active than the wild-type. However, an increase in the rate constant of the release of the enzyme-bound glycolyl moiety was observed and related to a stabilization of the "active glycolaldehyde" (alpha-carbanion) by histidine 263. Chemically synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate is bound to wild-type transketolase with an apparent K(D) of 4.3 +/- 0.8 microm (racemate) calculated from titration experiments using circular dichroism spectroscopy. Both enantiomers are cleaved by the enzyme at different rates. In contrast to the enzyme-generated alpha-carbanion of (alpha,beta-dihydroxyethyl)thiamin diphosphate formed by decarboxylation of hydroxylactylthiamin diphosphate after incubation of transketolase with beta-hydroxypyruvate, the synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate did not work as donor substrate when erythrose 4-phosphate is used as acceptor substrate in the coupled enzymatic test according to Sprenger (Sprenger, G. A., Schörken, U., Sprenger, G., and Sahm, H. (1995) Eur. J. Biochem. 230, 525-532).
The study of medicine in Germany is in need of reform. Oversubscribed courses, the lack of practical reference, scarce patient contact and cancelled lessons define the educational landscape for many students. Since at present we cannot carry out global reforms, the quality of medical education is highly dependent on the commitment of those responsible in the individual institution. The aim of the revised surgical curriculum in Göttingen is to demonstrate how medical education with a high didactic level including multimedia teaching can be realised despite large student numbers. Duties both in patient care and education are coordinated through integrated organisation. Educational content and structure are made transparent by online guidebooks. In the surgical examination course and the practical course in surgery, patient-oriented tuition takes place consistently in small groups. The Teaching Studio provides additional aids such as interactive CD-ROMs, online research, textbooks, ultrasound equipment, examination and suturing dummies. Curriculum-relevant information, including the weekly case examples and instruction guides for clinical practical experience, is presented on an Internet website. Constant evaluation of the curriculum not only documents student motivation, but also serves to improve the educational concepts on a continuous basis. On completion of the practical course in surgery, 65% of participants maintain that they have reached the defined learning objectives "well" or "very well" (prior to reform only 17%). In the summer semester 2000, 46% of students gave top marks in the practical course in surgery with respect to tuition in patient contact. The results of the evaluations verify the positive feedback from the student body.
UNLABELLED: Do children who remain in remission with epilepsy after withdrawal of anticonvulsive therapy still exhibit a different behaviour and performance-related abilities compared to healthy children? It was the aim of this study to investigate whether these children showed deviating psychiatric behaviour and/or neuropsychological function deficits as compared with a healthy control group and with children following occasional seizures which had never been treated. The study comprised 39 children with epilepsy after withdrawal of anticonvulsive therapy, 37 children with occasional seizures, and 39 matched healthy control children who were tested with regard to psychiatric and neuropsychological criteria. The patients were aged 6-15 years; 28 had a primarily generalised epilepsy, and 11 had a focal epilepsy. The patients remained free from seizures for more than 1 year. Psychiatric testing comprised the Child Behavior Checklist by Achenbach and Edelbrock and the Mannheim Parent Interview. Neuropsychological testing included non-verbal intelligence, visual perception, short-term memory and learning ability, arithmetics, movement coordination and motor skills, fine motor skills and verbal functions. After withdrawal from anticonvulsive therapy, the children with epilepsy did not exhibit any psychiatric disturbances, whereas those with occasional seizures frequently showed behaviour problems. After withdrawal from anticonvulsive therapy, no neuropsychological deficits were found in the children with epilepsy as compared to those with occasional seizures or to healthy children. Risk factors for poorer test results were first seizures occurring before the age of 3 years, delayed development, and neonatal problems. CONCLUSION: Children with epilepsy who remained without relapse after withdrawal from the anticonvulsive therapy did not show any psychiatric abnormalities and their performance was comparable to that of healthy control children, provided no risk factors were present.
The Raf kinases serve as central intermediates to relay signals from Ras to ERK. Cell-specific effects of these signals on growth, differentiation and survival can be observed due to the recruitment of different isoenzymes of the Raf family. The in vitro phosphorylation of a site unique to B-Raf (Ser429) has been proposed to be responsible for the negative regulation of the isoenzyme by Akt. Using phosphopetide mapping and site-directed mutagenesis we showed that Ser429 is phosphorylated upon cAMP elevation in PC12 cells and proposed that PKA is a major kinase phosphorylating the B-Raf-specific site in vivo.
In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6-calcyclin whose expression in granulocytes has not been described so far.
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.
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.
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.
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.
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.
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.
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.
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.
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.