Search PubMed⌕ Search

Biomedical subjects

R Kuhn

Publications and source records attributed to R Kuhn.

At least 127 records · Page 7Linked to original sources
PubMed1996

Distribution of metabotropic glutamate receptor 1a in the rat hypothalamus: an immunocytochemical study using monoclonal and polyclonal antibody.

L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is mediated via ionotropic and metabotropic glutamate receptors (mGluR). Eight mGluRs have already been cloned. In the present study the hypothalamic distribution of mGluR1a has been investigated by immunocytochemistry using monoclonal antibodies recently produced by some of the present authors (T. J. G., R. K., T. K.). The observations have been compared with findings obtained with a polyclonal antibody. A widespread and heterogeneous distribution of mGluR1a was found with the monoclonal antibodies. Intense immunolabelling of perikarya and dendrites occurred in several hypothalamic cell groups including the suprachiasmatic, anterior periventricular, anterior hypothalamic (posterior part), paraventricular, supraoptic, arcuate, tuberal magnocellular, dorsomedial and mammillary nuclei (particularly in the medial). It was only the ventromedial nucleus in which several perikarya were stained by the polyclonal antibody but appeared to be negative by the monoclonal antibodies. The findings fit extremely well with the data on the hypothalamic distribution of mGluR1 mRNA with the exception of the ventromedial nucleus. It remains to be elucidated whether alternatively spliced variants of mGluR1 (mGluR1b and 1c) are expressed in this nucleus. Further, they confirm the results of former immunohistochemical studies. In addition, they indicate that a significant part of the neuroendocrine region of the hypothalamus (including the paraventricular, supraoptic and arcuate nuclei) also contains mGluR1 suggesting that this receptor may play a role also in neuroendocrine regulation.

Animals↗
PubMed1995

The agonist selectivity of a class III metabotropic glutamate receptor, human mGluR4a, is determined by the N-terminal extracellular domain.

To test the hypothesis that the determinants for agonist selectivity of class III metabotropic glutamate receptors (mGluRs) are localized in the N-terminal extracellular domain, a chimaeric cDNA was constructed where 519 amino acids of the N-terminal extracellular domain of human mGluR1b were exchanged with the corresponding region of human mGluR4. The pharmacological profile of the chimaera, designated hmGlu(R4)1-519/1b, was analysed by recordings of intracellular calcium concentration ([Ca2+]i) in transiently transfected HEK 293 cells and compared with that of human mGluR1b and human mGluR4a stably expressed in Chinese hamster ovary cells. Application of 100 microM L-2-amino-4-phosphonobutyrate (L-AP4), a class III mGluR-specific agonist, induced a rise in [Ca2+]i in hmGlu(R4)1-519/1b but not in hmGluR1b expressing cells. In contrast, application of quisqualate (100 microM) induced a rise in [Ca2+]i at hmGluR1b but not at hmGlu(R4)1-519/1b. Dose-response analysis with L-AP4 and L-glutamate at hmGlu(R4)1-519/1b revealed a half-maximal effect (EC50) of 16.0 microM and 196 microM, respectively. The EC50 values for quisqualate, glutamate and (1S,3R)-ACPD at hmGluR1b were 10.25 microM, 225 microM and 3060 microM, respectively. The rank order of agonist potency of hmGlu(R4)1-519/1b corresponds to that of hmGluR4 (L-AP4 > L-glutamate > (1S,3R)-ACPD > quisqualate) but is different from that of hmGluR1b (quisqualate > glutamate >> (1S,3R)-ACPD).

Amino Acid Sequence↗
PubMed1995

Evaluation of an optically active crown ether for the chiral separation of di- and tripeptides.

The direct optical resolution of a number of di- and tripeptides was achieved by capillary zone electrophoresis using an enantioselective crown ether as buffer additive. The protonated primary amines form inclusion complexes with the crown ether. Chiral resolution is based on different stability constants of the diastereomeric complexes thereby changing the electrophoretic mobilities of the enantiomers. Enantioselectivity is strongly affected by the distance between the amine functionality and the chiral carbon atom. This effect was studied using di- and tripeptides especially synthesized for this purpose. In general, baseline resolution was obtained for those peptides with the amine group located as far as four bonds from the stereogenic center. Additionally, tripeptides possessing two chiral centers were separated to investigate the potential of the chiral selector for the analysis of complex analytes with related structures. Experimental factors such as crown ether concentration, buffer pH and temperature also show a strong influence on the resolution. These factors can be successfully employed for method optimization.

Amino Acid Sequence↗
PubMed1995

[Narrow-lumen mesocaval interposition shunt in liver cirrhosis and recurrent esophageal varices hemorrhage. Standard surgery of the future in failure of sclerotherapy?].

In a prospective study, conducted between 1.1.1987 and 31.12.1992, 72 consecutive patients (50 men, 22 women; mean age 59.8 [16-72] years) with cirrhosis of the liver and recurrent bleeding from oesophageal or fundal varices, were treated with a mesocaval interposition shunt, made of reinforced Goretex 10 or 12 mm in diameter. Further selection criteria were Child-Pugh class A (n = 39) or B (n = 33), liver volume (by ultrasound) between 1000 and 2500 ml, portal vein perfusion index of at least 10% and histological exclusion of activity or progression of the cirrhosis. Four patients (5.6%) died during the first 30 days after the intervention, and four had recurrent bleeding from the varices, three of them during the postoperative period, one during the second postoperative year. The cumulative shunt patency rate was 94.4% (up to 84 months). An acute encephalopathy occurred in six patients (8.3%), which was successfully treated with protein restriction and neomycin administration. Intra- and post-operative hemodynamic investigation confirmed maintenance of portal-vein perfusion in all patients, but there was a significant tendency towards reduction after 3 years in all patients. The overall survival rate after 7 years was 75%, with 85% for patients in Child-Pugh class A and 65% for those in class B. The results indicate that the described method can achieve satisfactory portal decompression with maintenance of portal-vein flow. It is especially suitable as a standard method if sclerotherapy has failed.

Adolescent↗
PubMed1995

Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 2.

A cDNA encoding the human metabotropic glutamate receptor type 2 (hmGluR2) was isolated from human brain cDNA libraries by cross-hybridization with rat mGluR2 probes. The deduced amino acid sequence of the human mGluR2 receptor consists of 872 residues and shows a sequence identity of 97% to the amino acid sequence of rat mGluR2. Northern blot analyses showed that hmGluR2 is widely expressed in different regions of the adult brain as well as in fetal human brain. Genomic Southern blotting localized the mGluR2 gene to human chromosome 3. Chinese hamster ovary (CHO) cells stably transfected with the cloned hmGluR2 cDNA exhibit agonist induced depression of forskolin-stimulated cAMP accumulation. A direct comparison of CHO cells stably expressing human and rat mGluR2 with five agonists revealed the same rank order of potency [(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine >> (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid = L-glutamate >> quisqualate = L-2-amino-4-phosphonobutyric acid] and similar EC50 values for both homologous receptors. (R,S)-alpha-methyl-4-carboxyphenylglycine, a reported antagonist at some mGluR subtypes, reduced the depression of forskolin-induced cAMP accumulation by (1S,3R)-ACPD in both human and rat mGluR2.

Amino Acid Sequence↗
PubMed1995

Profiling of trans-azetidine-2,4-dicarboxylic acid at the human metabotropic glutamate receptors mGlu1b, -2, -4a and -5a.

We have tested the two enantiomers of trans-azetidine-2,4-dicarboxylic acid, (2S,4S)-azetidine-2,4-dicarboxylic acid ((2S,4S)-ADA) and (2R,4R)-azetidine-2,4-dicarboxylic acid ((2R,4R)-ADA) for activity at the human metabotropic glutamate receptors mGlu1b, mGlu2, mGlu4a and mGlu5a expressed in mammalian cells. In Chinese hamster ovary (CHO) cells expressing human mGlu2 receptors, 500 microM (2S,4S)-ADA inhibited forskolin-stimulated cAMP accumulation by 33 +/- 3% while 100 microM (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid induced an inhibition by 66 +/- 5%. The (2R,4R)-ADA enantiomer was inactive at human mGlu2 receptors. In CHO cells expressing human mGlu4a receptors, 10 microM L-AP4 inhibited forskolin-stimulated cAMP levels by 37 +/- 4% whereas both ADA enantiomers of trans-azetidine-2,4-dicarboxylic acid (500 microM) had no such effect. In CHO cells expressing human mGlu1b receptors and L cells expressing human mGlu5a receptors, both enantiomers, applied at 500 microM or 1 mM, were ineffective in stimulating inositolmonophosphate accumulation and did not affect quisqualate-stimulated inositolmonophosphate accumulation. We conclude that (2S,4S)-azetidine-2,4-dicarboxylic acid is a weak human mGlu2 receptor agonist and that (2R,4R)-azetidine-2,4-dicarboxylic acid is inactive at human mGlu2 receptors. Trans-azetidine-2,4-dicarboxylic acid has no significant agonistic effect on human mGlu4a receptors and neither agonistic nor antagonistic effects on human mGlu1b and mGlu5a receptors.

Animals↗
PubMed1995

Mesocaval interposition shunt with small-diameter polytetrafluoroethylene grafts in sclerotherapy failure.

Fifty-seven patients with failed sclerotherapy received a mesocaval interposition shunt with an externally supported, ringed polytetrafluoroethylene prosthesis of either 10 or 12 mm diameter. Thirty-one patients had Child-Pugh grade A disease and 26 grade B; all had a liver volume of 1000-2500 ml. Follow-up ranged from 16 months to 6 years 3 months. Three patients (5 per cent) died in the postoperative period. There were two postoperative recurrences of variceal haemorrhage and one recurrent bleed in the second year after surgery. The cumulative shunt patency rate was 95 per cent and the incidence of encephalopathy 9 per cent; the latter was successfully managed by protein restriction and/or lactulose therapy. The actuarial survival rate for the whole group at 6 years was 78 per cent, for those with Child-Pugh grade A 88 per cent and for grade B 67 per cent. Small-lumen mesocaval interposition shunting achieves portal decompression, preserves hepatopetal flow, has a low incidence of shunt thrombosis, prevents recurrent variceal bleeding and is not associated with significant postoperative encephalopathy.

Anastomosis, Surgical↗
PubMed1995

Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 4.

A cDNA encoding the human metabotropic glutamate receptor type 4 (hmGluR4) was isolated from human brain cDNA libraries by cross-hybridization with rat mGluR4 probes. The deduced amino acid sequence of human mGluR4 consists of 912 residues and shows a sequence identity of 96% to the amino acid sequence of rat mGluR4. Northern blot analyses indicate that hmGluR4 is strongly expressed in the cerebellum of the adult human brain but also at low levels in hippocampus, hypothalamus and thalamus. Stimulation of hmGluR4 with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP), L-glutamate or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas quisqualate (0.5 mM) was ineffective. The rank order of agonist potencies is: L-AP4 > L-SOP > L-glutamate > (1S,3R)-ACPD >> quisqualate. (R,S)-alpha-methyl-4-carboxyphenylglycine (1 mM), a reported antagonist at some mGluR subtypes, did not reduce the depression of forskolin-induced cAMP accumulation by L-AP4.

Animals↗
PubMed1995

Activation of metabotropic glutamate receptors coupled to inositol phospholipid hydrolysis amplifies NMDA-induced neuronal degeneration in cultured cortical cells.

We have studied the influence of class I metabotropic glutamate receptors (mGluRs) on excitotoxic neuronal degeneration in cultured murine cortical neurons grown on a monolayer of astrocytes. These cultures expressed high levels of mGluR5 mRNA, which were comparable to those found in RNA extracts from cerebral cortex. Cortical neurons in mixed cultures were heavily stained with antibodies raised against mGluR5 and were also stained--albeit to a much lower extent--with mGluR1a but not with mGluR1b or c antibodies. Preferential agonists of class I mGluRs, such as quisqualate, 3,5-dihydroxyphenylglycine (DHPG), and trans-azetidine-2,4-dicarboxylic acid (t-ADA), as well as the mixed mGluR agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) all stimulated PPI hydrolysis in cultured cortical cells. The potency of N-methyl-D-aspartate (NMDA) in inducing neuronal degeneration was substantially enhanced when the drug was coincubated with quisqualate, DHPG or t-ADA during a 10-min pulse (paradigm of "fast" toxicity). None of the mGluR agonists influenced neuronal viability by itself. The amplification of NMDA toxicity by quisqualate or DHPG was attenuated by a series of protein kinase C (PKC) inhibitors, suggesting that class I mGluRs operate, at least in part, through activation of PKC. Quisqualate and, in particular, DHPG enhanced excitoxic neuronal degeneration even when applied after the toxic pulse with NMDA. This action is likely to occur early in the maturation of excitotoxic damage, because the functional activity of class I mGluRs was substantially reduced at 2 or 3 hr after the NMDA pulse. These results suggest that activation of class I mGluRs enhances NMDA-receptor mediated neuronal toxicity and encourage the search for selective antagonists for the experimental therapy of acute or chronic neurodegenerative diseases.

Amino Acid Sequence↗
PubMed1995

Pharmacological characterization of MCCG and MAP4 at the mGluR1b, mGluR2 and mGluR4a human metabotropic glutamate receptor subtypes.

The two reported metabotropic glutamate receptor (mGluR) antagonists, alpha-methyl-cyclopropyl glycine (MCCG) and alpha-methyl-aminophosphonobutyrate (MAP4) were tested on the mGluR1b, mGluR2 and mGluR4a subtypes of human mGluRs. Neither MCCG (500 microM) nor MAP4 (500 microM) antagonized the activation of mGluR1b by 10 microM quisqualate. MCCG was found to potently antagonize the action of 30 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] at mGluR2 (IC50 = 87.5 microM; apparent Kd = 25 microM) but did not block the action of 1 microM S-2-amino-4-phosphonobutyric acid at mGluR4a (IC50 >> 1 mM). MAP4 was found to be a weak antagonist or partial agonist at mGluR4a (IC50 > 500 microM) and, less potently, also antagonized the action of 30 microM (1S,3R)-ACPD) at mGluR2 (IC50 approximately 2 mM).

Amino Acids, Dicarboxylic↗
PubMed1994

Cellular and subcellular localization of the mGluR5a metabotropic glutamate receptor in rat spinal cord.

The cellular and subcellular distribution of the mGluR5a metabotropic glutamate receptor was studied in the spinal cord of the rat using an antibody raised against a mGluR5a-specific carboxy-terminal peptide. Strong mGluR5a-immunoreactivity (mGluR5a-ir) was found in the laminae I-II of the dorsal horn, which gradually decreased towards the deeper layers. At the electron microscopical level, mGluR5a-ir was present exclusively in neuronal somata and dendrites. Immunometal labelling revealed that mGluR5a-ir is concentrated at the periphery of postsynaptic densities of asymmetrical synapses or localized extrasynaptically at dendritic and somatic membranes. The mGluR5a-immunoreactive dendritic profiles were often targeted by synaptic boutons with the morphological characteristics of C-fibre terminals. These observations provide evidence for mGluR5a being involved in the nociceptive transmission at the dorsal horn.

Amino Acid Sequence↗
PubMed1994

Association of the CAMP phenomenon in Actinobacillus pleuropneumoniae with the RTX toxins ApxI, ApxII and ApxIII.

A non-hemolytic mutant of Actinobacillus pleuropneumoniae serotype 5 has a deletion spanning the entire apxI operon. Therefore it does not produce ApxI and is unable to secrete ApxII. This mutant also has lost the co-hemolytic CAMP effect which is characteristic of the species A. pleuropneumoniae. The CAMP effect is restored when the mutant is complemented in trans by the apxIBD genes cloned in a broad host range vector, thus permitting secretion of ApxII, or when the entire apxI operon is cloned in the mutant, thus restoring the original toxin phenotype ApxI+ ApxII+. When the toxins ApxI, ApxII or ApxIII individually are expressed and secreted from E. coli harboring recombinant plasmids containing the genes apxICA and apxIBD or apxIICA and apxIBD or apxIIICABD, respectively, the distinct CAMP phenomenon is produced by the recombinant strains. The CAMP phenomenon is strongest by the recombinant E. coli strain expressing the non-hemolytic ApxIII, somewhat less when ApxI is expressed, and weak when ApxII is expressed. In A. pleuropneumoniae the CAMP phenomenon is also strongest in those serotypes which express ApxIII. The CAMP phenomenon of A. pleuropneumoniae is assumed to be directly caused by any of the RTX-toxins ApxI, ApxII or ApxIII. A previously reported gene from A. pleuropneumoniae, named cfp or hlyX, which provides E. coli strains with a hemolytic character and a CAMP phenomenon, shows high similarity to the E. coli global regulation gene fnr, and which is able to complement a delta fnr mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus pleuropneumoniae↗
PubMed1994

Differential cellular localization of three splice variants of the mGluR1 metabotropic glutamate receptor in rat cerebellum.

Antibodies were raised against C-terminal peptides of the splice variants a, b and c of the rat mGluR1 metabotropic glutamate receptor. Affinity purified antibodies each specifically reacting with mGluR1a, mGluR1b and mGluR1c were used to study the cellular localization of these receptors in rat cerebellum. The mGluR1a antibody strongly labelled Purkinje cells at their cells bodies, portions of their dendritic trees and numerous small punctate elements reminiscent of dendritic spines. Also labelled were some stellate, basket, Golgi and Lugaro cells. Granule cells were devoid of staining. The mGluR1b antibody strongly labelled Purkinje cell bodies and their dendrites at levels which varied within the same lobule of the vermis or the hemispheres. No significant labelling was observed at stellate, basket, Golgi and granule cells, while occasionally a fraction of basket cells and cerebellar glomeruli was moderately immunoreactive. The mGluR1c antibody strongly labelled cell bodies and thick principal dendrites of Purkinje cells but not dendritic spines. Immunonegative Purkinje cells were intermingled with strongly labelled ones in lobules 4-10, while in lobules 1, 2 and 3, no stained Purkinje cells were detected. The mGluR1c antibody also labelled stellate, basket, some Golgi and some Lugaro cells as well as granule cells.

Animals↗
PubMed1994

Potential and limitations of an optically active crown ether for chiral separation in capillary zone electrophoresis.

Capillary zone electrophoresis using optically active 18-crown-6 tetracarboxylic acid (18C6H4) as chiral selector was studied for the enantiomeric separation of primary amines. From the separation of a variety of pharmaceutical drug substances, amino alcohols and amino acids, conclusions could be made concerning the influence of the chemical structure of the analytes on the separation. In addition, the effects of experimental parameters such as pH, proportion of organic modifier and buffer composition on the separation are discussed. A synergistic effect obtained by the joint application of 18C6H4 and a cyclodextrin was exploited to resolve analytes which were separated neither by the crown ether nor by the cyclodextrin.

Amines↗
PubMed1994

Use of capillary affinity electrophoresis for the determination of lectin-sugar interactions.

Capillary affinity electrophoresis was used to study the interactions of lectins from Tetragonolobus purpuras with L-(-)-fucose 1-phosphate. The electrophoretic mobilities of three lectin peaks were influenced by the complexation with the negatively charged fucose derivative. A model that allows the calculation of the complex formation constant from the change in mobility at different concentrations of fucose 1-phosphate has been derived. Calcium ions added to the buffer solution promoted the complex formation of the three lectins. This technique required only minute amounts of the lectins and enabled the simultaneous study of the complex formation of several proteins.

Calcium↗