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E Molnár

Publications and source records attributed to E Molnár.

At least 37 records · Page 2Linked to original sources
PubMed1996

Isoforms of endoplasmic reticulum Ca(2+)-ATPase are differentially expressed in normal and diabetic islets of Langerhans.

Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic beta-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco-(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F beta-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in beta-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in the beta-cell may contribute to the decreased insulin secretion seen in non-insulin dependent diabetes mellitus (NIDDM). We therefore investigated SERCA 2 and SERCA 3 mRNA expression by quantitative RT-PCR in islets prepared from Goto-Kakizaki (GK) rats, a non-obese spontaneous model of NIDDM. We found a significant reduction (about 68%) in SERCA 3 isoform expression. Since SERCA 2 expression was not significantly reduced, these genes are independently regulated and probably play distinct roles in islets of Langerhans. The marked decrease of SERCA 3 expression may constitute a defect in Ca2+ signalling in GK rat islets which could be a component of NIDDM.

Animals↗
PubMed1996

Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

Animals↗
PubMed1996

A unique combination of plasma membrane Ca2+-ATPase isoforms is expressed in islets of Langerhans and pancreatic beta-cell lines.

Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic beta-cells. The existence of steep Ca2+ gradients within the beta-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic alpha- and beta-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since beta-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain alpha, beta, delta and polypeptide-secreting cells, PMCA 4a and 4b were simultaneously present. No evidence was obtained for the expression of PMCA 3. Characterization of the beta-cell Ca2+-pump protein showed that it shared several similarities with the erythrocyte PMCA. It is a P-type ATPase; its phosphorylated intermediate was stabilized by La3+; it reacted with a PMCA-specific antibody; and it was not N-glycosylate. However, the beta-cell PMCA had a higher molecular mass than that of the erythrocyte; this difference could be explained by either predominant translation of the PMCA2 form, which has a molecular mass 3-8 kDa higher than the erythrocyte PMCA 1 and 4 proteins, or by a possible sequence insertion. Thus a unique combination of functionally distinct PMCA isoforms (1b, 2b, 4b) participates in Ca2+ homoeostasis in the beta-cell.

Alternative Splicing↗
PubMed1996

Therapy with pulsed electromagnetic fields in aseptic loosening of total hip protheses: a prospective study.

Aseptic loosening is the most common problem of hip arthroplasties, limiting its long term success. We report a study of pulsed electromagnetic field (PEMF) treatment in 24 patients with this complication. At the end of treatment, six months and one year later, pain and hip movements improved significantly with the exception of flexion and extension. There was significant improvement in both isotope scans and ultrasonography, but not in plain X-ray. The decreased pain and improved function suggest that PEMF is effective in improving symptoms of patients with loose hip replacement. No improvement, however, can be expected in patients with severe pain due to gross loosening.

Adult↗
PubMed1996

Cell surface expression of the human N-methyl-D-aspartate receptor subunit 1a requires the co-expression of the NR2A subunit in transfected cells.

Cell surface expression of the NR1a subunit has been examined in mouse L cell lines permanently transfected with the complementary DNA for human NR1a or with the complementary DNAs for NR1a and NR2A. The expression of the subunits was under the control of the murine mammary tumour virus promoter and following induction of expression by dexamethazone both cell lines expressed high levels of the NR1a subunit as determined by immunofluorescence using permeabilized cells and immunoblotting of cell membranes with subunit specific antibodies. However, cell surface expression of the NR1a subunit was found only in the cells expressing both the NR1a and NR2A subunits. This was confirmed by cell surface biotinylation of the two cell lines and affinity isolation of the receptor subunits. To determine if this result was solely due to the use of a particular cell line and or the choice of expression vector, Cos-7 cells were transiently transfected with either NR1a or NR1a plus NR2A. Here too cell surface expression was only found following co-transfection of both subunits.

Animals↗
PubMed1996

[Advanced-stage ankylosing spondylitis in a subject in the 8th century].

Paleopathological study of an adult male skeleton, coming from a Hungarian archeological site from the 8th century, revealed lesions of the spine (ankylosing vertebral syndesmophytosis, costo-vertebral ankylosis, discal calcifications), of the sacro-iliacs (bilateral ankylosis) and several extraspinal changes (abnormalities of symphysis pubis, enthesopathies). Radiological and CT investigations confirmed the diagnosis of advanced stage ankylosing spondylitis.

Adult↗
PubMed1996

Toxicity of the herbicides Flubalex, Fusilade S and Maloran 50 WP to chicken embryos after administration as single compounds or in combination.

The teratogenic effects of three herbicides (Flubalex, Fusilade S and Maloran 50 WP) were studied in chicken embryos. Each of the three test substances was administered on days 0 and 12 of incubation. Treatment was followed by evaluation on day 19. The compounds were injected into the air-chamber of eggs at three different concentrations. The medium concentration corresponded to that usually applied in chemical plant protection. In order to determine the combined toxicity of the three herbicides, the medium concentration of Maloran 50 WP and three different concentrations of Flubalex of Fusilade S each were administered simultaneously at a final volume of 0.1 ml per egg, at similar times. Evaluation was done on day 19. In tests of individual toxicity, after injection on day 0 of incubation Maloran 50 WP and Flubalex caused a significant reduction in body mass, while Maloran 50 WP and Fusilade S resulted in marked embryonic mortality. After injection on day 12, the medium and the highest concentration of Flubalex and the highest concentration of Fusilade S caused a marked increase in embryonic mortality. The developmental anomalies were of sporadic nature: their incidence increased only after Flubalex treatment, irrespective of the time of administration. The combined administration of Maloran 50 WP and Flubalex on day 0 resulted in a significant or marked body mass reduction in all groups. Embryonic mortality increased substantially after treatment with the highest dose of Flubalex, while all three concentrations of the other two herbicides led to similar results. When treatment was performed on day 12, the two highest concentrations of Flubalex and the highest concentration of Fusilade S caused expressed embryonic mortality. The developmental anomalies did not show a dose-dependent effect in any of the test series.

Animals↗
PubMed1995

Identification of functional ionotropic glutamate receptor proteins in pancreatic beta-cells and in islets of Langerhans.

The presence of ionotropic glutamate receptor proteins in islets of Langerhans and pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) was investigated. For this purpose immunoblot analysis of beta-cell membranes was performed with subunit-specific antibodies. We identified NMDAR1 subunits of the NMDA and KA-2 subunits of the kainate receptors, but did not detect GluR1 subunits of the AMPA receptor. The receptor subunits present were shown to be glycosylated. beta-cell membranes contained specific binding sites for glutamate receptor ligands, and NMDA increased insulin secretion. These results demonstrate that ionotropic glutamate receptor proteins, similar to those in the central nervous system, are expressed in rat pancreatic beta-cells.

Amino Acid Sequence↗
PubMed1995

Characterisation of endoplasmic reticulum and plasma membrane Ca(2+)-ATPases in pancreatic beta-cells and in islets of Langerhans.

We have investigated the plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca(2+)-ATPases involved in active transport of Ca2+ in pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) and in islets of Langerhans. Under selective membrane phosphorylation conditions (at low ATP concentration, in the presence of Ca2+ and La3+ and in the absence of Mg2+ at 4 degrees C) the only labelled proteins are the phosphoenzyme intermediates of the Ca(2+)-ATPases. Under these conditions, beta-cell membranes incorporated 32P from [gamma-32P]ATP into two proteins with molecular mass on acidic SDS-polyacrylamide gels of around 115 and 150 kDa. The 150 kDa band was identified as PMCA (i) by reaction with a monoclonal anti-human erythrocyte plasma membrane Ca(2+)-ATPase antibody; (ii) by its typical tryptic cleavage pattern which generated an 80 kDa band; (iii) by lack of inhibition of its autophosphorylation by SERCA-specific inhibitors. The 115 kDa band was identified as SERCA (i) by reaction with a polyclonal anti-rat fast skeletal muscle Ca(2+)-ATPase antibody; (ii) by the concentration-dependent inhibition of its autophosphorylation by thapsigargin and 2,5-di(t-butyl)-1,4-benzohydroquinone (tBHQ), which are specific inhibitors of SERCA. The 115 kDa band was further characterised as the SERCA-2b isoform by reaction with a polyclonal rabbit antibody against the 12 C-terminal amino acids of SERCA-2b.

Animals↗
PubMed1995

High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus.

The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the GluRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the GluRD subunit. At the subcellular level, silver intensified immunogold particles for the GluRA, GluRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CA1 and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type 1 synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata. The results demonstrate that the GluRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

Animals↗
PubMed1994

The cisternal organelle as a Ca(2+)-storing compartment associated with GABAergic synapses in the axon initial segment of hippocampal pyramidal neurones.

The axon initial segment of cortical principal neurones contains an organelle consisting of two to four stacks of flat, membrane-delineated cisternae alternating with electron-dense, fibrillar material. These cisternal organelles are situated predominantly close to the synaptic junctions of GABAergic axo-axonic cell terminals. To examine the possibility that the cisternal organelle is involved in Ca2+ sequestration, we tested for the presence of Ca(2+)-ATPase in the cisternal organelles of pyramidal cell axons in the CA1 and CA3 regions of the hippocampus. Electron microscopic immunocytochemistry using antibodies to muscle sarcoplasmic reticulum ATPase revealed immunoreactivity associated with cisternal organelle membranes. The localisation of Ca(2+)-ATPase in cisternal organelles was also confirmed by enzyme cytochemistry, which produced reaction product in the lumen of the cisternae. These experiments provide evidence for the presence of a Ca2+ pump in the cisternal organelle membrane, which may play a role in the sequestration and release of Ca2+. Cisternal organelles are very closely aligned to the axolemma and the outermost cisternal membrane is connected to the plasma membrane by periodic electron-dense bridges as detected in electron micrographs. It is suggested that the interface acts as a voltage sensor, releasing Ca2+ from cisternal organelles upon depolarisation of the axon initial segment, in a manner similar to the sarcoplasmic reticulum of skeletal muscle. The increase in intra-axonal Ca2+ may regulate the GABAA receptors associated with the axo-axonic cell synapses, and could affect the excitability of pyramidal cells.

Animals↗
PubMed1994

Membrane topology of the GluR1 glutamate receptor subunit: epitope mapping by site-directed antipeptide antibodies.

In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N-terminal residues 253-267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877-889 increased about 1.6-fold following permeabilization. Our data suggest that the N- and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross-reactivities in different species and their effect on ligand binding activity were also established.

Amino Acid Sequence↗
PubMed1994

The value of different serological tests in serotyping of Actinobacillus pleuropneumoniae strains.

Immune sera were raised in rabbits against 12 serotypes of Actinobacillus pleuropneumoniae biotype I in order to compare serological tests for simplicity and sensitivity in serotype determination. The serological tests included the tube agglutination test with and without 2-mercaptoethanol addition, the indirect haemagglutination test with and without 2-mercaptoethanol, the immunodiffusion test and the coagglutination test. All these tests proved suitable for serotype determination with the reference strains; however, cross-reactions of varying degree were observed in all of them. Owing to its simplicity, rapidity and specificity, the coagglutination test appears to be the most suitable for primary serotyping. A more definite answer to the question could be obtained by serotyping strains isolated from field cases.

Actinobacillus pleuropneumoniae↗
PubMed1994

Synaptic and nonsynaptic localization of the GluR1 subunit of the AMPA-type excitatory amino acid receptor in the rat cerebellum.

The cellular and subcellular distribution of the GluR1 subunit of the AMPA-type excitatory amino acid receptor was determined in the cerebellar cortex of rat using immunocytochemistry. Two polyclonal antibodies were raised against the N- and C-terminal regions of the subunit. They both labeled a band in immunoblots of rat cerebellar membranes with a molecular weight corresponding to that predicted for this subunit of 105 kDa molecular mass. In light microscopy the distribution of immunoreactivity for the two antibodies was very similar. The molecular layer was strongly immunoreactive whereas no labeling was observed in the granular layer. Electron microscopy revealed that the antibody raised against the N-terminal part of the subunit recognizes an extracellular epitope(s), whereas the antibody against the C-terminal part recognizes an intracellular epitope(s) along the plasma membrane. In Bergmann glial cells the endoplasmic reticulum, Golgi apparatus, and multivesicular bodies were labeled, presumably demonstrating sites of synthesis and degradation for the GluR1 subunit, respectively. Immunoreactivity was associated with Bergmann glial processes surrounding Purkinje cell dendrites, spines, and the glutamate-releasing axon terminals of the parallel and climbing fibers. This suggests that the neurotransmitter glutamate and the AMPA-type glutamate receptors are involved in neuronal/glial communication. The GluR1 subunit was also found at glial membranes in contact with other glial cells. Purkinje cells showed immunoreactivity in the endoplasmic reticulum and multivesicular bodies. No immunoreaction was detected in basket and stellate cells. Immunoreactivity was observed at type 1 synaptic junctions, including the synaptic cleft. These synaptic junctions were between spines, often originating from Purkinje cell dendrites, and parallel or climbing fiber terminals. Our results demonstrate that the GluR1 subunit of the AMPA-type ionotropic excitatory amino acid receptor is present at both parallel and climbing fiber synapses, which are surrounded by glial processes containing the same receptor subunit.

Amino Acid Sequence↗
PubMed1993

Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluR1 glutamate receptor subunit: cellular and subcellular distribution in the rat forebrain.

Antibodies were made to synthetic peptides corresponding to residues 253-367, 757-771 and 877-889 of the published amino acid sequence of the rat brain glutamate receptor GluR1 subunit [Hollmann et al. (1989) Nature 342, 643-648]. The peptides were synthesized both as multiple copies on a branching lysyl matrix (multiple antigenic peptides) and conventional linear peptides using solid-phase synthesis. Rabbits were immunized with these peptides either without conjugation (multiple antigenic peptides) or following coupling to ovalbumin with glutaraldehyde (monomeric peptides). The antibodies from immune sera were then purified by affinity chromatography using reactigel coupled monomeric peptides. All the rabbits produced good antipeptide responses, and were characterized by immunoprecipitation of solubilized alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainate binding activity and by their staining patterns on immunoblots. Antibody to peptide 253-267 specifically immunoprecipitated 12 +/- 3, 50 +/- 3 and 44 +/- 4% of solubilized alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding activity from cortex, hippocampus and cerebellum, respectively. Under identical conditions, antibody against the 877-889 peptide removed 23 +/- 4, 9 +/- 4 and 15 +/- 9% of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites from these areas. On immunoblots of rat brain membrane samples separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, antibodies labelled a 105,000 mol. wt immunoreactive band. GluR1 was immunoaffinity-purified using subunit-specific antibodies against both N-terminal (253-267) and C-terminal (877-889) residues, covalently attached to protein A-agarose. Analysis of the purified product from each column showed a major immunoreactive band, recognized by both sera at 105,000 mol. wt and silver staining identified the same major protein. After exhaustive immunoprecipitation of solubilized membrane samples with antibody against the C-terminal of the subunit, a subpopulation of GluR1 was labelled with antibodies specific for the N-terminal part of the receptor. These observations suggest that the GluR1 subunit consists of at least two isoforms possessing a common N-terminal region but a distinct C-terminus. Immunocytochemistry, using immunoperoxidase staining, was performed for the GluR1 subunit in rat forebrain with antisera raised against the N-terminal (253-267) and the C-terminal parts (877-889) of the molecule. Both antisera gave a similar distribution of immunoreactivity at the light-microscopic level. Immunoreactivity for the GluR1 subunit was selectively distributed throughout the rat forebrain. The hippocampus, septum, amygdala and olfactory bulb exhibited the strongest immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

[Quality control of radiographic imaging of the lumbar spine].

The qualitative control of the roentgenographic findings was performed independently by the assessment of five x-ray specialists on 40 two-directional lumbosacral roentgenograms. The findings were based on questioners including all details and a table of the internationally used "Radiological Diagnosis". In earlier publications the anatomical and structural changes were considered the risk factors of the waist. (Osteoporosis, changes in the position of the vertebral body, change in the shape of the spine, restriction of the intervertebral cleft and deformation of the small articulations). The views of the research workers were compared by means of statistical analysis. Compared with other authors the methods of these findings are in agreement with the international norms. These findings drew attention to the necessity of an objective assessment of the subjective findings.

Diagnostic Imaging↗
PubMed1992

Different sensitivity of the sarcoplasmic reticulum Ca(2+)-ATPase enzyme to fluorescein-isothiocyanate in rabbit and carp muscles.

1. Carp and rabbit sarcoplasmatic reticulum Ca(2+)-ATPase enzymes were compared with respect to their sensitivity to FITC labelling. 2. The carp enzyme showed much lower sensitivity to FITC in the Ca(2+)-Mg2+ activated ATPase activity. Fifty percent inhibition was observed at 20 microM labelling FITC concentration; in rabbit enzyme this inhibition was already achieved at 2 microM FITC. 3. The tryptic cleavage products of the carp enzyme identified with immunoblot analysis as well as with FITC fluorescence, suggest multiple cleavage, yielding different fragments from the ones well known in rabbit and in rat enzyme. 4. The present results indicates major structural differences with respect to the FITC binding, and tryptic cleavage between the SR Ca(2+)-ATPase enzymes from carp and rabbit, despite the cross-reactivity with polyclonal antibodies.

Animals↗