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T Bartfai

Publications and source records attributed to T Bartfai.

At least 37 records · Page 2Linked to original sources
PubMed2001

Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin.

The neuropeptide galanin has been shown to suppress epileptic seizures. In cortical and hippocampal areas, galanin is normally mainly expressed in noradrenergic afferents. We have generated a mouse overexpressing galanin in neurons under the platelet-derived growth factor B promoter. RIA and HPLC analysis revealed up to 8-fold higher levels of galanin in transgenic as compared with wild-type mice. Ectopic galanin overexpression was detected especially in dentate granule cells and hippocampal and cortical pyramidal neurons. Galanin-overexpressing mice showed retardation of seizure generalization during hippocampal kindling, a model for human complex partial epilepsy. The high levels of galanin in mossy fibers found in the transgenic mice were further increased after seizures. Frequency facilitation of field excitatory postsynaptic potentials, a form of short-term synaptic plasticity assessed in hippocampal slices, was reduced in mossy fiber-CA3 cell synapses of galanin-overexpressing mice, indicating suppressed glutamate release. This effect was reversed by application of the putative galanin receptor antagonist M35. These data provide evidence that ectopically overexpressed galanin can be released and dampen the development of epilepsy by means of receptor-mediated action, at least partly by reducing glutamate release from mossy fibers.

Animals↗
PubMed2001

Circadian regulation of diverse gene products revealed by mRNA expression profiling of synchronized fibroblasts.

Genes under a 24-h regulation period may represent drug targets relevant to diseases involving circadian dysfunctions. As a testing model of the circadian clock system, we have used synchronized rat fibroblasts that are known to express at least six genes in a circadian fashion. We have determined the expression patterns of 9957 transcripts every 4 h over a total period of 76 h using high density oligonucleotide microarrays. The spectral analysis of our mRNA profiling data indicated that approximately 2% (85 genes) of all expressed genes followed a robust circadian pattern. We have confirmed the circadian expression of previously known clock or clock-driven genes, and we identified 81 novel circadian genes. The majority of the circadian-regulated gene products are known and are involved in diverse cellular functions. We have classified these circadian genes in seven clusters according to their phase of cycling. Our pathway analysis of the mRNA profiling data strongly suggests a direct link between circadian rhythm and cell cycle.

Animals↗
PubMed2001

VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions.

Cell-penetrating peptides, CPPs, have been shown to translocate into living cells by a receptor-independent mechanism and to carry macromolecules over the plasma membrane. This article reports studies of the internalization of pVEC, an 18-amino acid-long peptide derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin, amino acids 615-632. Fluorophore-labeled pVEC entered four different cell lines tested: human aortic endothelial cells, brain capillary endothelial cells, Bowes melanoma cells, and murine brain endothelial cells. In order to evaluate the translocation efficiency of pVEC, we performed a side-by-side comparison with penetratin, a well-characterized CPP. The cellular uptake of pVEC was highest for murine brain endothelial cells. All cell lines tested contained equal or slightly higher concentrations of pVEC than penetratin. pVEC mainly accumulated in nuclear structures but was also found throughout the cells. Furthermore, pVEC functioned as a transporter of both a hexameric peptide nucleic acid molecule of 1.7 kDa and a 67-kDa protein, streptavidin-FITC, and cellular uptake of fluorophore-labeled pVEC took place at 4 degrees C, suggesting a nonendocytotic mechanism of translocation. In conclusion, our results indicate that pVEC is efficiently and rapidly taken up into cells and functions as a potent carrier peptide.

Active Transport, Cell Nucleus↗
PubMed2001

Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells.

Fluorescence correlation spectroscopy (FCS) allows the study of interactions of fluorescently labeled ligand with receptors in living cells at single-molecule detection sensitivity. From the autocorrelation functions of fluorescence intensity fluctuations, the diffusion time of molecules through the confocal volume is analyzed, and from that, the molecular weights of free and bound molecules can be calculated. We have applied FCS to study the receptor diversity for the neuropeptide galanin (GAL) in cultured cells. FCS measurement of the fluorophore rhodamine-labeled GAL (Rh-GAL) has been performed in 0.2-fL confocal volume elements of the laser beam. The analysis of autocorrelation functions of Rh-GAL in solution above cells and at cell membranes demonstrates that the diffusion time of unbound Rh-GAL is 0.16 ms, whereas diffusion times of membrane-bound Rh-GAL are 22 and 700 ms. Because both of the diffusion times (22 and 700 ms) are much longer as compared to that of unbound Rh-GAL, they correspond to slow-diffusing complexes when Rh-GAL is bound to the cell membranes. Addition of excess nonlabeled GAL is accompanied by competitive displacement. Full saturation of the GAL binding is obtained at nanomolar concentrations. Scatchard analysis of binding data reveal one binding process, assuming one binding site per Rh-GAL (n = 1). On the other hand, the appearance of two diffusion times, 22 and 700 ms, suggests the existence of two subpopulations of GAL receptor complexes or two subtypes of GAL receptor not detected before. This makes an important point that FCS permits the identification of receptors, which were not possible to detect before by conventional binding techniques. The inhibitory effect of pertussis toxin on the GAL binding considers a G-protein-involved allosteric system, important for the clarification of essential steps in the G-protein-related signal transduction. This study is of pharmaceutical significance, since it will provide insights into how FCS can be used as a rapid technique for studying ligand-receptor interactions in living cells, which is one step forward for large-scale drug screening in cell cultures.

Animals↗
PubMed2001

Microdialysis in freely moving mice: determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice.

In the present study, we describe micro-surgical methods for simultaneous implantation of a microdialysis probe and an intraventricular injection cannula via their respective guide cannulas into the mouse brain. Basal and stimulated release of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (NA) was determined in the ventral hippocampus of freely moving mice. NA and 5-HT were determined in one run by a newly developed HPLC method based on precolumn derivatization with benzylamine and fluorescence detection. The mice with a loss-of-function mutation of the galanin gene (KO) and the mice that over-expressed galanin (OE) were studied. No significant differences in basal, potassium-stimulated or scopolamine-induced extracellular ACh levels were observed in 4-month-old wild-type (WT) and KO mice. In the aged, 10-month-old animals, the basal extracellular ACh levels were significantly reduced in both WT and KO groups. Galanin (1 nmol i.c.v.) caused a significant reduction of basal extracellular NA by about 40% in both WT and galanin OE mice, however, in the latter group the effect was delayed by almost 2 h. A 10-min forced swimming stress caused a higher increase in release of NA and 5-HT in the OE group than in the corresponding WT mice. Finally, venlafaxin (10 mg/kg i.p.) increased extracellular NA to 400% of the control values in the CBA mice, but only to 250% in the C57BL mice. It is concluded that galanin may play an important role in the cholinergic mechanisms underlying cognitive disorders. Furthermore, modulation by galanin and by behavioral activation, of NA and 5-HT neurotransmission in galanin over-expressing mice indicates its possible role in the aetiology of mood disorders.

Acetylcholine↗
PubMed2001

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif.

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.

Animals↗
PubMed2001

Intrathecal administration of PNA targeting galanin receptor reduces galanin-mediated inhibitory effect in the rat spinal cord.

Peptide nucleic acids (PNA) are nucleic acid analogues containing neutral amide backbone, forming stable and tight complexes with complementary DNA/RNA. However, it is unclear whether unmodified PNA can efficiently penetrate neuronal tissue in order to act as antisense reagent. Here we show that intrathecal (i.t.) injection of an unmodified antisense PNA complementary to the rat galanin receptor type 1 (GalR1) mRNA is able to block the inhibitory effect of i.t. administered galanin on spinal nociceptive transmission. Autoradiographic ligand binding studies using [125I]galanin show that the unmodified PNA is able to reduce the density of galanin binding sites in the dorsal horn. Thus, unmodified PNA applied i.t. appears to function as an effective antisense reagent in rat spinal cord in vivo.

Animals↗
PubMed2001

Mice over-expressing galanin have elevated heat nociceptive threshold.

The neuropeptide galanin may have a role in modulation of nociceptive input at spinal level. Here we report that mice over-expressing galanin exhibit significant elevation of nociceptive threshold to thermal stimulation in comparison to wild-type mice as assessed by the tail flick and paw heat irradiation tests. No change in response to mechanical or cold stimulation was seen. The elevated heat nociceptive threshold in the galanin over-expressing mice was reversed by intrathecal application of the putative galanin receptor antagonist M-35, galanin-(1-12)-pro-bradykinin-(2-9). The results thus support that galanin has an inhibitory function in rodent spinal cord.

Animals↗
PubMed2001

Ca2+ signaling via the neuronal calcium sensor-1 regulates associative learning and memory in C. elegans.

On a radial temperature gradient, C. elegans worms migrate, after conditioning with food, toward their cultivation temperature and move along this isotherm. This experience-dependent behavior is called isothermal tracking (IT). Here we show that the neuron-specific calcium sensor-1 (NCS-1) is essential for optimal IT. ncs-1 knockout animals show major defects in IT behavior, although their chemotactic, locomotor, and thermal avoidance behaviors are normal. The knockout phenotype can be rescued by reintroducing wild-type NCS-1 into the AIY interneuron, a key component of the thermotaxis network. A loss-of-function form of NCS-1 incapable of binding calcium does not restore IT, whereas NCS-1 overexpression enhances IT performance levels, accelerates learning (faster acquisition), and produces a memory with slower extinction. Thus, proper calcium signaling via NCS-1 defines a novel pathway essential for associative learning and memory.

Animals↗
PubMed2001

PNA oligomers as tools for specific modulation of gene expression.

Small synthetic molecules that can specifically inhibit translation and/or transcription have shown great promise as potential antisense/antigene drugs. Peptide nucleic acid (PNA), an oligonucleotide mimic, has a non-charged achiral polyamide backbone to which the nucleobases are attached. PNA oligomers are extremely stable in biological fluids and they specifically hybridise to DNA or RNA in a complementary manner, forming very strong heteroduplexes. Some of the mRNAs have yet undetermined and possibly long half-lives, successful down regulation of gene expression by antisense oligonucleotides (ON) requires that the antisense agent is long lived. PNA fulfils this requirement better than phosphodiester or phosphorothioate ONs. PNA can inhibit transcription and translation of respective genes by tight binding to DNA or mRNA. First in vitro experiments to specifically down regulate protein expression by PNA have been followed by successful antisense and antigene application of PNA oligomers in vivo. This review discusses the principles of the in vitro and in vivo use of PNA oligonucleotides.

Animals↗
PubMed2001

Galanin: neurobiologic mechanisms and therapeutic potential for Alzheimer's disease.

The neuropeptide galanin (GAL) is widely distributed in the mammalian CNS. Several lines of evidence suggest that GAL may play a critical role in cognitive processes such as memory and attention through an inhibitory modulation of cholinergic basal forebrain activity. Furthermore, GAL fibers hyperinnervate remaining cholinergic basal forebrain neurons in Alzheimer's disease (AD). This suggests that GAL activity impacts cholinergic dysfunction in advanced AD. Pharmacological and in vitro autoradiographic studies indicate the presence of heterogeneous populations of GAL receptor (GALR) sites in the basal forebrain which bind GAL with both high and low affinity. Interestingly, we have recently observed that GALR binding sites increase in the anterior basal forebrain in late-stage AD. Three G protein-coupled GALRs have been identified to date that signal through a diverse array of effector pathways in vitro, including adenylyl cyclase inhibition and phospholipase C activation. The repertoire and distribution of GALR expression in the basal forebrain remains unknown, as does the nature of GAL and GALR plasticity in the AD basal forebrain. Recently, GAL knockout and overexpressing transgenic mice have been generated to facilitate our understanding of GAL activity in basal forebrain function. GAL knockout mice result in fewer cholinergic basal forebrain neurons and memory deficits. On the other hand, mice overexpressing GAL display hyperinnervation of basal forebrain and memory deficits. These data highlight the need to explore further the putative mechanisms by which GAL signaling might be beneficial or deleterious for cholinergic cell survival and activity within basal forebrain. This information will be critical to understanding whether pharmacological manipulation of GALRs would be effective for the amelioration of cognitive deficits in AD.

Alzheimer Disease↗
PubMed2001

Galanin: an endogenous anticonvulsant?

Galanin is a neuroendocrine peptide involved in the regulation of feeding, pain, sexual behavior, learning, and memory. The recent discovery, that galanin antagonized excitatory glutamatergic neurotransmission in the hippocampus, provided a rationale for its possible antiepileptic effects. Here we summarize the data on the effects of galanin on seizure activity in several animal models of epilepsy. Pharmacological and molecular biological evidence suggest potent anticonvulsant effects of galanin. Exogenous administration of galanin receptor agonists attenuated seizures, whereas application of galanin receptor antagonists potentiated seizure expression. Genetically engineered mice, with either deletion or overexpression of galanin gene, showed altered resistance to seizures, which was in direct correlation with galanin gene expression. Possible mechanisms of the anticonvulsant action of galanin include its effects on synaptic potentiation in hippocampal circuits and inhibition of the release of the excitatory neurotransmitter glutamate from principal hippocampal neurons.

Animals↗
PubMed2001

Increased gene expression of interleukin-1alpha and interleukin-6 in rat primary glial cells induced by beta-amyloid fragment.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques. The main constituent of the amyloid plaques is the amyloid beta-peptide (A beta) shown to activate glial cells in vitro. A growing body of evidence suggests that these cells contribute to neurotoxicity through production of inflammatory cytokines, chemokines, and neurotoxic substances, such as reactive oxygen species (ROS). In this study, mRNA levels of the inflammatory cytokines interleukin (IL)-1alpha and beta, and IL-6 were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) in rat primary mixed glial cells after treatment with A beta(25-35), a biologically active fragment of A beta peptide with neurotoxic properties. Clear morphological changes of the astrocytes, as well as proliferation and clustering of microglial cells were observed by light and immunofluorescence microscopy after 24 h treatment. Significant increases in IL-1alpha and IL-6 mRNA levels were detected after 24 and 72 h, whereas significantly increased levels of IL-1beta mRNA could only be detected after 4 h treatment. The most pronounced effect was seen on IL-6 mRNA expression, which increased approx two- to threefold after treatment. In addition, increased secretion of IL-6 was detected after 96 h exposure. Recently, association of IL-1alpha and IL-6 gene polymorphism with AD was reported, suggesting that these cytokines may play an important role in the development of the disease. The increased mRNA levels of IL-1alpha and IL-6 in parallel with the morphological changes in the mixed glial-cell cultures support that these cytokines may be involved in A beta-induced gliosis and in the pathogenesis of AD.

Amyloid beta-Peptides↗
PubMed2000

Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice.

IL-1beta and its endogenous receptor antagonist (IL-1Ra) are rapidly induced by seizures in the rodent hippocampus. Exogenously applied IL-1beta prolongs seizures in an IL-1R type I-mediated manner. This effect depends on N-methyl-d-aspartate receptor activation. We report here that intrahippocampal application of recombinant IL-1Ra or its selective endogenous overexpression in astrocytes under the control of glial acidic fibrillary protein promoter potently inhibits motor and electroencephalographic seizures induced by bicuculline methiodide in mice. Accordingly, transgenic mice show a reduced seizure-related c-fos mRNA expression in various forebrain areas compared with their wild-type littermates. Recombinant IL-1Ra was ineffective in mice deficient in IL-1R type I, having per se a delayed onset to generalized convulsions. These results demonstrate that IL-1Ra mediates potent anticonvulsant effects acting on IL-1R type I and suggest that the balance between brain IL-1beta and IL-1Ra represents a crucial mechanism to control seizure generalization.

Animals↗
PubMed2000

Homophilic interaction of junctional adhesion molecule.

Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.

Animals↗
PubMed2000

Inducible expression of the long pentraxin PTX3 in the central nervous system.

PTX3 is a prototypic long pentraxin consisting of a C terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. PTX3 is induced by primary proinflammatory signals in various cell types, most prominently macrophages and endothelial cells. Other long pentraxins, such as murine or rat neuronal pentraxin 1 (NP1) and human neuronal pentraxin 2 (NPTX2), are expressed in the central nervous system (CNS). The present study was designed to investigate whether PTX3 is expressed in the brain and to define the structures and cells involved. Intracerebroventricular (i.c.v.), but not i.v., injection of LPS induced high levels of PTX3 mRNA in the mouse brain. In contrast NP1 is constitutively expressed in the murine CNS and is not modulated by LPS administration. I.c.v. IL-1beta was also a potent inducer of PTX3 expression in the CNS, whereas TNFalpha was substantially less effective and IL-6 induced a barely detectable signal. Central administration of LPS and IL-1 induced PTX3 also in the periphery (heart), whereas the reverse did not occur. Expression of PTX3 was also observed in the brain of mice infected with Candida albicans (C. albicans) or Cryptococcus neoformans. (C. neoformans). The kinetics of PTX3 gene induction were consistently different between C. albicans- and C. neoformans-infected mice, according to the diverse outcome of the CNS immune reaction. In situ hybridization revealed that i.c.v. injection of LPS induced a strong PTX3 expression in presumptive glial cells, in the white matter (corpus callosum, fimbria) and meningeal pia mater as well as in dentate gyrus hilus and granule cells. No constitutive expression of PTX3 was detected. Central expression of PTX3 may amplify mechanisms of innate resistance and damage in the CNS. The possibility of a direct interaction of PTX3 with neuronal cells, as suggested for NPTX2, remains to be explored.

Animals↗