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W E Müller

Publications and source records attributed to W E Müller.

At least 127 records · Page 7Linked to original sources
PubMed1998

Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death.

Sponges (Porifera) represent the lowest metazoan phylum; they have been shown to be provided with the characteristic metazoan structural and functional molecules. One autapomorphic character of sponges is the presence of high levels of telomerase activity in all cells (or almost all cells, including somatic cells). In spite of this fact previous attempts to cultivate sponge cells remained unsuccessful. It was found that dissociated sponge cells do not replicate DNA and lose their telomerase activity. In addition, no nutrients or metabolites have been detected that would stimulate sponge cells to divide. In the present study we report the culture conditions required for the formation of multicellular aggregates from dissociated single cells of Suberites domuncula, termed primmorphs. These primmorphs are formed in seawater without addition of further supplements, and have an organised tissue-like structure; they have been cultured for more than 5 months. Cross-sections revealed a distinct external layer covered by a continuous pinacoderm, and a central zone composed primarily of spherulous cells. After their association into primmorphs, the cells turn from the telomerase-negative state into the telomerase-positive state; a telomerase level of 4.7 total product generated (TPG) units/5 x 10(3) cell equivalents has been determined. Moreover, a major fraction of the cells in the primmorphs undergoes DNA synthesis and hence has the capacity to grow. Applying the BrdU-labelling and detection assay it is demonstrated that up to 33.8% of the cells in the primmorphs are labelled with BrdU after an incubation period of 12 h. It is proposed that the primmorph system described here is a powerful novel model system to study basic mechanisms of cell proliferation and cell interaction, as well as of morphogenesis, ageing and apoptosis.

Animals↗
PubMed1998

Characterization and phylogenetic analysis of a cDNA encoding the Fes/FER related, non-receptor protein-tyrosine kinase in the marine sponge sycon raphanus.

In search of ancient versions of phylogenetically conserved genes/proteins, which are typical for multicellular animals, we have decided to analyse marine sponges (Porifera), the most ancient and most primitive metazoan organisms. We report here the complete nucleotide sequence of Sycon raphanus cDNA coding for a 879 aa long protein, which displays high overall similarity in primary structure and organization of domains with non-receptor tyrosine kinases (TKs) from the Fes/FER family. The encoded protein, which we named Fes/FER_SR, has a highly conserved, 260 aa long tyrosine kinase domain at the C-terminus. Amino-terminal to the catalytic domain is an 85 aa long SH2 domain. The N-terminus is over 500 aa long and displays homology only with N-terminal domains of protein-tyrosine kinases (PTKs) from the Fes/FER family. Mammalian Fes/FER proteins show around 58% overall homology with Fes/FER_SR (identity and similarity) and lower homology was found with Drosophila melanogaster Fps (FER) protein (49%). Homologies in TK, SH2 and N-terminal domains are on average 78%, 65% and 49%, respectively. Fes/FER_SR shows next to best homology with the Abl family of non-receptor PTKs, while Src-related PTKs from the fresh-water sponge Spongilla lacustris are related only distantly to Fes/FER_SR. Phylogenetic analysis shows that the S. raphanus TK is indeed the most ancient known member of the Fes/FER family of non-receptor PTKs. The role of these PTKs in signal transduction in higher animals is still enigmatic; they are present in the nucleus as well as in the cytoplasm and FER is found in all cell types examined. The function of Fes/FER_SR in sponge, the most primitive multicellular animal which lacks specialized organ systems, remains to be elucidated.

Amino Acid Sequence↗
PubMed1998

Enhanced vulnerability to apoptotic cell death in sporadic Alzheimer's disease.

All important neuropathological features like neurodegeneration and amyloid deposition occur similarly in familial and in sporadic Alzheimer's disease (AD) patients, suggesting a common pathogenetic mechanism. We investigated whether defective vulnerabilty to the induction of the apoptotic cell death pathway might be one of the underlying mechanisms leading to progressive cell death in sporadic AD. Our test could prove useful in supporting the diagnosis of AD and in predicting individuals predisposed to AD.

Aged↗
PubMed1998

Pharmacological intervention in age-associated brain disorders by Flupirtine: Alzheimer's and prion diseases.

Alzheimer's disease, a major form of dementia in the elderly has become an increasingly important health problem in developed countries. In vitro studies on primary neurons demonstrate that Flupirtine (Katadolon) at a concentration of 1 microg/ml, significantly reduces the neurotoxic (apoptotic) effect displayed by A beta25-35, a segment of the amyloid beta-protein precursor the etiologic agent of Alzheimer's disease. Flupirtine, which has been in clinical use since 10 years ago, prevents the toxic effect of PrP, the presumed etiologic agent of the Creutzfeldt-Jakob disease as well as the excitatory amino acid glutamate on cortical neurons. Flupirtine displays a bimodal activity. Its strongest cytoprotective effect against glutamate-induced neurotoxicity was measured if administered at least 120 min prior to the addition of the glutamate. A likewise potent anti-apoptotic activity was measured if cells were simultaneously incubated with Flupirtine and the apoptotic inducers. Administration of Flupirtine during postincubation time in the experiments with glutamate did not result in neuroprotection. In parallel with the determination of the effect of Flupirtine on the toxin (A beta, PrP or glutamate)-induced neuronal death the effect of the drug on the intracellular Ca2+ level [Ca2+]i, was measured. It is well established that incubation of neurons with glutamate causes an increase in [Ca2+]i. It was found that a simultaneous administration of Flupirtine and glutamate did not reduce the glutamate-induced high Ca2+ level. Only if the cells had been preincubated for approximately 30 min with the drug the intracellular Ca2+ level was significantly lower. Experimental evidence given here shows that the molecular basis for the antiapoptotic effect of Flupirtine against glutamate, triggered during pre-incubation, is an increased expression of the protooncogene bcl-2. The neuroprotective effect determined during coincubation with the inducer is attributed to a normalization of the glutathione level which dropped in the presence of the inducers. It is concluded that Flupirtine is a promising drug to treat neurodegenerative disorders occurring with age, e.g. Alzheimer's disease and prion based diseases, like Creutzfeldt-Jakob disease. This conclusion is corroborated by the favourable pharmacokinetic profile of Flupirtine.

Aging↗
PubMed1998

Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium that is conserved in higher metazoans.

Species belonging to the lowest metazoan phylum, the sponges (Porifera), exhibit a surprisingly complex and multifaceted Bauplan (body plan). Recently, key molecules have been isolated from sponges which demonstrate that the cells of these animals are provided with characteristic metazoan adhesion and signal transduction molecules, allowing tissue formation. In order to understand which factors control the spatial organization of these cells in the sponge body plan, we screened for a cDNA encoding a soluble modulator of the behaviour of endothelial cells. A cDNA encoding a putative protein, which is highly similar to the human and mouse endothelial monocyte-activating polypeptide (EMAP) II has been isolated from a library of the marine sponge Geodia cydonium. The sponge EMAP-related polypeptide (EMAPR) has been termed EMAPR1_GC. The full-length cDNA clone, GCEMAPR1, has a size of 592 nucleotides (nt) and contains a 447 nt-long potential open reading frame; the molecular weight (MW) of the deduced amino acid sequence, 16,499 Da, is close to that of mature mammalian EMAP II (ca. 18 kDa). The sponge polypeptide is also closely related to a deduced polypeptide from the cosmid clone F58B3 isolated from Caenorhabditis elegans. A phylogenetic analysis revealed that the sponge and the nematode EMAPR molecules form a cluster which is significantly separated from the corresponding mammalian EMAP molecules. The function of the first cloned putative soluble modulator of endothelial cells in sponges remains to be determined.

Amino Acid Sequence↗
PubMed1998

Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells.

Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium. It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell equivalents and the one from G. cydonium 8.3 TPG units; hence the activity reached approximately 30-20% of the activity seen in telomerase-positive reference cells. In contrast, dissociated spherulous cells from G. cydonium, after an incubation period of 24 h, contained no detectable telomerase activity. From earlier studies it is known that isolated sponge cells do not proliferate. Based on these findings it is assumed that the separation of the senescent sponge cell lineage from the immortal germ/somatic cell lineage is triggered by the loss of contact with cell adhesion factors. First evidence is included which suggests that the final progress of the senescent, telomerase-negative cells to cell death is caused by apoptosis.

Animals↗
PubMed1998

Multiple Ig-like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium.

The receptor tyrosine kinase of the marine sponge Geodia cydonium features two extracellular Ig-like domains in which we recently documented RT-PCR polymorphism among individuals. Genomic-PCR analysis presented here revealed 14 unique sequences from four sponges, differing predominantly in the sequence of an intron which splits the Ig-like domains. Nevertheless, analysis of putative coding regions in 19 distinct clones (156-159 aa) from seven sponges revealed 69 positions of nucleotide substitutions, 67.6% of them non-synonymous, translating into 43 positions of divergent residues. Excluding aa deletions, these 19 sequences share pairwise aa identities of 89-99%. In three sponges, four or five unique Ig-like coding regions were scored. PCR amplification across this intron revealed multiple bands, polymorphic among five of six sponges. Further substantiated by Southern and Northern blots, it is evident that the genome of G. cydonium harbors multicopies of moderately divergent Ig-like domains. Presently, this only appears paralleled by the human KIR multigene family of NK cells MHC class I-specific receptors, which consist of two or three moderately divergent extracellular Ig-like domains.

Amino Acid Sequence↗
PubMed1998

Evolutionary analysis of G-proteins in early metazoans: cloning of alpha- and beta-subunits from the sponge Geodia cydonium.

G-protein-coupled (seven-transmembrane segment)-receptors represent a major group of metazoan receptors, involved in transduction of extracellular signals. The G-proteins, which are made up of Galpha/beta/gamma-subunits, link the receptors to the effector system(s). To analyze the phylogenetic relationships among the metazoan alpha-subunits of G-proteins, cDNAs of alpha-subunits were isolated from Geodia cydonium, a marine sponge belonging to the lowest metazoan phylum, Porifera. One encodes a putative isotype of a stimulator of the adenylyl cyclase (Galpha s), another one a putative inhibitor of the adenylyl cyclase (Galpha i/o) and the third one a putative activator of phospholipase C (Galpha q). In addition one putative beta-subunit was cloned from the same species. The deduced amino acid sequences of the sponge Galpha s -(putative Mr 44749), the Galpha i/o -(Mr 41064) and the Galpha q subunits (Mr 41363) were found to display high similarity with the corresponding sequences from higher Metazoa, and are only distantly related to those of slime mold, yeast or plants. Of lower similarity are the sequences of the beta-subunits among animals and plants, thus not allowing robust grouping. These data demonstrate that the phylogenetic relationships, obtained from analyses of the alpha subunits from metazoan G-proteins, support the conclusion that all metazoan phyla, including the Porifera are of monophyletic origin.

Amino Acid Sequence↗
PubMed1998

Induction of digoxin-like material production, and the digoxin binding in the unicellular organism Tetrahymena by digitoxin.

Thin layer chromatographic, and laser-confocal microscopic analyses with a monoclonal antibody to digoxin also displaying high affinity to digoxigenin, were used to determine the presence and localization of cardioactive glycosides. Tetrahymena pyriformis was found to possess digitoxigenin-like material, but digoxin, digitoxin, digoxigenin, gitoxin and lanatoside C were not detected. Digitoxin treatment elicited the appearance of a digoxin-like material in the progeny generations. Digoxin was taken up by untreated Tetrahymena, especially strongly 24 h after digitoxin treatment. While the cardenolide was localized in vesicles of the cell body in untreated Tetrahymena, the engulfed digoxin appeared in the epiplasmic layer and also in the cilia after digitoxin pretreatment. Digoxin pretreatment did not increase digoxin uptake. These data indicate that Tetrahymena has: (1) the capacity to discriminate between closely related molecules; (2) the ability to induce digoxin-like material production; and/or (3) enzymes that can effect a digitoxin-digoxin transformation.

Animals↗
PubMed1998

Changes of intracellular calcium regulation in Alzheimer's disease and vascular dementia.

Free intracellular calcium ([Ca2+]i) represents probably the most important intracellular messenger for many signal transduction pathways. Due to this crucial role of [Ca2+]i, it has been assumed that alterations of [Ca2+]i are critically involved in brain aging and in the pathophysiology of Alzheimer's disease (AD). This hypothesis is corroborated by several studies demonstrating changes of [Ca2+]i in peripheral cells from AD patients. However, the findings are still controversial. Using blood lymphocytes and neutrophils as two different peripheral model systems, we evaluated several parameters of intracellular Ca2+ regulation in a very large group of AD patients and non-demented controls. We found no major difference in Ca2+ homeostasis, since neither the basal [Ca2+]i, nor the activation-induced Ca2+ responses differed among neutrophils or lymphocytes from aged controls and AD patients. However, we observed a delayed Ca2+ response of AD lymphocytes after phytohemagglutinin (PHA) stimulation indicating an impaired function of Ca2+ influx-controlling mechanisms. Furthermore, we studied whether differences exist in Ca2+ regulation between lymphocytes from patients with vascular dementia and AD patients, to define AD-specific alterations and to distinguish between the two dementia groups and non-demented control subjects respectively. First evidences indicate that Ca2+ mobilization in lymphocytes is specifically impaired in lymphocytes from patients with vascular dementia.

Aged↗
PubMed1998

Lymphocytes as cell model to study apoptosis in Alzheimer's disease: vulnerability to programmed cell death appears to be altered.

Recent evidence indicates that programmed cell death (apoptosis) may contribute to neuronal death in Alzheimer's disease (AD). In situ data derived from post mortem brain tissue indicate that DNA fragmentation which represents an important and typical apoptotic feature is markedly increased in brain cells of AD patients compared to controls. Furthermore, in vitro studies demonstrate that the peptide beta-amyloid (A beta) and its fragments induce apoptosis in neuronal cell cultures. One possible mechanism initiating apoptosis could be free radical generation by the peptide leading to oxidative stress. In a wide range of cell types common morphological and molecular events occur during apoptosis and several genes appear to be involved. Particularly in lymphocytes, apoptosis plays an important physiological role. Our data demonstrate that similar oxidative stressors induce apoptosis in mature human lymphocytes as in neurons. In addition, first evidence indicates that susceptibility to apoptosis is altered in lymphocytes from AD patients compared to non-demented controls. Our preliminary findings suggest that changes of the individual sensitivity to undergo cellular apoptosis are already detectable in lymphocytes from AD patients, probably as a consequence of genetic as well as other risk factors. Therefore, this biochemical marker might have the potential for identifying individuals at risk of the diseases.

Aged↗
PubMed1998

Phylogenetic position of the Hexactinellida within the phylum Porifera based on the amino acid sequence of the protein kinase C from Rhabdocalyptus dawsoni.

Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin. However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida. We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni. The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses. Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R. dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later. The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge. This finding was also confirmed by comparing the regulatory part of the kinase gene. We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges.

Amino Acid Sequence↗
PubMed1998

Identification and expression of the SOS response, aidB-like, gene in the marine sponge Geodia cydonium: implication for the phylogenetic relationships of metazoan acyl-CoA dehydrogenases and acyl-CoA oxidases.

Sponges (Porifera) are the phylogenetically oldest metazoan organisms. From one member of the siliceous sponges, Geodia cydonium, the cDNA encoding a putative SOS protein, the AidB-like protein of the Ada system from bacteria, was isolated and characterized. The cDNA, GCaidB, comprises an open reading frame of 446 amino acid (aa) residues encoding a polypeptide with a calculated Mr of 49,335. This molecule shows high similarity to the bacterial AidB proteins from Mycobacterium tuberculosis and Escherichia coli and somewhat lower similarities to acyl-CoA dehydrogenases (ADHs) and acyl-CoA oxidases (AOXs). Northern blot analysis confirmed the presence of the complete transcript. The deduced sponge aa sequence, GC_aidB, possesses the two characteristic acyl-CoA dehydrogenase signatures 1 and 2. Incubation of the sponge with N-methyl-N'-nitro-N-nitrosoguanidine causes a strong increase in the 2.1-kb large transcript of GCaidB; maximal expression is seen after 24 h of incubation with this DNA methylating agent. ADHs and AOXs can be grouped, depending on the position of the catalytically important Glu residue, into the Glu-Gly (Glu adjacent to Gly) class and the Glu-Arg (Glu adjacent to Arg) class. The phylogenetically oldest metazoan AidB-like molecule, GC_aidB of G. cydonium, belongs to the Glu-Gly class of ADHs. Phylogenetic analyses of the Glu-Gly class enzymes, with the described AidB-like protein from G. cydonium and the bacterial AidB polypeptides, together with metazoan ADHs and AOXs, revealed that the AidB(-like) proteins diverged first from a common ancestor, while the eukaryotic AOX and ADA polypeptides as well as the GHDs appeared later. According to the analyses, the very long-chain ADHs are older than the medium-chain, short-chain, and branched-chain ADHs. Inclusion of the phylogenetical oldest member of the Glu-Arg class of enzymes, the bacterial ADH-CaiA sequence in these analyses, revealed that this class of enzymes appeared later in evolution and arose from the Glu-Gly class perhaps after gene duplication.

Acyl-CoA Dehydrogenase↗
PubMed1998

Origin of Metazoa: sponges as living fossils.

The phylogenetic position of the phylum Porifera (sponges) is at the base of the kingdom Metazoa. During the past few years not only rDNA sequences but--and this was a major advance--even cDNAs/genes have been isolated and characterized from sponges, especially from the marine demosponge Geodia cydonium, which code for proteins. The analyses of their deduced amino acid sequences allowed a molecular biological approach to solve the problem of monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin, metabotropic glutamate receptor), and homologs/modules of an immune system (immunoglobulin like molecules, scavenger receptor cysteine-rich, and short consensus repeats, rhesus system) classify the Porifera as true Metazoa. As living fossils, provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion as well as processes of signal transduction as known in a more complex manner from higher Metazoa, they also show peculiarities not known in other metazoan phyla. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as model will not only help to understand the evolution of Protoctista to Metazoa but also the complex, hierarchial regulatory network of cells in higher Metazoa.

Amino Acid Sequence↗