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R Bühler

Publications and source records attributed to R Bühler.

At least 19 recordsLinked to original sources
PubMed2006

HESS observations of the galactic center region and their possible dark matter interpretation.

The detection of gamma rays from the source HESS J1745-290 in the Galactic Center (GC) region with the High Energy Spectroscopic System (HESS) array of Cherenkov telescopes in 2004 is presented. After subtraction of the diffuse gamma-ray emission from the GC ridge, the source is compatible with a point source with spatial extent less than 1.2;{'}(stat) (95% C.L.). The measured energy spectrum above 160 GeV is compatible with a power law with photon index of 2.25+/-0.04(stat)+/-0.10(syst) and no significant flux variation is detected. It is finally found that the bulk of the very high energy emission must have non-dark-matter origin.

Journal Article↗
PubMed2006

Fast variability of tera-electron volt gamma rays from the radio galaxy M87.

The detection of fast variations of the tera-electron volt (TeV) (10(12) eV) gamma-ray flux, on time scales of days, from the nearby radio galaxy M87 is reported. These variations are about 10 times as fast as those observed in any other wave band and imply a very compact emission region with a dimension similar to the Schwarzschild radius of the central black hole. We thus can exclude several other sites and processes of the gamma-ray production. The observations confirm that TeV gamma rays are emitted by extragalactic sources other than blazars, where jets are not relativistically beamed toward the observer.

Journal Article↗
PubMed2005

Quantification of central motor conduction deficits in multiple sclerosis patients before and after treatment of acute exacerbation by methylprednisolone.

OBJECTIVE: To compare the effects of intravenous methylprednisolone (IVMP) in patients with relapsing-remitting (RR-MS), secondary progressive (SP-MS), and primary progressive multiple sclerosis (PP-MS). METHODS: Clinical and neurophysiological follow up was undertaken in 24 RR-MS, eight SP-MS, and nine PP-MS patients receiving Solu-Medrol 500 mg/d over five days for exacerbations involving the motor system. Motor evoked potentials (MEPs) were used to measure central motor conduction time (CMCT) and the triple stimulation technique (TST) was applied to assess conduction deficits. The TST allows accurate quantification of the number of conducting central motor neurones, expressed by the TST amplitude ratio. RESULTS: There was a significant increase in TST amplitude ratio in RR-MS (p<0.001) and SP-MS patients (p<0.02) at day 5, paralleling an increase in muscle force. TST amplitude ratio and muscle force remained stable at two months. In PP-MS, TST amplitude ratio and muscle force did not change. CMCT did not change significantly in any of the three groups. CONCLUSIONS: In RR-MS and SP-MS, IVMP is followed by a prompt increase in conducting central motor neurones paralleled by improvement in muscle force, which most probably reflects partial resolution of central conduction block. The lack of similar clinical and neurophysiological changes in PP-MS corroborates previous clinical reports on limited IVMP efficacy in this patient group and points to pathophysiological differences underlying exacerbations in PP-MS.

Adult↗
PubMed2002

Hemispheric asymmetry in visuospatial attention assessed with transcranial magnetic stimulation.

Transcranial magnetic stimulation (TMS) was used to study visuospatial attention processing in ten healthy volunteers. In a forced choice recognition task the subjects were confronted with two symbols simultaneously presented during 120 ms at random positions, one in the left and the other in the right visual field. The subject had to identify the presented pattern out of four possible combinations and to press the corresponding response key within 2 s. Double-pulse TMS (dTMS) with a 100-ms interstimulus interval (ISI) and an intensity of 80% of the stimulator output (corresponding to 110-120% of the motor threshold) was applied by a non-focal coil over the right or left posterior parietal cortex (PPC, corresponding to P3/P4 of the international 10-20 system) at different time intervals after onset of the visual stimulus (starting at 120 ms, 270 ms and 520 ms). Double-pulse TMS over the right PPC starting at 270 ms led to a significant increase in percentage of errors in the contralateral, left visual field (median: 23% with TMS vs 13% without TMS, P=0.0025). TMS applied earlier or later showed no effect. Furthermore, no significant increase in contra- or ipsilateral percentage of errors was found when the left parietal cortex was stimulated with the same timing. These data indicate that: (1) parietal influence on visuospatial attention is mainly controlled by the right lobe since the same stimulation over the left parietal cortex had no significant effect, and (2) there is a vulnerable time window to disturb this cortical process, since dTMS had a significant effect on the percentage of errors in the contralateral visual hemifield only when applied 270 ms after visual stimulus presentation.

Adult↗
PubMed2001

The triple stimulation technique to study central motor conduction to the lower limbs.

OBJECTIVE: To quantify the percentage of motor units of a foot muscle that can be activated by transcranial magnetic stimulation (TMS) in normal subjects and patients. METHODS: We adapted the recently described triple stimulation technique (TST) for recordings from abductor hallucis (AH). Conventional motor evoked potentials (MEPs) of this muscle are usually small and variable in shape, because of an important temporal desynchronization of the TMS induced spinal motor neuron discharges. The TST allows 'resynchronization' of these discharges and thereby a quantification of the proportion of motor units activated by TMS. The lower limb (LL-) TST was applied to 33 sides of 18 normal subjects and 51 sides of 46 patients with multiple sclerosis, amyotrophic lateral sclerosis, or spinal cord disorders. RESULTS: In healthy subjects, the LL-TST demonstrated that TMS achieves activation of virtually all motor neurons supplying the AH. In 33 of 51 patient sides, abnormal LL-TST responses suggested corticospinal conduction failures of various degrees. The LL-TST was 2.54 times more sensitive to detect central conduction failures than the conventional LL-MEPs. Combining the LL-TST with TST of the upper limbs further increased the sensitivity to detect a conduction failure by 1.50 times. CONCLUSION: The LL-TST markedly improves the examination of corticospinal pathways.

Adult↗
PubMed1996

Prevention of hyperacute rejection by removal of antibodies to HLA immediately before renal transplantation.

BACKGROUND: Many patients with circulating antibodies to human leucocyte antigens (anti-HLA) are highly sensitised against renal transplantation and are liable to immediate graft loss through hyperacute rejection. Our aim was to find out whether removal of anti-HLA immediately before renal transplantation prevented hyperacute graft rejection. METHODS: 13 highly sensitised patients underwent cadaveric renal transplants immediately after immunoadsorption (IA) treatment to remove anti-HLA. Before IA, 12 patients had a positive crossmatch against donor cells either by cytotoxic or flow-cytometric assay; results for one patient were equivocal. FINDINGS: Renal biopsy samples were obtained 20 min after removal of the vascular clamps in nine patients. There was no evidence of hyperacute rejection in six of the nine patients; the other three patients showed glomerular thrombosis but no other evidence of hyperacute rejection. Two of these three grafts were functioning at 31 months of follow-up. Six episodes of acute rejection occurred in five patients during the first month after transplantation and overall there were 13 rejection episodes in nine patients. At latest follow-up (median 26 months, range 9-42), 12 of 13 patients were alive and seven of 13 grafts were surviving with a median plasma creatinine concentration of 185 mumol/L (range 106-296) in the functioning grafts. No graft was lost as a result of classic hyperacute rejection. INTERPRETATION: Immediate pretransplant IA can prevent hyperacute rejection and provide an opportunity for successful transplantation in highly sensitised patients.

Adolescent↗
PubMed1995

Sequential potassium binding at the extracellular side of the Na,K-pump.

Ion binding at the extracellular face of the Na,K-ATPase is electrogenic and can be monitored by the styryl dye RH 421 in membrane fragments containing a high density of the Na,K-pumps. The fluorescent probe is noncovalently bound to the membrane and responds to changes of the local electric field generated by binding or release of cations inside the protein. Due to the fact that K+ binding from the extracellular side is an electrogenic reaction, it is possible to detect the amount of ions bound to the pump as function of the aqueous concentration. The results are in contradiction to a second order reaction, i.e., a simultaneous binding of two K+ ions. A mathematical model is presented to discuss the nature of the two step binding process. On the basis of this model the data allow a quantitative distinction between binding of the first and the second K+ ion. The temperature dependence of ion binding has been investigated. At low temperatures the apparent dissociation constants differ significantly. In the temperature range above 20 degrees C the resulting apparent dissociation constants for both K+ ions merge and have values between 0.2 and 0.3 mM, which is consistent with previous experiments. The activation energy for the half saturating concentration of K+ is 22 kJ/mol. Additional analysis of the titration curve of K+ binding to the state P-E2 by the Hill equation yields a Hill coefficient, nHill, of 1.33, which is in agreement with previously published data.

Animals↗
PubMed1995

Improved detection of proteolytically cleaved high molecular weight kininogen by immunoblotting using an antiserum against its reduced 47 kDa light chain.

Several ligand blotting or immunoblotting assays for the detection of single-chain and proteolytically cleaved two-chain high molecular weight kininogen (HK) in whole plasma have been described. Since they may suffer from poor sensitivity for the light chain species of cleaved HK on reduced blots, an antiserum against the reduced and alkylated 47 kDa light chain of HK was raised in rabbits allowing improved immunodetection of HK species on blots of reduced electropherograms. This immunoblotting method is highly specific and sensitive, permitting detection of 0.2 ng single-chain HK or the light chains of 2 ng proteolytically cleaved HK in whole plasma. Thus, this immunoblotting technique is at least 50-100 times more sensitive than ligand blotting with radiolabelled factor XI overlay. A similar cleavage pattern was observed following in vitro activation of normal human plasma by dextran sulphate and after plasma kallikrein-induced proteolysis of purified HK. However, bands of different molecular weights were generated after HK had been cleaved by purified leukocyte elastase. During acute attack in a patient with hereditary angioedema, high levels of in vivo cleaved HK were noticed, whereas concentration of cleaved HK in plasma samples and synovial fluids from patients suffering from various inflammatory conditions were not substantially higher than those in normal plasma. During in vitro cold activation of plasma samples of pregnant women concomitant HK cleavage and plasma kallikrein generation were observed.

Angioedema↗
PubMed1994

Inactivation of the Na, K-ATPase by radiation-induced free radicals. Evidence for a radical-chain mechanism.

Free radicals produced by water radiolysis were used to study the inactivation of the enzymatic activity of the Na,K-ATPase. A decrease of the activity to virtually zero with a mono-exponential dependence on the radiation dose was observed. The inactivation process is initiated by hydroxyl radicals. This was shown by the effect of appropriate radical scavengers such as t-butanol, formate and vitamin C. In all cases a significant increase in the characteristic D37 dose of inactivation was observed. Inactivation was found to show a so-called inverse dose-rate effect, i.e., the sensitivity of the enzyme to radical attack is increased if the dose rate is reduced. The data were found to agree with the relationship 1/D37 approximately 1/D1/2, which is known to be a strong indicator of a radical chain mechanism. This means that the inactivation, after initiation by single radicals, is amplified by a subsequent chain mechanism.

Cell Membrane↗
PubMed1993

Comparison of a phospholipid-based protein-free surfactant and a natural bovine surfactant (SURVANTA) during pressure and volume-controlled ventilation in an improved rabbit fetus model.

During pressure- or volume-controlled ventilation different surfactant preparations were compared in an improved rabbit fetus model. Based on a self-designed software program, this model enables on-line registration of lung mechanics and heart rate in up to ten fetuses. Using a commercially available bovine lung surfactant (SURVANTA) as standard, we compared animals treated with a protein-free surfactant preparation containing only phospholipids, PL (dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol++ +, DPPC:POPG 70:30) plus palmitic acid (PA) with an untreated ventilated control group. During pressure-controlled ventilation the insufflation pressure (IP) was decreased and increased stepwise with and without positive end-expiratory pressure (PEEP). SURVANTA was significantly more potent than PL plus PA and both differed significantly from the untreated controls. With additional PEEP the differences between SURVANTA and PL+PA disappeared but the differences to the controls were still present. We found that, with additional PEEP, active natural surfactants lead to ECG-irregularities, which indicates that PEEP influences pulmonary and cardiovascular function and compromises the benefits of surfactant therapy. Also during volume-controlled ventilation SURVANTA was superior to PL+PA and the untreated controls. In order to raise the level of activity of pure PL mixtures to that of natural bovine surfactants, we suggest that a surface active protein (probably SP-C) must be added to such mixtures.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Zonation of cytochrome P450 isozyme expression and induction in rat liver.

The regional expression of six different cytochrome P450 (CYP) forms in rat liver under constitutive and induced conditions was compared using immunological techniques. Immunostaining of consecutive thin sections from control liver revealed that the same hepatocytes, forming a 6-8 cells thick layer surrounding the terminal hepatic venules, were stained for CYP2B1/2, CYP2E1 and CYP3A1. Staining of CYP2A1 extended further into the midzonal region, whereas all cells of the acinus stained for CYPEtOH2. These results were supported by Western blot analysis of cell lysates from the periportal or perivenous region obtained by zone-restricted digitonin treatment during in situ perfusion. The data suggest three distinct patterns of constitutive P450 expression: perivenous-restricted (CYP2B1/2, CYP2E1 and CYP3A1); perivenous-dominated (CYP2A1) and panacinar (CYPEtOH2). Chronic exposure to ethanol caused induction of CYP2E1 in the same cells already being constitutively expressed, whereas CYPEtOH2 was more induced in the periportal area. The relative induction of CYP2B1/2, CYP3A1 and CYPEtOH2 after treatment with phenobarbital was stronger in periportal hepatocytes, resulting in levelling out of the initial perivenous dominance of CYP2B1/2 and CYP3A1, whereas CYPEtOH2 became periportal-dominated. Acetone induced CYP2E1, CYP2C11 and CYP3A1 selectively in the perivenous area. These studies indicate that a particular P450 isozyme is generally induced in the same cells where it is constitutively expressed, and that this regional selectivity is independent of the kind of inducer. The data suggest that, during maturation, the hepatocytes acquire various phenotypes in the periportal and perivenous region, to respond differently to endogenous and exogenous signals in the control of P450 expression.

Acetone↗
PubMed1992

Regional distribution of low-Km mitochondrial aldehyde dehydrogenase in the rat central nervous system.

To clarify the regional capacity of the brain to oxidize biogenic aldehydes and ethanol-derived acetaldehyde, a quantitative immunohistochemical study of the microregional and cellular expression of low Km mitochondrial aldehyde dehydrogenase (mALDH; EC 1.2.1.3) in the rat central nervous system was undertaken, using antiserum raised in rabbit against low-Km aldehyde dehydrogenase purified from rat liver mitochondria. mALDH-specific immunoreactivity (IR) was observed to various extent in the majority of structures in all brain and spinal cord areas. Staining was strong in the extranuclear cytoplasm of neuronal and glial cell bodies but less pronounced in their processes and terminals, the conducting tracts, white matter and neuropile and in blood vessels. Immunostaining density was 2 to 3 times higher in neuronal perikarya as compared with neuropile. mALDH-positive neurons were found in all brain regions, being strongest in the inferior olive and hippocampus stratum pyramidale and weakest in substantia nigra. The percentage of morphologically identifiable ALDH-positive neurons ranged from 40% in the arcuate hypothalamic nucleus to 88% in the cerebellar Purkinje cells. A comparison of the heterogeneous expression of mALDH in various rat CNS regions and cells, as observed in the present study, with the corresponding previously published distributions of the potential acetaldehyde-producing enzymes ADH and cytochrome P450 2E1 indicates major differences, which may help in understanding potential acetaldehyde-mediated CNS effects of ethanol. Knowledge of the regional distribution of high-affinity aldehyde dehydrogenase should also throw light on the neurophysiological role of local regulation of the metabolism of biogenic aldehydes in the brain.

Aldehyde Dehydrogenase↗
PubMed1991

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.

Antibodies, Monoclonal↗
PubMed1991

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements.

Membrane fragments containing a high density of Na,K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na,K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.

Adenosine Triphosphate↗
PubMed1991

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face.

In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na,K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na+ are electroneutral steps, and that release of Na+ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K+ indicate that binding and occlusion of K+ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na+ or K+ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel ("ion well"). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP+) or tetraphenylborate (TPB-), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP+ leads to a decrease of binding affinity for Na+ and K+, which is thought to result from the TPP(+)-induced change of electric field strength in the access channel.

Adenosine Triphosphate↗
PubMed1991

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.

Base Sequence↗
PubMed1991

The use of protease inhibitors in experimental allergic neuritis.

In experimental allergic neuritis (EAN) break-down of myelin is attributed to macrophages, which among other factors contain and secrete proteases. In vitro studies have shown that cathepsin D, an acidic aspartyl endopeptidase, and plasmin can degrade myelin proteins. In order to elucidate a potential therapeutic effect of protease inhibitors we treated Lewis rats, immunized with bovine peripheral nervous system myelin, with epsilon-amino-caproic acid (EACA) or pepstatin. EACA or pepstatin was administered twice daily by intraperitoneal injection beginning on day 6 postimmunization or from the onset of disease (on day 12) through day 24. Compared to saline-treated controls, animals treated with either of the inhibitors showed delayed development of clinical signs and electrophysiological abnormalities. Maximal severity and the further course of disease, however, were not different in control and treated groups. Immunohistological evaluation of sciatic nerve specimens on day 24 postimmunization showed equal numbers of cells positive for ED1 (macrophages) and cathepsin D in all animal groups. There was also no difference in the spontaneous proteolytic activity of the sciatic nerve homogenates at pH 2.8, 5.0, and 7.4. Incubation of the homogenates with pepstatin, however, significantly reduced proteolytic activity at pH 2.8 and 5.0, while EACA had no effect at any pH tested. These results imply that treatment to limit the infiltration of cathepsin D-positive cells or to reduce the induction or activity of cathepsin D may provide a therapeutic avenue for treating inflammatory demyelination of the peripheral nervous system.

Aminocaproic Acid↗