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Biomedical subjects

W Fischer

Publications and source records attributed to W Fischer.

At least 127 records · Page 7Linked to original sources
PubMed1993

Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.

Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.

Bacteriorhodopsins↗
PubMed1993

Brain-derived neurotrophic factor enhances function rather than survival of intrastriatal dopamine cell-rich grafts.

Brain-derived neurotrophic factor (BDNF) has been shown to promote the survival of dopaminergic neurons from the substantia nigra in cell culture. In order to assess whether a similar survival-promoting effect is present also in vivo, we grafted fetal nigral tissue to the dopamine-depleted striatum of 6-hydroxydopamine-lesioned rats receiving two-week intraventricular infusions or daily intrastriatal injections of BDNF, NGF, or vehicle. When infused chronically at a high dose (12 micrograms/day) into the lateral ventricle, BDNF caused a behavioral syndrome of reduced food and water intake, body weight loss, and locomotor hyperactivity in comparison to NGF- and vehicle-infused graft recipients. NGF-infused graft recipients displayed a transient weight loss during the first week of infusion. At 15 days, amphetamine-induced turning was significantly attenuated to 3% of pregraft values in BDNF-infused recipients, whereas functional graft effects were not present in NGF- or vehicle-infused animals. Survival of tyrosine hydroxylase-immunoreactive graft cells, however, was similar in all treatment groups. Notably, NGF- and BDNF-infusions led to a significant size increase of cholinergic host neurons in the medial septal nucleus and the vertical limb of the diagonal band ipsilateral to the infusion, whereas there was no cholinergic neuron hypertrophy in vehicle-infused animals. Daily intrastriatal injections of BDNF (2 micrograms) produced no weight loss or locomotor hyperactivity, but also enhanced functional graft effects in BDNF-injected, as compared to vehicle-injected animals. Survival rates of grafted tyrosine hydroxylase-immunoreactive cells were, however, similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphetamine↗
PubMed1993

Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine.

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.

Alkaline Phosphatase↗
PubMed1993

Teichoic acid and lipoteichoic acid of Streptococcus pneumoniae possess identical chain structures. A reinvestigation of teichoid acid (C polysaccharide).

Teichoic acid (C polysaccharide) was extracted and purified from Streptococcus pneumoniae R6 with standard procedures except that lipoteichoic acid was extracted first. The dephosphorylated repeating unit was isolated after hydrolysis with 48% (by mass) HF, the bis(phosphocholine)-containing repeating unit was isolated by alkali hydrolysis, anion-exchange chromatography and phosphomonoester cleavage. On the basis of compositional analysis, fast-atom-bombardment mass spectrometry and NMR spectroscopy the following structure is proposed: [formula: see text] where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. The repeating units are linked to each other by phosphodiester bonds between O5 of the ribitol and O6 of the glucopyranosyl residue of adjacent units. This chain structure is identical with that previously established for pneumococcal lipoteichoic acid [Behr, T., Fischer, W., Peter-Katalinić, J. & Egge, H. (1992) Eur. J. Biochem. 207, 1063-1075]. This represents a unique situation because in other Gram-positive bacteria teichoic and lipoteichoic acids are structurally unrelated.

Carbohydrate Sequence↗
PubMed1993

Protection by phosphodiesterase inhibitors against endotoxin-induced liver injury in galactosamine-sensitized mice.

Phosphodiesterase inhibitors were used as a tool to manipulate cellular nucleotide levels in vitro and in vivo. The lipopolysaccharide (LPS)-induced release of tumor necrosis factor alpha (TNF-alpha) from mouse peritoneal macrophages was inhibited by prostaglandin E2 with an IC50 of 0.05 microM and by dibutyryl-cAMP with an IC50 of 180 microM. In the presence of the phosphodiesterase inhibitors zardaverine or rolipram the intracellular cAMP concentration of LPS-stimulated macrophages was significantly increased. In these cells, LPS-inducible TNF release was inhibited by zardaverine (IC50 = 1.5 microM) or by rolipram (IC50 = 0.35 microM). In a model of septic shock, i.e. LPS challenge of galactosamine-sensitized mice, a dose-dependent protection against liver injury was observed following oral application of rolipram (ED50 = 0.55 mg/kg) or of zardaverine (ED50 approximately 30 mg/kg). The adenylate cyclase activator forskolin was also protective. Rolipram also protected against TNF-induced liver injury in mice while zardaverine failed to do so. It is concluded that the intracellular cAMP level of macrophages is a critical determinant of LPS-inducible TNF release and therefore modulates the susceptibility to septic shock.

Animals↗
PubMed1993

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Basic structure.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic-interaction chromatography. The preparations from both strains have an identical structure: a 1,3-linked poly(glycerophosphate) chain phosphodiester-linked to Glc-(alpha 1-2)Glc(alpha 1-3)acyl2Gro as the lipid anchor. The chain is substituted with D-alanine ester and glycosyl residues which comprise mono-, di-, tri- and tetra-alpha-D-glucopyranosyl residues with (1-6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H-NMR and 13C-NMR spectroscopy. The alpha-isomalto-oligosaccharides add a novel motif to lipoteichoic-acid chain substituents. 1H-NMR and 13C-NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3-glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor. Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).

Carbohydrate Sequence↗
PubMed1993

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Microheterogeneity and distribution of chain substituents.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 contains a poly(glycerophosphate) chain, with 49% of the glycerophosphate residues being substituted with D-alanine ester, 35% with alpha-D-glucopyranosyl and alpha-isomalto-oligosaccharide residues. Analysis of molecular species by affinity chromatography on concanavalin A showed all chains to be substituted and alanine ester and glycosyl residues to be present on the same rather than on separate chains. Molecular species varied in the length of the poly(glycerophosphate) chain, the extent of glycosylation, and had a constant alanine-ester content. An alkali-hydrolysis procedure revealed a distribution pattern between random and regular for the glycosyl substituents and suggested a similar distribution for the alanyl residues which occupy the free positions between the glycosyl substituents.

Chromatography, Affinity↗
PubMed1993

[Comparative study of the pharmacokinetics of amitriptyline oxide and trimipramine after single administration in healthy male probands and patients with renal failure].

The pharmacokinetics of the antidepressants amitriptyline oxide and trimipramine and their major metabolites amitriptyline, nor-triptyline and desmethyltrimipramine, were studied in twelve healthy male subjects (aged from 22 to 62 years) and twelve patients (aged from 25 to 73 years) with severe renal impairment (glomerular filtration rate < 10 ml/min). Oral single doses of 60 mg amitriptyline oxide and 50 mg trimipramine, separated by a washout period, were administered to all study participants. Blood and urine samples were collected up to 120 hours after administration. For trimipramine and desmethyltrimipramine, a new HPLC method was developed. The "Fischer Somatic and Undesired Effects Check List" was used for the assessment of adverse events. The mean plasma half-life and AUC of amitriptyline oxide and its metabolites were significantly higher in patients than in healthy adults. For trimipramine the AUC was significantly higher in patients. The plasma half-life of trimipramine was longer in patients, but statistically not significant. The maximum plasma concentrations for both drugs and metabolites were at an average distinctly higher in patients. Clearance rate of amitroptylinoxide and trimipramine also differed between the two groups. Correlating with these results a high incidence and a longer persistence (in most cases > 12 hours) and more pronounced adverse effects were noted in the patient group, whereas in volunteers adverse events were only observed up to approximately eight hours.

Adult↗
PubMed1993

Molecular analysis of lipid macroamphiphiles by hydrophobic interaction chromatography, exemplified with lipoteichoic acids.

Analyzed were (I) the oligoglucosyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Enterococcus hirae containing Glc(alpha 1-2)Glc(alpha 1-3)acyl2-Gro and a phosphatidyl derivative thereof as lipid anchor, (II) the poly(digalactosyl, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing the same glycolipid and an acyl derivative of it, and (III) the N-acetylglucosaminyl-, alanyl-substituted poly(glycerophosphate)lipoteichoic acid of Staphylococcus aureus with Glc(beta 1-6)Glc(beta 1-3)acyl2Gro as the sole lipid moiety. Hydrophobic interaction chromatography on octyl-Sepharose separated lipoteichoic acids I and II into two peaks according to the number of fatty acids. Within each peak further fractionation occurred in the order of decreasing length of the hydrophilic chain. A similar fractionation was observed within the single peak of lipoteichoic acid III. With lipoteichoic acid I and III the extent of glycosylation decreased with decreasing length of the hydrophilic chain whereas the content of alanine ester remained either constant or increased. Variations in the oligosaccharide pattern of lipoteichoic acid I and in the fatty acid composition of all lipoteichoic acids could also be observed. Collectively, the data provide for the first time a detailed picture of the complex polydispersity of lipoteichoic acids comprising the number of fatty acids, the length of the hydrophilic chain, the kind and extent of chain substitution, and the fatty acid composition. The procedure will also be applicable to the molecular analysis of lipopolysaccharides and bacterial lipoglycans. Moreover, the results are of physicochemical interest because they demonstrate for lipid macroamphiphiles an inverse relationship between hydrophobicity and the size of the hydrophilic headgroup.

Animals↗
PubMed1993

Molecular analysis of the lipoglycans of Mycobacterium tuberculosis.

Lipoglycans were extracted from disrupted cells of Mycobacterium tuberculosis with hot phenol-water. Hydrophobic interaction chromatography on octyl-Sepharose of the crude extract separated nucleic acids and lipoglycans. The latter were retained on the column and fractionated on elution with a propanol gradient into six peaks primarily according to decreasing size of the hydrophilic head groups which dropped from 70 to 5 or 4 monosaccharide residues per molecule. The number of fatty acids per molecule rose from 2.1 to 3.4 over the elution profile, suggesting species containing two, three, and four fatty acids. Peaks I and II and peaks III and IV represented two pairs of lipoarabinomannan and lipomannan, those of peak III and IV containing as a whole smaller hydrophilic head groups and more fatty acids. Peak V and VI are reminiscent of the oligomannosyl derivatives of phosphatidylinositol discovered earlier in mycobacteria. The lipid anchor of all molecular species was shown to be phosphatidylinositol with the extra acyl groups not yet localized. This and an accompanying report complement each other in demonstrating the potential of hydrophobic interaction chromatography for molecular analysis of lipid macroamphiphiles. Lipoteichoic acids, due to continuous small variations in the size of their hydrophilic head groups, separate primarily according to the number of fatty acids, whereas mycobacterial lipoarabinomannans, lipomannans, and phosphatidylinositol mannosides differ as entities so greatly in the size of the hydrophilic headgroup that this quality becomes predominant for separation.

Chromatography, Agarose↗
PubMed1993

Phosphorylation-contraction coupling in smooth muscle: role of caldesmon.

In intact smooth muscle strips from chicken gizzard, carbachol elicited brief, phasic contractions which were associated with a very rapid, transient phosphorylation of the 20 kDa myosin light chains. Phosphorylation was not significantly different from basal levels after 30 s while force still amounted to 50% of the peak value. The rate of tension decline could be increased by addition of atropine, even at apparently basal phosphorylation levels suggesting a phosphorylation independent regulation. The force, at a given level of phosphorylation, could also be modulated by addition of the actin binding, putative regulatory protein, caldesmon. Caldesmon, inhibits phosphorylation dependent force in skinned fiber bundles of chicken gizzard without affecting myosin light chain phosphorylation. This suggests that caldesmon might modulate contraction in smooth muscle. Moreover our results suggest that caldesmon does not function to maintain passive tension.

Animals↗
PubMed1993

[Continuity and discontinuity of psychopathology: a study of patients examined as children and as adults. II. Childhood antecedents of drug dependent adults].

The demographic stability of the Swiss Canton of Geneva provided us with the necessary conditions for a follow-up study of the entire childhood population who, having consulted the Child Psychiatry Services between 1963 and 1967, later consulted the adult public psychiatric services (720 cases). We looked for specific clusters of clinical signs which would enable us to differentiate, statistically speaking, the groups of children according to their adult diagnosis. We present here the results for the group of children who were diagnosed at adulthood as drug addicts (DSM III R). We found a specific cluster significantly differentiating these children from the other populations. It includes: a personality disorder (undifferentiated behaviour, and solitary and aggressive behaviour), environmental factors (monoparental, rejecting family) and a lack of mental deficiency.

Adolescent↗
PubMed1993

Anticonvulsant and related effects of U-54494A in various seizure tests.

The anticonvulsant activity of (+-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benz am ide monohydrochloride (U-54494A), a benzamide derivative chemically related to kappa opioid receptor agonists, was investigated in three selected seizure models of experimental epilepsy. In the maximal electroshock seizure test in mice, U-54494A (ED50 28 mg/kg i.p.) was effective, with a potency somewhat less than phenobarbital. In combination with clinically used antiepileptics, especially phenobarbital and carbamazepine, the anticonvulsant activity of the latter was significantly increased. More detailed studies with phenobarbital showed additive anticonvulsant effects. The anticonvulsant activity of U-54494A was partially antagonized by naloxone. On the other hand, this compound did not elevate the pentylenetetrazol seizure threshold (at high doses a tendency of proconvulsant action was seen). Furthermore, in unrestrained rats with chronically implanted electrodes, U-54494A (> or = 10 mg/kg) significantly reduced the duration of electrically evoked hippocampal afterdischarges. However, the focal stimulation threshold was not markedly increased. With respect to the possible mode of action, whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes showed that U-54494A depressed the fast sodium inward current in a concentration- and frequency-dependent manner. In summary, our results agree with earlier reports that demonstrated marked anticonvulsant effects of U-54494A in grand mal-analogous seizure tests. Moreover, in combination with some standard antiepileptics, additive effects can be found. It is suggested that, in addition to kappa opioid and excitatory amino acid receptor related effects, modulations of Na+ membrane currents may contribute to the mechanisms of action.

Animals↗
PubMed1993

[Effectiveness and tolerance of amitriptyline oxide in chronic tension headache--a multicenter double-blind study versus amitriptyline versus placebo].

Tricyclic antidepressants, especially amitriptyline, are the medication of first choice in the treatment of chronic tension headache. Few previous studies meet modern standards of study design and statistical analysis. Tolerability and efficacy of 60-90 mg amitriptyline oxide (AO) as a single dose in the evening were compared with 50-75 mg amitriptyline (AM) and placebo (PL) in a double-blind, parallel-group trial consisting of a 4-week baseline phase and 12 weeks of treatment. The 3-armed study was conducted in 7 centers. The inclusion criterion was tension-type headache on at least 15 days monthly with a duration of at least 6 months. Exclusion criteria were a migraine history, previous participation in another clinical trial within the last 3 months, drug abuse, medication with other antidepressants or tranquilizers, current use of other acknowledged prophylactic headache medication, lack of compliance, major psychiatric disorder according to DSM-III and medical contra-indications against tricyclic antidepressants. The primary study endpoint was a reduction at least 50% of the product of headache duration and frequency and a reduction at least 50% in headache intensity. Statistics used were Fisher's Exact Test and an analysis of variance. A total of 211 patients were included in this trial. One hundred ninety-seven cases, 87 males and 110 females, with a mean age of 38 +/- 13 (18-68) years, could be analysed completely (66 AO, 67 AM, 64 PL). With regard to the strictly defined primary study endpoint, no significant difference emerged between AO, AM and PL: treatment responders were 30.3% with AO, 22.4% with AM and 21.9% with PL (PAO-PL = 0.3210, PAM-PL = 1.000, PAO-AM = 0.3299 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

[Screening for risk factors in agricultural schools of 5 Austrian federal counties ].

While screening activities have already been performed in a wide range in adults in Austria, extensive investigations in children and juveniles have so far only been done in a very limited number. In 1094 out of 1732 juveniles attending agricultural schools all lipid parameters were fully documented and thus could be evaluated. On an average, the total cholesterol (169 mg/dl) was too high; the worst findings for lipid parameters cholesterol, HDL-cholesterol, triglycerides, apolipoprotein-A1 were found in pupils of Burgenland. The cholesterol/HDL-cholesterol ratio in Burgenland was significantly (p < 0.01) higher than in Lower Austria. Female participants had a higher total cholesterol, HDL-cholesterol, and apolipoprotein-A1, but lower triglycerides and also lower blood pressure. Juveniles with a positive family history showed lower total cholesterol, but higher triglycerides as well as HDL-cholesterol. In heavy smokers, but also in occasional smokers, a lower total cholesterol, but decreased HDL-cholesterol and apolipoprotein-A1 and increased triglycerides were found. These findings underline the high prevalence of hypercholesterolemia in juveniles justifying screening activities and possibly therapeutic intervention at this young age.

Adolescent↗
PubMed1992

The structure of pneumococcal lipoteichoic acid. Improved preparation, chemical and mass spectrometric studies.

Pneumococcal lipoteichoic acid was extracted and purified by a novel, quick and effective procedure. Structural analysis included methylation, periodate oxidation, Smith degradation, oxidation with CrO3, and fast-atom-bombardment mass spectrometry. Hydrolysis with 48% (by mass) HF and subsequent phase partition yielded the lipid anchor (I), the dephosphorylated repeating unit of the chain (II) and a cleavage product of the latter (III). The proposed structures are: (I) Glc(beta 1----3)AATGal(beta 1----3)Glc(alpha 1----3)acyl2Gro, (II) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc(beta 1----1)ribitol and (III) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose, and all sugars are in the pyranose form and belong to the D-series. Alkaline phosphodiester cleavage of lipoteichoic acid, followed by treatment with phosphomonoesterase, resulted in the formation of II and IV, with IV as the prevailing species: [sequence: see text] The linkage between the repeating units was established as phosphodiester bond between ribitol 5-phosphate and position 6 of the glucosyl residue of adjacent units. The chain was shown to be linked to the lipid anchor by a phosphodiester between its ribitol 5-phosphate terminus and position 6 of the non-reducing glucosyl terminus of I. The lipoteichoic acid is polydisperse: the chain length may vary between 2 and 8 repeating units and variations were also observed for the fatty acid composition of the diacylglycerol moiety. Preliminary results suggest that repeating units II and IV are enriched in separate molecular species. All species were associated with Forssman antigenicity, albeit to a various extent when related to the non-phosphocholine phosphorus. Owing to its unique structure, the described macroamphiphile may be classified as atypical lipoteichoic acid.

Carbohydrate Sequence↗
PubMed1992

[The medical record at the center of medical informatics].

Several areas in medical institutions, particularly the technical and administrative ones have been governed by computers for a long time. The proper medical work field (diagnosis, treatment, follow-up, correspondence and billing) however has hitherto mostly been ignored by data processing. We describe an electronic medical record introduced 4 years ago. All aspects of medical practise and all the specialists involved in patient care are integrated in the electronic document. In the center is a medical base of knowledge adaptable to all specialties as well as an expert system for direct control of the data entered by all concerned persons.

Ambulatory Surgical Procedures↗