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Biomedical subjects

A Maelicke

Publications and source records attributed to A Maelicke.

At least 37 records · Page 2Linked to original sources
PubMed1999

Expression of nicotinic acetylcholine receptor subunits in the cerebral cortex in Alzheimer's disease: histotopographical correlation with amyloid plaques and hyperphosphorylated-tau protein.

Impairment of cholinergic transmission and decreased numbers of nicotinic binding sites are well-known features accompanying the cognitive dysfunction seen in Alzheimer's disease (AD). In order to elucidate the underlying cause of this cholinoceptive dysfunction, the expression of two pharmacologically different nicotinic acetylcholine receptor (nAChR) subunits (alpha4, alpha7) was studied in the cerebral cortex of Alzheimer patients as compared to controls. Patch-clamp recordings of 14 dissociated neurons of control cortices showed responses suggesting the existence of alpha4- and alpha7-containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the pattern of distribution and the number of alpha4 and alpha7 mRNA-expressing neurons were similar, whereas at the protein level a decrease in the density of alpha4- and alpha7-expressing neurons of approximately 30% was observed in Alzheimer patients. The histotopographical correlation of nAChR expression with accompanying pathological changes, e.g. accumulation of hyperphosphorylated-tau (HP-tau) protein and beta-amyloid showed that neurons in the vicinity of beta-amyloid plaques bore both nAChR transcripts. Neurons heavily labelled for HP-tau, however, expressed little or no alpha4 and alpha7 mRNA. These results point to an impaired synthesis of nAChRs on the protein level as a possible cause of the cholinoceptive deficit in AD. Further investigations need to elucidate whether interactions of HP-tau with nAChR mRNA, or alterations in the quality of alpha4 and alpha7 transcripts give rise to decreased protein expression at the level of individual neurons.

Aged↗
PubMed1999

Expression of functional alpha7 nicotinic acetylcholine receptor during mammalian muscle development and denervation.

We have studied, on the transcriptional, protein and functional level, the expression of alpha7 nicotinic acetylcholine receptors (nAChR) in the course of rat muscle development, denervation and renervation. At foetal day 13, alpha7 nAChR expression was observed in somites and developing muscles of the back, but not yet in migrating myoblasts. Two days later, concomitant with myoblast aggregation, the alpha7 isoform began to be expressed in isolated myoblasts, with the highest level of expression in the frontal zone of the migrating wave. On foetal day 18, a time when the myoblasts in the upper hindleg have fused, alpha7 nAChR expression was most prominent in the outer layer of muscle tissue. The highest level of expression was observed in the first postnatal week, when practically all muscle cells stained positively for alpha7 protein. During the following weeks, alpha7 nAChR expression slowly decreased and practically disappeared in adult hindleg muscle. Following chronic denervation of adult Soleus muscle fibres, expression of alpha7 nAChR returned within 2-4 weeks. Electrophysiological measurements showed that the alpha7 nAChR of chronically denervated soleus muscle fibres were functional and, in particular, that they could be activated by choline. The presence of the alpha7 nAChR in developing and denervated muscle suggests a role for this nicotinic receptor in neuronal pathfinding and/or endplate stabilization.

Animals↗
PubMed1998

Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.

The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.

Amino Acid Sequence↗
PubMed1998

The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes.

We have studied the expression of the nuclear orphan receptor GCNF (germ cell nuclear factor) on the mRNA and protein level in pubertal and adult mouse testes. We show by Northern and Western blot analyses and by in situ hybridization that GCNF is expressed in spermatocytes and round spermatids of adult mouse testis suggesting that GCNF may be a transcriptional regulator of spermatogenesis. Since the GCNF protein is accumulated in the XY body of late pachytene spermatocytes, it may be involved in transcriptional inactivation of sex chromosomes.

Animals↗
PubMed1998

Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy.

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

Animals↗
PubMed1998

Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression.

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding alpha subunits, and the other is a structural beta subunit. The existence of at least one more nAChR gene, probably encoding a beta subunit, is indicated. Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. localpha1, localpha3, and locbeta1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Loc<alpha3 binds alpha-bungarotoxin with high affinity, it may form a homomeric nAChR subtype such as the mammalian alpha7 nAChR. Localpha1 and Locbeta1 may then form the predominant heteromeric nAChR in the locust brain. localpha4 is mainly expressed in optic lobe ganglionic cells and localpha2 in peripherally located somata of mushroom body neurons. localpha3 mRNA was additionally detected in cells interspersed in the somatogastric epithelium of the locust embryo, suggesting that this isoform may also be involved in functions other than neuronal excitability. Transcription of all nAChR subunit genes begins approximately 3 days before hatching and continues throughout adult life. Electrophysiological recordings from head ganglionic neurons also indicate the existence of more than one functionally distinct nAChR subtype. Our results suggest the existence of several nAChR subtypes, at least some of them heteromeric, in this insect species.

Animals↗
PubMed1998

Cell fate specification in an in vitro model of neural development.

We have studied in an in vitro model of neural development the effect of neighboring cells on the fate of single fluorescently labeled precursor cells. In one line of experiments, PCC7-Mz1 embryonal carcinoma cells were transiently transfected with "green fluorescent protein" (GFP) and, following incubation with 0.1 microM all-trans retinoic acid (RA), the number and morphology of derivatives (neuronal or non-neuronal) was determined that form groups of GFP-expressing cells in a surrounding of unlabeled cells. Because single PCC7-Mz1 cells can produce single-lineage and mixed-lineage derivatives, they are individually pluripotent. In another line of experiments, we have analyzed the fate of GFP-expressing PCC7-MzN cells in different cellular environments. Whereas in the absence of other cells, PCC7-MzN cells exclusively differentiated to neuronal derivatives following RA induction (Lang, E., M. L. Mazauric-Stüker, A. Maelicke, J. Cell Biol. 109, 2481-2493 (1989)), they differentiated also to non-neuronal phenotypes (astrocytes and fibroblasts) when co-cultured with either PCC7-Mz1 stem cells or freshly RA-induced cells. The fate of PCC7-MzN cells could also be shifted in the absence of other cells when the cells were grown on laminin-coated surfaces. These results suggest that a putative fate-shifting activity (FSA) is released by PCC7-Mz1 and PCC7-MzN cells which requires, at least in the case of MzN cells, presentation by extracellular matrix-like structures in order to function in cell fate specification. Very few other cell types, in particular primary cultures of mouse forebrain cells of embryonic day 13, were capable of shifting the developmental potential of PCC7-MzN cells in a similar manner as PCC7-Mz1 cells do. We conclude that cell type specification in this model of neural development may occur by similar mechanisms as have been established in Drosophila neurogenesis. A default pathway (neuronal) is modulated by lateral signaling between neighboring cells so that cellular diversity can arise from initially homogeneous populations of progenitor cells.

3T3 Cells↗
PubMed1998

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro.

We used primary cultures of rat hippocampal neurons and PCC7-Mz1 cells to correlate the expression of the protein kinase C (PKC) gene family with specific events during neural differentiation. Multipotent PCC7-Mz1 embryonic carcinoma stem cells develop into a tissue-like pattern of neuronal, fibroblast-like and astroglial cells by all-trans retinoic acid (RA) treatment. Western blot analyses demonstrate that PKCalpha, betaI, gamma, theta, mu, lambda, and zeta were constitutively expressed but the expression of PKCbetaII, delta, epsilon, and eta was up-regulated three days after addition of RA when cells mature morphologically. While the protein levels of the PKC isoforms betaII, delta and eta decreased after d6, when the major phenotypical alterations of the developing neurons were completed, PKCepsilon expression remained at a high level. Immunofluorescence studies demonstrated that PKCalpha, lambda and zeta were constantly expressed in stem cells and the arising cell types. PKCdelta was detected in all differentiated cell types, whereby PKCbetaII, gamma, epsilon, and zeta were solely found in the neuronal derivatives with PKCgamma predominantly located in the nuclei. PKCeta was weakly expressed at the Golgi complex of stem cells but expanded throughout the entire somata of all developing neurons. In contrast, PKCbetaII was abundant only in the somata of a minor fraction of all neurons (approximately 2.5%). Also, PKCepsilon was exclusively synthesized by a subpopulation of neurons (40+/-5%), where it was localized in the somata and in the axons. PKCzeta was persistently expressed in two forms, the full-length PKCzeta and the constitutively active, proteolytic product PKMzeta, reasoning that permanent PKCzeta activity is important for PCC7-Mz1 physiology. Fractionation of extracts from undifferentiated and differentiating PCC7-Mz1 cells revealed that the conventional cPKCalpha was partly and the cPKCbetaI and the novel nPKCs delta and epsilon were mainly membrane bound, implying that they were also in an active state. However, when using the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate) to monitor cellular PKC activity, we observed that activation of PKC by phorbol ester was required for complete MARCKS phosphorylation and its translocation from the membrane to the cytoplasm. Our data show that the cell type-specific expression, subcellular localization and activation of PKCs are regulated in an isoform-specific manner during neurogenesis suggesting that they are involved in the control of neural development and in particular in neuronal differentiation.

Animals↗
PubMed1998

Retinoic acid induces apoptosis-associated neural differentiation of a murine teratocarcinoma cell line.

Incubation with all-trans retinoic acid (RA) induces PCC7-Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue-like pattern of neuronal, astroglial, and fibroblast-like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA-induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan-caspase inhibitor zVAD-fmk (benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10(-7) M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron-specific protein kinase C substrate GAP-43. RA-induced apoptosis increased with the plating density of PCC7-Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA-damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle-arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis-inhibiting factors by the cultured cells is indicated. Neither ApoI/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl-2 proto-oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13-dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long-term treatment with phorbol 12,13-dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of apoptosis of PCC7-Mz1 cells. We conclude that the apoptotic activity following RA treatment of cultured PCC7-Mz1 cells probably is controlled by the same cascade of gene regulatory events that govern the early cell lineage determinations in this in vitro model system of neural development.

Animals↗
PubMed1998

Cloning of the human NCNF gene.

We have cloned from a cDNA library of human testis tissue the human homologue to the mouse nuclear orphan receptor NCNF (neuronal cell nuclear factor). The open reading frame encodes a protein of 480 amino acids, the sequence of which (EMBL accession no. X99975) is 98.3% identical to the mouse homologue. Northern blot analysis of adult human tissues revealed a broad pattern of tissue expression. Similar to NCNF expression in mouse testis, two transcript forms of the single copy gene are expressed in human tissues. The two transcript forms which differ only in their 3'UTR, result in human from differential polyadenylation, in mouse from alternative splicing. Based on the high level of sequence identity of human and murine NCNF, it is likely that also the human nuclear receptor is involved in the control of neurogenesis and gametogenesis.

Adult↗
PubMed1997

Neuronal cell nuclear factor--a nuclear receptor possibly involved in the control of neurogenesis and neuronal differentiation.

We have cloned from a cDNA library of neuronal derivatives of retinoic-acid-induced embryonic carcinoma cells a nuclear receptor that may be involved in the control of late neurogenesis and early neuronal differentiation. The receptor which is practically identical in sequence with germ cell nuclear factor, has been designated neuronal cell nuclear factor (NCNF). NCNF is exclusively expressed in the neuronal derivatives of PCC7-Mz1 cells, with the expression beginning within hours of exposure to retinoic acid. In the developing mouse brain, NCNF is expressed in the marginal zones of the neuroepithelium which are known to contain young postmitotic neurons. NCNF binds to the DR0 sequence thereby silencing transcription. Because NCNF does not recognize hormone response elements of other nuclear receptors tested and does not heterodimerize with these, it probably binds exclusively as a homodimer. NCNF may induce neuronal differentiation by repressing the activity of genes that permit cell fates other than the neuronal one.

Animals↗
PubMed1997

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

Adenosine Triphosphate↗
PubMed1997

Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons.

In the present study, we demonstrate that choline, a precursor of acetylcholine (ACh) and a product of acetylcholine hydrolysis by acetylcholinesterase (AChE), acts as an efficient and relatively selective agonist of alpha7-containing nicotinic acetylcholine receptors (nAChR) in neurons cultured from the rat hippocampus, olfactory bulb and thalamus as well as in PC12 cells. Choline was able to activate postsynaptic and presynaptic alpha7 nAChRs, with the latter action resulting in the release of other neurotransmitters. Although choline was approximately one order of magnitude less potent than ACh (EC50 of 1.6 mM for choline and 0.13 mM for ACh), it acted as a full agonist at alpha7 nAChRs. In contrast, choline did not activate alpha4beta2 agonist-bearing nAChRs on hippocampal neurons, and acted as a partial agonist at alpha3beta4-containing nAChRs on PC12 cells. The ethyl alcohol moiety of choline is required for the selective action on alpha7 nAChR. Exposure of cultured hippocampal neurons for 10 min to choline (10-100 microM) resulted in desensitization of the native alpha7 nAChRs. Moreover, chronic exposure (10 days) of the cultured hippocampal neurons to a desensitizing concentration of choline (approximately 30 microM) decreased their responsiveness to ACh. The selective action of choline on native alpha7 nAChRs suggests that this naturally occurring compound may act in vivo as an endogenous ligand for these receptors. Putative physiological actions of choline include retrograde messenger activity during the development of the mammalian central nervous system and during periods of elevated synaptic activity that leads to long-term potentiation.

Acetylcholine↗
PubMed1997

Methamidophos: an anticholinesterase without significant effects on postsynaptic receptors or transmitter release.

Methamidophos (O,S-dimethyl phosphoroamidothiolate, Tamaron), an organophosphate (OP) anticholinesterase of limited toxicity, is widely used as an insecticide and acaricide. To provide additional insight into the molecular basis of its action, we have used electrophysiological and biochemical techniques to study the effects of methamidophos on the neuromuscular junction of rat and frog and on the central nervous system of rat. Methamidophos has a relatively weak inhibitory action on cholinesterases in rat diaphragm muscle, brain and hippocampal homogenates, with IC50 values on the order of 20-20 microM. An even weaker anticholinesterase activity was found in frog muscle homogenates, with the IC50 being above 300 microM. As further evidence of anticholinesterase activity, methamidophos (1-100 microM) was able to reverse the blockade by d-tubocurarine (0.5-0.7 microM) of neuromuscular transmission in rat phrenic nerve-hemidiaphragm preparations. Inhibition of cholinesterase activity by methamidophos was long lasting, which is consistent with the formation by the agent of a covalent bond with the enzyme's active serine residue. The action was also slowly reversible, which suggests spontaneous reactivation of the enzyme. electrophysiological studies at the rat neuromuscular junction showed that, due to its anticholinesterase activity, methamidophos increased the amplitude and prolonged the decay phase of nerve-evoked and spontaneous miniature end-plate potentials. In contrast to other OP compounds, e.g., paraoxon (Rocha et al., 1996a), methamidophos did not affect neurotransmitter release, nor did it interact directly with the muscle nicotinic acetylcholine receptor. Moreover, it contrast to paraoxon, methamidophos did not affect the whole-cell currents induced by application of acetylcholine, glutamate or gamma-aminobutyric acid recorded to cultured hippocampal neurons. Based on these data, methamidophos appears to have a selective effect on cholinesterase.

Animals↗
PubMed1997

De novo acquisition of neuronal polarity in retinoic acid-induced embryonal carcinoma cells.

The mouse embryonal carcinoma cell line PCC7-Mz1 represents an advantageous model to study acquisition of polarity by neurons. During the first two days after differentiation is induced by the addition of retinoic acid, the neuronal derivatives develop extensions which for at least four more days do not differ from each other in growth characteristics, morphology, and marker expression. Beginning around differentiation day 6 and following the relocation of the nucleus from a central to a polar position in the cell soma, the morphology and marker expression changes dramatically: expression of MAP2 diminishes and eventually disappears in the thinner neurite (future axon), which originates at the nucleated pole, but remains strong in the branched, broad based neurite(s). The opposite changes in expression are observed for synaptophysin, together with a clustering of the vesicle protein in varicosity-like areas. Complete segregation of expression of the two markers is achieved around day 12, shortly followed by dendrite-specific location of MAP2 mRNA and the ability to generate and conduct action potentials. Our studies add several aspects to the process of neuronal polarity acquisition, as it was previously studied in primary cultures of embryonic neurons: (i) we monitored neuronal differentiation from the birth of neurons, rather than from later and less defined maturation stages, (ii) cell nucleus relocation may be associated with the induction of neuronal polarity, and (iii) functional competence of neurons is closely associated with previous acquisition of polarity. Acquisition of polarity by PCC7-Mz1 neuronal derivatives probably refers to de novo acquisition rather than to reestablishment of polarity.

Animals↗
PubMed1996

Stable expression in HEK-293 cells of the rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor.

The alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor (nAChR) was stably expressed in human embryonic kidney (HEK) 293 cells that co-expressed a voltage-gated Ca2+ channel. alpha3/beta4-nAChR-expressing clones were identified using the fura-2 Ca2+ imaging technique, and were further characterised by single-cell and whole-cell patch-clamp studies. Acetylcholine (ACh) induced fast activating currents which showed desensitisation and inward rectification. The conductance of the ACh-activated channel was 29 pS. The order of potency of the nicotinic agonists tested was cytisine approximately = nicotine > acetylcholine. The EC50 value for ACh was 145 microM; the Hill coefficient was close to 2. The currents elicited by ACh were effectively blocked by nicotinic antagonists, but not by the muscarinic antagonist atropine. These properties are comparable to the pharmacological and physiological profile of ganglionic nicotinic receptors and type III currents of cultured hippocampal neurons.

Acetylcholine↗
PubMed1996

Expression of alpha subunit genes of nicotinic acetylcholine receptors in human lymphocytes.

Using immunohistochemistry and in situ hybridisation, we have studied whether alpha-subunits of nicotinic acetylcholine receptors (nAChRs) are expressed in human lymphocytes. Cells were isolated by differential low speed gradient centrifugation from heparinised venous blood of 10 healthy volunteers. Receptor sites were visualised using the monoclonal antibody WF6 which specifically recognises alpha-isoforms from several species including man. For visualisation of transcripts, digoxigenin-labelled cRNA probes for alpha 4- and alpha 3-subunits were used. Immunostaining revealed specific binding of WF6 to isolated human lymphoid cells. The antibody was bound to most cells and concentrated preferentially in the perinuclear/surface region. The immunoreactivity resembled that observed after application of an antibody recognising CD4 surface proteins which was conducted for comparison. In situ-hybridisation revealed that the alpha 4-subunit genes of nAChRs was expressed in lymphocytes of all probands. The alpha 3-subunit was found, with lower intensity than alpha 4-transcripts, in eight of the 10 individuals. Control incubations with corresponding sense cRNAs were negative. It is concluded that human lymphocytes are able to express alpha-subunit genes of nAChRs.

Adult↗