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Biomedical subjects

B Schmid

Publications and source records attributed to B Schmid.

At least 55 records · Page 3Linked to original sources
PubMed1997

Quantitative determination of pentaerythrityl tetranitrate and its metabolites in human plasma by gas chromatography/mass spectrometry.

Up to now, there has been no data available on the pharmacokinetics of pentaerythrityl tetranitrate (PETN, CAS 78-11-5) and its metabolites, pentaerythrityl-trinitrate (PE-tri-N), pentaerythrityl-dinitrate (PE-di-N), pentaerythrityl-mononitrate (PE-mono-N) in human plasma. Therefore, in order to determine PETN and its metabolites in plasma sensitive and highly selective GC/MS methods had to be developed and validated. PETN and its metabolite PE-tri-N were validated in the concentration range 50 pg/ml to 10 ng/ml. Isosorbide dinitrate (ISDN) was used as the internal standard and the analytes were extracted with dichloromethane from the plasma. The mass spectrometric tests were carried out using chemical ionization in the negative mode (NlCl) with the application of ammonia as a reagent gas. The nitrate ion m/z 62 was determined in the analytes and internal standard. The accuracy of the mean of the quality control samples during the three days (between days) was between 100 and 110% (PETN), as well as 90 and 106% (PE-tri-N). After an oral application of 100 mg PETN in a pilot study, unchanged PETN and PE-tri-N was measured in plasma. Both metabolites PE-di-N and PE-mono-N were validated at the concentration range of 0.25 ng/ml to 25 ng/ml plasma. After extraction, these analytes were derivatized with BSTFA (N,O-bis[trimethylsilyl]trifluoro-acetamide). The applied internal standard was isosorbide-5-mononitrate (IS-5-MN). The mass spectrometric tests were carried out in the same manner as for PETN and PE-tri-N with chemical ionization in the NlCl mode. The detected masses were m/z 324 for PE-di-N, m/z 351 for PE-mono-N and m/z 217 for IS-5-MN. The accuracy of the mean of the quality control samples during 5 days were between 104 and 107% (PE-di-N) and 102 and 106% (PE-mono-N). The maximum concentration of these analytes in the subject samples were on the average all over 5 ng/ml plasma after the oral administration of 100 mg PETN.

Calibration↗
PubMed1996

Experimental verification of the secondary structures of guide RNA-pre-mRNA chimaeric molecules in Trypanosoma brucei.

RNA editing in kinetoplastid organisms is an RNA-processing reaction that adds and deletes U nucleotides at specific sites in mitochondrial pre-mRNAs. The edited sequence is specified by guide RNAs and the processing presumably occurs within a high-molecular-mass ribonucleoprotein complex containing several enzymatic activities. Although the mechanism is not currently known, potential intermediates or by-products of the editing process are chimaeric RNAs where guide (g) RNAs are covalently attached, via their non-encoded U-tail, to their cognate pre-mRNAs. We determined the secondary structures of three different ATPase 6 chimaeras of Trypanosoma brucei using a set of structure-sensitive chemical and enzymatic probes. The experiments revealed a bipartite domain structure consisting of a gRNA/pre-mRNA interaction hairpin and an independently folding mRNA stem/loop in all three RNAs. The connecting U-tail was a determinant for the length of the interaction stems with the oligo(U) nucleotides base pairing to internal gRNA sequences. The probed structures have calculated delta G27o values of -92 kJ/ mol to -134 kJ/mol, somewhat less stable than the predicted minimal free energy structures and support previously proposed models for the interaction between gRNAs and pre-mRNAs. Optical melting studies indicated additional, higher order structural features for all three molecules with four defined melting transition between 10 degrees C and 90 degrees C. A comparison of CD spectra in the absence and presence of mitochondrial protein extracts demonstrated no gross structural changes of the RNA structures induced by the association with polypeptides.

Adenosine Triphosphatases↗
PubMed1996

Expression of porin from Rhodopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure.

The homotrimeric membrane channel porin from Rhodopseudomonas blastica was expressed without signal sequence in Escherichia coli. The protein assembled in inclusion bodies in the cytosol, from which it could be recovered using urea and detergents. After purification by anion-exchange chromatography, the protein crystallized under wild-type conditions. The X-ray structure was determined at 2.2 angstroms resolution, and a comparison with the known wild-type structure showed that the recombinant porin is identical at the atomic level. The method yields porin and designed mutants thereof in 100 mg amounts, allowing for detailed functional and mechanistic studies.

Cloning, Molecular↗
PubMed1996

Protein phosphotyrosine phosphatases in Ascaris suum muscle.

Two forms of protein tyrosine phosphatases were partially purified from the musculo-cutaneous layer of Ascaris suum. A 50-55-kDa soluble form of the phosphatase cross-reacted with antisera raised against human PTP-1B and TC-PTP. Like the enzyme of human origin the phosphatase from Ascaris exhibited a preference for anionic substrates (tyrosine-phosphorylated carboxymethylated and maleylated lysozyme) and was inhibited by micromolar concentrations of vanadate, molybdate, Zn2+, heparin, and poly(Glu4Tyr). As revealed by immuno-cytochemistry, the phosphatase was mainly localized and appeared equally distributed in the cytoplasm, apart from the myofibrils, possibly in loose association with cytoskeletal elements. A second tyrosine phosphatase of 180 kDa molecular mass was mainly found in detergent extracts from a microsomal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and dephosphorylated a basic substrate (Tyr-phosphorylated myelin basic protein). It was resistant to common inhibitors of mammalian tyrosine phosphatases except Zn2+ and thiol reagents.

Animals↗
PubMed1996

A gradient of cytoplasmic Cactus degradation establishes the nuclear localization gradient of the dorsal morphogen in Drosophila.

Dorsoventral axis formation in the Drosophila embryo is established by a signal transduction pathway that comprises the products of at least 12 maternal genes. Two of these genes, dorsal and cactus, show homology to the mammalian transcription factor NF-kappa B and its inhibitor I kappa B, respectively. As in the case for I kappa B and NF-kappa B, Cactus inhibits Dorsal by retaining it in the cytoplasm. In response to the signal produced and transmitted by the products of the other genes, Dorsal translocates to the nucleus preferentially on the ventral side of the embryo. Here, we show that Cactus forms a cytoplasmic concentration gradient inversely correlated to the nuclear translocation gradient of Dorsal. Deletions of the N-terminus and C-terminus of Cactus reveal that two modes of degradation control cactus activity: signal-induced degradation and signal-independent degradation, respectively. Genetic evidence indicates that degradation of Cactus is required, but not sufficient to translocates Dorsal completely into the nucleus.

Animals↗
PubMed1995

Effects of angiotensin II and angiotensin-converting enzyme inhibitors on human myocardium.

Human myocardial angiotensin II receptors and the angiotensin AT1 and AT2 receptor subtypes were characterised using the partial angiotensin II receptor agonist [125I][Sar1,IIe8]angiotensin II and the selective antagonists losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'((1H-tetrazol-5-yl)biphen yl-4-yl)- methyl]imidazole) and PD 123177 (1-[(4-amino-3-methylphenyl)methyl]-5-(diphenyl-acetyl)- 4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxylic acid). The density of angiotensin II receptors was higher in atrial than in ventricular myocardium. Angiotensin AT2 receptors were predominant in atria and ventricles (80-85% of total angiotensin II receptors). Only in isolated, electrically driven atrial trabeculae but not in ventricular preparations, angiotensin II did produce a concentration-dependent positive inotropic effect, which was antagonized exclusively by the angiotensin AT1 receptor antagonist losartan and which amounted to about 20% of the positive inotropic effect of milrinone and isoprenaline. The application of the angiotensin-converting enzyme inhibitors captopril, enalaprilat and ramiprilat had no inotropic effect in either tissue. It is concluded that angiotensin AT1 receptors exclusively mediate direct positive inotropic effects in atrial myocardium. Since angiotensin-converting enzyme inhibitors do not produce any inotropic effect, tonic regulation of basal force of contraction by angiotensin II does not occur.

Adolescent↗
PubMed1995

The secondary structure of guide RNA molecules from Trypanosoma brucei.

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.

Animals↗
PubMed1995

The safety assessment of over-the-counter (OTC) products.

In recent years many compounds have become available as Over-The-Counter (OTC) medicine, for self-medication, which were originally marketed as prescription medicines only. This has been coupled with changes in the consumers' behaviour to self treatment and the governments commitment to restrict rising health costs. The safety and safe use of OTC products are important considerations for their wide distribution which is paralleled by diminishing medical supervision. OTC products were generally registered when toxicological requirements differed from the stringent guidelines of today, and their safety assessment thus often involves ongoing evaluation as new data become available. For this, human data must also be integrated for these assessments. However, in some areas, such as mutagenicity and carcinogenicity testing, human data are hardly obtainable and testing in animals or cells, is ultimately the only reliable source for extrapolation to man. Routine toxicological methods have mainly been developed for the assessment of new pharmaceutical entities and often provide 'yes/no' answers only. Data of such tests are, therefore, not directly applicable to the safety assessment of OTC products. Additional information on the kinetics in animal and man are indispensable for such extrapolation. On the other hand, modifications of original test protocols for the assessment of new formulations are indispensable. It was the aim of this workshop to discuss general approaches of safety assessments within the important new area of OTC products, as well as to present specific cases of recently arisen safety issues.

Animals↗
PubMed1995

Bone cement implantation syndrome. A prospective randomised trial for use of antihistamine blockade.

Bone cement implantation syndrome (BCIS) is characterised by hypotension, hypoxaemia, cardiac arrhythmias, cardiac arrest or any combination of these, leading to death in 0.6-1% of patients. One of the mechanisms suggested to explain these complications is diffuse microembolisation of the lungs as a consequence of extrusion of the bone marrow content by the pressurised bone cement. By reducing intramedullary pressure and changing the operative technique, BCIS can be diminished, but deaths still occur. An anaphylactoid mechanism as a major factor in BCIS is receiving renewed attention since increased plasma histamine levels were recently demonstrated after the implantation of bone cement and a prosthesis. Therefore, we conducted a prospective, randomised study to demonstrate the potential benefit of histamine-receptor-blocking agents in patients undergoing cemented hip arthroplasty. Thirty patients were divided into two groups: group 1, the control group, received no histamine-receptor-blocking agents; group 2, the antihistamine group, received H1 and H2-receptor-blocking agents in standard dosages preoperatively. Both groups were comparable concerning age, sex and physical status (ASA criteria). There was no hospital mortality in either group. Thirteen patients of group 1 demonstrated a sudden fall by more than 10% of their blood pressure, level of PaO2 or both. Fourteen patients of group 2 showed similar changes. The mean decrease of blood pressure in group 1 was 14.6 mmHg (SD 36.8) and in group 2 20.5 mmHg (SD 33.43). The difference is not significant (P = 0.65). The mean decrease of PaO2 in group 1 was 30.5 mmHg (SD 30.5) and in group 2 33.4 mmHg (SD 34.1). The difference is not significant (P = 0.81). Overall, we found even a slight disadvantage for patients receiving antihistamine drugs (statistically not significant). Therefore, histamine-receptor-blocking agents do not have a prophylactic potential in BCIS.

Bone Cements↗
PubMed1995

Alterations of cardiac alpha- and beta-adrenoceptors and inotropic responsiveness in hypertensive transgenic rats harbouring the mouse renin gene (TGR(mREN2)27).

In the present study, we investigated the alpha- and beta-adrenoceptor-mediated effects on myocardial force of contraction (using phenylephrine in the presence of propranolol and isoprenaline) and receptor densities (binding studies using [3H]-prazosin and [125I]-iodocyanopindolol) in hypertensive transgenic rats (TGR(mREN2)27) and age-matched Sprague-Dawley rats (SP) as controls. In TGR(mREN2)27 the positive inotropic effects of isoprenaline and phenylephrine were reduced, while the effect of Ca2+ was unchanged. The EC50-values did not differ in both groups. A down-regulation of the beta-adrenoceptors was observed in the hypertrophied left ventricles of transgenic rats, which is postulated to be involved in the reduced beta-adrenoceptor-mediated positive inotropic effect. The alpha-adrenoceptor density was increased, which could represent a compensatory mechanism for the impaired effectiveness of the beta-adrenergic pathway. However, since the effect of alpha-adrenoceptor agonist is not enhanced but even reduced, an uncoupling of alpha-adrenoceptors from post receptor events could play a role in the observed effects.

Animals↗
PubMed1994

Ylt1, a highly repetitive retrotransposon in the genome of the dimorphic fungus Yarrowia lipolytica.

A highly repetitive composite element, Ylt1, was detected in the genome of the dimorphic fungus Yarrowia lipolytica. Ylt1 resembles retrotransposons found in other eukaryotes. It is about 9.4 kb long and can transpose in the genome. The Ylt1 element is bounded by a long terminal repeat (LTR), the zeta element. Several copies of zeta were isolated and sequenced. The sequence of this element is well conserved. It is 714 bp long and is bounded by nucleotides 5'-TG...CA-3', which are part of a short inverted repeat, a feature conserved in the LTRs of retroviruses and retrotransposons. Sequence analysis revealed motifs commonly found in LTR elements, like signals for the start and termination of transcription. The zeta element exists as part of retrotransposon Ylt1, as well as a solo element in the genome. Ylt1 and solo zeta elements are flanked by a 4-bp directly repeated genomic sequence. The copy numbers of Ylt1 and solo zeta are dependent on the strain examined, but at least 35 copies of the composite Ylt1 element and more than 30 copies of the solo zeta element per haploid genome have been observed.

Base Sequence↗
PubMed1994

Beta-adrenergic neuroeffector mechanisms in cardiac hypertrophy of renin transgenic rats.

We studied neuroeffector defects in hypertrophied myocardium of hypertensive transgenic rats harboring the mouse Ren-2d gene. In transgenic rats, epinephrine and neuropeptide Y concentrations were reduced. A heterologous desensitization of adenylyl cyclase was observed, which was accompanied by a downregulation of beta 1-adrenergic receptors, an increase of inhibitory G protein alpha-subunits, and a mildly depressed catalyst activity of adenylyl cyclase, whereas the bioactivity of stimulatory G protein alpha-subunits and beta 2-adrenergic receptors was unchanged. Desensitization of adenylyl cyclase was accompanied by a reduced positive inotropic response to isoproterenol, whereas the effect of Ca2+ was unchanged. We conclude that sympathetic neuroeffector defects occur in transgenic rats similar to those observed in human failing myocardium. These alterations occur in the stage of hypertrophy and could contribute to contractile dysfunction in later stages.

Adenylyl Cyclases↗
PubMed1993

[The Gailingen Scaffold Test--a stress trial for occupations with a risk of falling].

The Jugendwerk Gailingen's rehabilitees with severe craniocerebral trauma placed in open employment include some 10% who work in construction, electric and metal-working industry occupations involving exposure to fall hazards, as a rule having returned to their previous occupations or following vocational training. During their rehabilitation, specific considerations have to be taken into account due to this hazard, both relative to work characterization and to rehabilitee remaining abilities and vocational assessment. Initially, the risk of falling must be identified, with subsequent estimation whether continuation of the hazard-generating activity will presumably be possible and/or justifiable. In the affirmative, this is followed by vocational assessment, which, beyond work tolerance and capacities, also deals with fall safety. The necessary strength, mobility, coordination, attention, concentration, and flexibility are assessed using the so-called Gailingen scaffold test. Although this test simulates but a fraction of possible work situations above ground and does look relatively harmless compared to real worklife, more than 100 rehabilitees have achieved successful, lasting integration in a variety of occupations which partly involve a rather high exposure to fall hazards, without any accidents having been reported at follow-up.

Accidental Falls↗
PubMed1993

Cardiac inotropic as well as coronary and pulmonary artery actions of epinine in human isolated tissues.

The present study was aimed to characterize the effects of epinine, the metabolite of the p.o. active dopamine derivate ibopamine in human cardiovascular tissues such as myocardium, coronary artery and pulmonary artery. Isometric force of contraction was studied in electrically driven papillary muscle strips from nonfailing (brain death), moderately failing (New York Heart Association class II-III, mitral valve replacement) and terminally failing human myocardium (New York Heart Association class IV, heart transplants) as well as in auricular trabeculae (aortocoronary bypass operation). Epinine increased force development in a concentration-dependent manner. In comparison to isoprenaline, epinine had a significantly lower potency but a similar efficacy to enhance force of contraction. Depending on the degree of myocardial failure, the effectiveness of epinine was reduced, whereas the potency was similar. Only in nonfailing myocardium, epinine increased force of contraction as effectively as Ca++. Prestimulation with forskolin or milrinone enhanced the potency of the epinine-mediated inotropic effect. In contrast, the beta-1-selective antagonist CGP 207.12A [2-hydroxy-5-(2-(hydroxy-3-(4-((1-methyl-4-trifluoromethyl)-1-H-imidazol -2-yl)-phenoxy)-propyl)-aminoethoxyl)-benzamide] and the beta-2-selective antagonist ICI 118.551 [erythro-(+-)-1-(7-methylindan-4-yloxy)-3- isopropylaminobutan-2-ol-hydrochloride] shifted the concentration-response curve of epinine significantly to the right, indicating action at both beta-2 and beta-1 adrenoceptors. Epinine exerted higher affinity at beta-2 compared to beta-1 adrenoceptors in radioligand binding experiments ([125I]iodocyanopinodolol). In human coronary artery rings and pulmonary artery rings epinine alone as well as epinine in the presence of propranolol initiated a concentration-dependent increase in tension development in precontracted (prostaglandin F2 alpha, 0.3 mumol/l) as well as in non-precontracted rings. These results suggest that epinine exerts no direct vasodilatory activity in human coronary and pulmonary arteries at concentrations which are capable to produce positive inotropic activity. The supposed beneficial effects of ibopamine in the treatment of heart failure may not be due to positive inotropic actions as the concentrations producing positive inotropy are much higher than the clinically observed plasma concentrations.

Adenylyl Cyclases↗
PubMed1992

Additional biochemical findings in a patient and fetal sibling with a genetic defect in the sphingolipid activator protein (SAP) precursor, prosaposin. Evidence for a deficiency in SAP-1 and for a normal lysosomal neuraminidase.

It has been shown that sphingolipid activator proteins (SAPs) 1 and 2 are encoded on the same gene along with two other putative activator proteins [Fürst, Machleidt & Sandhoff (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 and O'Brien, Kretz, Dewji, Wenger, Esch & Fluharty (1988) Science 241, 1098-1101]. We have undertaken further biochemical investigations on a patient and fetal sibling, who were previously shown to have a unique sphingolipid storage disorder associated with an SAP-2 deficiency [Harzer, Paton, Poulos, Kustermann-Kuhn, Roggendorf, Grisar & Popp (1989) Eur. J. Pediatr. 149, 31-39]. The severity of their disorder suggested that other products of the SAP precursor or prosaposin gene may also be deficient. The turnover of cerebroside sulphate and globotriaosylceramide were investigated and were both impaired in fibroblasts from the patient and fetus. However, the activities of cerebroside sulphate sulphatase and globotriaosylceramide alpha-galactosidase in vitro were normal in cells from the fetus and patient respectively. In addition, there was an increase in cerebroside sulphate concentration in the kidney of the affected fetus. These results indicate that, in addition to the SAP-2 deficiency, there was a defect in SAP-1 function in this disorder. Additional increases in the concentration of monohexosyl- and dihexosyl-ceramide in the fetal kidney probably reflect the deficiency of SAP-2 in the case of monohexosylceramides, and the combined activator deficiency in the case of dihexosylceramides. Lactosylceramide-loading studies confirmed that there was a defect in the turnover of this lipid in fibroblasts from the affected patient and fetus but not from a patient with an isolated SAP-1 deficiency, or from patients with Krabbe disease, GM1 gangliosidosis or galactosialidosis. It has been suggested [Potier, Lamontagne, Michaud & Tranchemontagne (1990) Biochem. Biophys. Res. Commun. 173, 449-456] that the prosaposin gene also codes for lysosomal neuroaminidase. However, we found normal neuraminidase activity in fibroblasts from our patient, using assay conditions which are diagnostic for sialidosis patients. The role of prosaposin gene products in sphingolipid metabolism is discussed in view of our biochemical findings in this genetic disorder.

Cells, Cultured↗
PubMed1992

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.

Animals↗