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Biomedical subjects

A Hermann

Publications and source records attributed to A Hermann.

At least 19 recordsLinked to original sources
PubMed2002

Trifluoroacetylsulfenyl trifluoroacetate, CF(3)C(O)-S-O-C(O)CF(3), a novel compound with a symmetrically substituted S-O bond: synthesis, spectroscopic characterization, and quantum chemical calculations.

The new compound trifluoroacetylsulfenyl trifluoroacetate, CF(3)C(O)SOC(O)CF(3), which possesses two identical carbonyl substituents attached to the S-O bond, has been synthesized. The IR and UV spectra of the gas phase as well as the (13)C NMR spectrum of the solution in CDCl(3) were recorded and assigned. Quantum chemical calculations were performed with the ab initio methods HF and MP2 and the density functional approach B3LYP. The 6-31G basis set was chosen in all calculations. The molecule possesses a skew structure, and according to all computational methods, the syn-syn structure (C=O bonds of both C(O)CF(3) groups synperiplanar to S-O bond) represents the most stable conformer. In agreement with the quantum chemical calculations, the presence of small amounts (< or =5%) of a second conformer (anti-syn) cannot be excluded on the basis of the IR spectrum. The calculated values for the torsional angle around the S-O bond (delta(C-S-O-C)) of the syn-syn form are smaller than 80 degrees (72-78 degrees). Comparison with theoretical results for the corresponding disulfide CF(3)C(O)SSC(O)CF(3) and peroxide CF(3)C(O)OOC(O)CF(3) indicates that the structural properties of sulfenyl compounds are more similar to those of disulfides than to those of peroxides.

Journal Article↗
PubMed2002

[Tolerance of high dosage carbamazepine monotherapy in treatment of epilepsy].

We examined whether high dose carbamazepine (CBZ) monotherapy (CBZ serum levels > 9 mg/l) is well tolerated. Forty-one patients with localisation-related epilepsy were enrolled. They received high dose monotherapy either from the time of recruitment (30/41), or their CBZ treatment was pushed into the high dose range (11/41) during the observation period (mean 145 days). CBZ and CBZ metabolite serum levels, full blood counts, liver function tests, electrolytes were measured in addition to clinical assessments. The mean daily CBZ dose was 1515 mg (median 1500, range 600-3600 mg). Mean CBZ serum level was 11.1 mg/l (median 11.1, range 5.3-16.4 mg/l). Eleven of 41 patients (26.8%) reported side effects (mean serum level 11.4, median 11.6, range 8.7-13.7 mg/l), but side effects were intolerable in only five patients so that the CBZ dose had to be reduced (mean CBZ serum level 11.4, median 11.6, range 8.7-13.7 mg/l). The total daily CBZ dose but not CBZ or CBZ metabolite levels were correlated with side effects. No treatment changes had to be undertaken because of abnormal laboratory parameters. High dose CBZ anticonvulsant monotherapy is well tolerated.

Adolescent↗
PubMed2001

Structures and conformations of trifluoromethyl fluoroformate and perfluorodimethyl carbonate.

The conformational properties and geometric structures of trifluoromethyl fluoroformate, CF(3)OC(O)F (1), and perfluorodimethyl carbonate, (CF(3)O)(2)CO (2), have been studied by matrix IR spectroscopy, gas electron diffraction (GED), and quantum chemical calculations (MP2 and B3LYP with 6-311G basis sets). In both compounds the synperiplanar orientation of the O-CF(3) groups relative to the C=O double bond is preferred. If heated Ar/1 and Ar/2 mixtures are deposited as a matrix at 14 K, new bands appear in the matrix IR spectra which are assigned to the anti form of 1 and to the syn/anti form of 2. At room temperature the contribution of the anti rotamer of 1 is 4% (DeltaH degrees = H degrees (anti) - H degrees (syn) = 1.97(5) kcal/mol), and the contribution of the syn/anti conformer of 2 is estimated to be less than 1%. These high-energy conformers are not observed in the GED experiment. The quantum chemical calculations reproduce the structural and conformational properties of both compounds satisfactorily.

Journal Article↗
PubMed2001

Biphasic response to human galanin of extracellular acidification in human Bowes melanoma cells.

The metabolic response of galanin GAL1 receptor subtype, endogenously expressed in human Bowes melanoma (HBM) cells, was investigated. Cytosensor microphysiometry was used to determine the extracellular acidification rate. A biphasic response, consisting of a rapid increase in the extracellular acidification rate followed by a decrease below the basal level, was observed after perfusion with human galanin. The magnitude and the rate of onset of both phases were dependent on the galanin concentration. The increase in the extracellular acidification rate (maximum of 25% of basal level; -log(EC(50))=7.23+/-0.14) was transient, whereas the following decrease (maximum of 40% of basal level; -log(EC(50))=7.77+/-0.23) was sustained. The EC(50) values for the increase and decrease were in a similar range. After consecutive galanin administration, the magnitude of the response was the same as for the unexposed cells, indicating the absence of galanin receptor desensitization or internalization in HBM cells. Responses were blocked by pretreatment with pertussis toxin and phorbol-12-myristate-13-acetate (PMA), indicating a G-protein/protein kinase C signalling pathway. Our microphysiometry results show a biphasic response of the extracellular acidification rate mediated by the galanin receptor expressed in HBM cells which has not been described previously for any other endogenously expressed neuropeptide receptor.

1-Methyl-3-isobutylxanthine↗
PubMed2001

The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA.

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.

5-Methylcytosine↗
PubMed2001

Bis(trifluoroacetyl) peroxide, CF(3)C(O)OOC(O)CF(3).

Pure, highly explosive CF(3)C(O)OOC(O)CF(3) is prepared for the first time by low-temperature reaction between CF(3)C(O)Cl and Na(2)O(2). At room temperature CF(3)C(O)OOC(O)CF(3) is stable for days in the liquid or gaseous state. The melting point is -37.5 degrees C, and the boiling point is extrapolated to 44 degrees C from the vapor pressure curve log p = -1875/T + 8.92 (p/mbar, T/K). Above room temperature the first-order unimolecular decay into C(2)F(6) + CO(2) occurs with an activation energy of 129 kJ mol(-1). CF(3)C(O)OOC(O)CF(3) is a clean source for CF(3) radicals as demonstrated by matrix-isolation experiments. The pure compound is characterized by NMR, vibrational, and UV spectroscopy. The geometric structure is determined by gas electron diffraction and quantum chemical calculations (HF, B3PW91, B3LYP, and MP2 with 6-31G basis sets). The molecule possesses syn-syn conformation (both C=O bonds synperiplanar to the O-O bond) with O-O = 1.426(10) A and dihedral angle phi(C-O-O-C) = 86.5(32) degrees. The density functional calculations reproduce the experimental structure very well.

Journal Article↗
PubMed2001

Gas-phase structures of acetyl peroxynitrate and trifluoroacetyl peroxynitrate.

The molecular structures and conformational properties of acetyl peroxynitrate (PAN, CH3C(O)OONO2) and trifluoroacetyl peroxynitrate (FPAN, CF3C(O)OONO2) were investigated in the gas phase by electron diffraction (GED), microwave spectroscopy (MW), and quantum chemical methods (HF/3-21G, HF/6-31G*, MP2/6-31G*, B3PW91/6-31G*, and B3PW91/6-311+G*). All experimental and theoretical methods show the syn conformer (C=O bond of acetyl group syn to O-O bond) to be strongly predominant relative to the anti conformer. The O-NO2 bonds are extremely long, 1.492(7) A in PAN and 1.526(10) A in FPAN, which correlates with their low bond energy and the easy formation of CX3C(O)OO* and *NO2 radicals in the atmosphere. The O-O bonds (1.418(12) A in PAN and 1.408(8) A in FPAN) are shorter than that in hydrogen peroxide (1.464 A). In both compounds the C-O-O-N dihedral angle is close to 85 degrees.

Journal Article↗
PubMed2001

Identification of multiple gephyrin variants in different organs of the adult rat.

The neurotransmitter receptor anchoring protein gephyrin is encoded by a highly mosaic gene whose primary transcript is subject to extensive alternative splicing. Gephyrin mRNAs are widely expressed in various mammalian tissues, and gephyrin has been implicated in neuron-specific and general metabolic functions. Using a novel affinity isolation procedure, we report the identification of different gephyrin variants in various organs of the adult rat. In particular, polypeptides of 52, 56, 60, and 91 kDa were detected in addition to the previously characterized 93-kDa protein. Our results suggest tissue-specific functional differences between gephyrin variants.

Animals↗
PubMed2001

Nitric oxide augments voltage-activated calcium currents of crustacea (Idotea baltica) skeletal muscle.

Invertebrate skeletal muscle contraction is regulated by calcium influx through voltage-dependent calcium channels in the sarcolemmal membrane. In present study we investigated the effects of nitric oxide (NO) donors on calcium currents of single skeletal muscle fibres from the marine isopod, Idotea baltica, using two-electrode voltage clamp recording techniques. The NO donors, S-nitrosocysteine, S-nitroso-N-acetyl-penicillamine or hydroxylamine reversibly increased calcium inward currents in a time dependent manner. The increase of the current was prevented by methylene blue. Our experiments suggest that NO increases calcium inward currents. NO, by acting on calcium ion channels in the sarcolemmal membrane, therefore, may directly be involved in the modulation of muscle contraction.

Action Potentials↗
PubMed2001

Nitric oxide activates voltage-dependent potassium currents of crustacean skeletal muscle.

Nitric oxide (NO), a radical gas, acts as a multifunctional intra- and intercellular messenger. In the present study we investigated the effects of NO on muscle membrane potassium currents of isolated single muscle fibers from the marine isopods, Idotea baltica, using two-electrode voltage clamp recording techniques. Voltage-activated potassium currents consist of an outward current with fast activation and inactivation kinetics and a delayed, persistent outward current. Both currents were blocked by extracellular 4-aminopyridine and tetraethylammonium; the currents were not blocked by charybdotoxin or apamin. Application of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine increased both the early and the delayed outward current in a dose- and time-dependent manner. PTIO, a NO scavenger, suppressed the effect of SNAP. N-Acetyl-dl-penicillamine, a related control compound which does not liberate NO, had no significant effect on outward currents. Methylene blue, a guanylyl cyclase inhibitor, prevented the increase of the outward current while 8-bromo-cGMP increased the current. Our experiments show that potassium currents of Idotea muscle are increased by NO donors. They suggest that NO by stimulating cGMP production mediates the effects on membrane currents involved in regulation of invertebrate muscle excitability.

Animals↗
PubMed2001

Effects of metabotropic glutamate receptor agonists and antagonists on D-aspartate release from mouse cerebral cortical and striatal slices.

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[3H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[3H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)alpha-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S,1'S,2'R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2001

Nitric oxide up-regulates ferritin mRNA level in snail neurons.

We cloned and sequenced the ferric ion-binding protein, ferritin, from the nervous system of the pulmonate snail, Helix pomatia. Helix H-ferritin cDNA contains a 519-bp open reading frame (ORF) and predicts an iron-responsive element (IRE) at the 5'-untranslated region (5'-UTR) of the ferritin mRNA. The deduced amino acid sequence revealed 86% similarity with Lymnaea stagnalis ferritin and about 70% similarity with vertebrate H-ferritin. While secreted ferritin isoforms contain a signalling sequence at their N-terminal end, Helix ferritin does not contain this sorting signal indicating that it is restricted to the cytoplasm. The amino acid ligands at positions Glu25, Tyr30, Glu59, Glu60, His63, Glu105 and Gln139 indicate an active ferroxidase site in Helix ferritin. In situ hybridization visualized ferritin mRNA in neuronal cell bodies but not in the neuropil. In contrast, ferritin-immunoreactive protein was localized in cell bodies and neurites. We further demonstrate that the NO donors S-nitroso-N-acetylpenicillamine (SNAP), or hydroxylamine (HA), increase the intracellular ferritin mRNA level by about 55%. In conclusion, our findings show that Helix neurons express an intracellular H-ferritin isoform and suggest that iron and NO metabolism are coupled.

Amino Acid Sequence↗
PubMed2001

Platelet CD40 ligand (CD40L)--subcellular localization, regulation of expression, and inhibition by clopidogrel.

This study compares the subcellular localization and the regulation of expression of the platelet activation markers CD62P and CD63 with CD40 ligand (CD40L) on the surface of washed human platelets. CD40L was expressed upon stimulation with a wide range of platelet activators. However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L expression was low. Upon stimulation with thrombin receptor-activating peptide (TRAP-6), all activation markers were expressed. In contrast, upon stimulation with low concentrations of collagen (1-3 microg/ml), CD40L, but not the granule proteins (CD62P, CD63), were expressed. Using immunofluorescence microscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic localization of CD40L was verified by Western blotting of subcellular platelet fractions. The staining of CD40L was different from that of filamentous actin and only little association of CD40L with platelet cytoskeleton was found. Surface expression of CD40L was dependent on internal Ca2+ stores and protein kinase C, while the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 microM)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level of CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in the promegakaryocytic cell line MEG-01.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2001

Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin.

The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.

Amino Acid Motifs↗
PubMed2000

Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons.

In gastropods, the interneuronal messenger, nitric oxide (NO), modulates spike frequency and synaptic transmission. We have characterized the effect of NO on ion currents underlying neuronal excitability, using current-clamp and two-electrode voltage-clamp techniques. Identified neurons of the pulmonate snail, Helix pomatia, respond to the NO donor sodium nitroprusside (SNP) by increasing the firing frequency and decreasing the latency. Voltage-clamp experiments revealed that SNP or S-nitro-N-acetylpenicillamine (SNAP) depressed the macroscopic outward current, while the control compound N-acetylpenicillamine (NAP) had no effect. Current voltage curves generated from voltage steps to different membrane potentials ranging from -40 to +180 mV showed an N-shaped outward current. Superfusion of ganglia with Ca(2+) free Helix solution abolished the N-shape, indicating the contribution of a Ca(2+) activated K(+) current (I(K,Ca)). Exposure of neurons to SNP or SNAP diminished the N-shape, indicating that NO affects I(K,Ca). The depressing effect of SNP on the outward current was slow and reached steady state in about 5 min. In conclusion, our findings indicate that NO enhances excitability in Helix nervous system by decreasing I(K,Ca).

Action Potentials↗
PubMed2000

Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins.

Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5' coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3-7 and 16-29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.

Alternative Splicing↗