Interstitial Concentrations of Amino Acids in the Rat Striatum During Global Forebrain Ischemia and Potassium-Evoked Spreading Depression

The early detection and appropriate treatment of brain ischemia is of paramount importance. The interstitial concentrations of neurotransmitter amino acids are often used as an index of neuronal injury. However, monitoring of non-neurotransmitter amino acids may be equally important. We have studied the behavior of 10 amino acids during K⁺-induced spreading depression (application of 70 mM KCl during 40 min) and global forebrain ischemia (two-vessel occlusion with hypotension during 20 min). The concentrations of glutamate, aspartate, taurine, GABA, glycine, and alanine, measured in the rat striatum by microdialysis, increased during both ischemia and spreading depression, whereas glutamine concentrations decreased in both cases. Only ischemia, but not spreading depression, led to enhanced release of serine, threonine, and asparagine. We thus conclude that an elevation in the interstitial concentrations of non-neurotransmitter amino acids is specific to deep ischemic injury to nervous tissue. We propose the monitoring of serine, asparagine, and threonine, together with excitatory amino acids, as an index of the degree of ischemic brain injury.     

Comparison of HPLC method and commercial ELISA assay for asymmetric dimethylarginine (ADMA) determination in human serum

The performance of a new ELISA assay kit (DLD Diagnostika GmbH, Hamburg, Germany) for the determination of asymmetric dimethylarginine (ADMA) was evaluated against a reversed phase HPLC method. ADMA concentrations of 55 serum samples were measured with both methods. The intra-assay CV for ADMA-ELISA was 19% (n=10). Inter-assay CVs for ADMA-ELISA were 9% for kit control 1 (0.410+/-0.037 microM) and 14% for kit control 2 (1.174+/-0.165 microM). The intra- and inter-assay CVs for HPLC assay for ADMA were 2.5% (0.586+/-0.015 microM) and 4.2% (0.664+/-0.028 microM), respectively. There was no correlation between these two methods (R(2)=0.0972). The effect of storage conditions of the samples on ADMA concentrations was investigated by HPLC. ADMA concentration was stable after four freezing and thawing cycles. Overall, the HPLC method offered better sensitivity, selectivity and, very importantly, simultaneous determination of ADMA, SDMA, l-homoarginine and l-arginine.     

Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase

1. Download high-res image (133KB)
2. Download full-size image

     

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[³H]glutamate. The apparent binding constant K _d was 135 nM and 393 nM and the maximal binding capacity B_max 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC₅₀ in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [³H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

Modification of chloride flux across brain membranes by inhibitory amino acids in developing and adult mice

The influx of³⁶Cl^– was studied in membrane vesicles prepared from different brain regions from 3-day-old and adult mice. In both age groups the influx was enhanced about threefold by -aminobutyric acid (GABA), which effect was blocked by bicuculline and picrotoxin but not by baclofen, characteristic of a GABA_A receptor-mediated event. In samples from the adult brain stem the GABA stimulation was smaller than in samples from the other brain regions. Most of the compounds studied apparently act at the same receptor site with the following order of efficacy: muscimol > GABA > -alanine > hypotaurine > taurine. A number of anticonvulsant taurine derivatives were not effective and glycine only in the brain stem. The weak modulatory effects of taurine could be of significance in vivo since depolarizing stimuli release massive amounts of taurine in developing brain tissue.     

Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

Human parechovirus 1 (HPeV-1) (family Picornaviridae) is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to αVβ1, αVβ3 and αVβ6 integrin receptor(s), the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of αVβ1, αVβ3, αVβ6 and α5β1 in susceptible cell lines (A549, HeLa and SW480) to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express αVβ1 integrin but not αVβ3 or αVβ6 integrins. To further study the role of β1 integrin, we used a mouse cell line, GE11-KO, which is deficient in β1 expression, and its derivate GE11-β1 in which human integrin β1 subunit is overexpressed. HPeV-1 (Harris strain) and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-β1 supported the internalization process. An integrin β1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with β1 integrin on the cell surface, and HPeV-1 and β1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of αVβ1 integrin without visible receptor clustering.     

Iron–Manganese Concretions Sustaining Microbial Life in the Baltic Sea: The Structure of the Bacterial Community and Enrichments in Metal-Oxidizing Conditions

The abundant deposits of spherical iron-manganese concretions in the Gulf of Finland are colonized by bacteria in vast numbers. Communities on the surface and in the porous interior have formed two separate clusters, in accordance with their genetic differences. The overall bacterial community in the concretions was highly diverse, representing 12 phyla. Half of the bacteria were affiliated with the most common classes of Proteobacteria, while a third of the bacteria were unclassified. Cloned 16S rRNA-gene sequences of the concretion bacteria showed high scores for similarity to the sequences obtained from sea sediments, metal-rich environments, and ocean crust. The clone library of native concretions was not dominated by known Fe- and Mn-oxidizing species. Known Mn-oxidizing bacteria Sphingomonas, Pseudomonas, and Bacillus were enriched in experiments with Mn²⁺-containing liquid media, whereas Prosthecobacter (Verrucomicrobia) and Rheinheimera were enriched in semisolid media possibly better simulating the natural conditions in the concretions. In a corresponding experiment, the Fe²⁺-oxygen gradient favored the enrichment of Shewanella baltica and Thalassolituus oleivorans, which are known to reduce Fe and to degrade petroleum hydrocarbons, respectively. An individual spherical concretion forms a microcosm for a diverse microbial community having potential to oxidize Fe and Mn as shown in cultivation experiments. Therefore, bacteria may significantly affect the formation of the concretions in the Gulf of Finland.