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Biomedical subjects

M Schweizer

Publications and source records attributed to M Schweizer.

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

Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA).

The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non-denaturing gels. Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-specific competitor or gel concentration and/or composition. We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp1 (stimulatory protein 1), NF-Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used. Furthermore, we show the influence of glycine concentration on Sp1 binding using recombinant glutathione S-transferase-Sp1 fusion protein expressed in Escherichia coli.

Base Sequence↗
PubMed2001

Two rat brain staufen isoforms differentially bind RNA.

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

Alternative Splicing↗
PubMed2001

Characterization of the promoter of PRS1 in Saccharomyces cerevisiae identifies three regions potentially involved in control of expression.

The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at -179 bp. Measurement of beta-galactosidase activity of the successively deleted PRS1 promoter linked to lacZ and integrated at the ura3 locus defined three DNA regions involved in the control of PRS1 expression. Gel shift analysis confirmed the data.

Base Sequence↗
PubMed2001

Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Both noncytopathic (ncp) and cytopathic (cp) biotypes of BVDV can be isolated from persistently infected cattle suffering from the lethal mucosal disease. The cp biotype correlates with the production of the NS3 nonstructural protein, which in the corresponding ncp biotype is present in its uncleaved form, NS23. Previously, we have shown that cp but not ncp BVDV induces the formation of alpha/beta interferons in bovine macrophages. In this study, we demonstrate that ncp BVDV inhibits the induction of apoptosis and the expression of interferon alpha/beta by poly(IC), a synthetic double-stranded RNA (dsRNA). Inhibition was observed only in cells which had been infected with ncp BVDV at least 12 h prior to the addition of dsRNA, which indicates that expression of viral proteins is necessary for the ncp virus to inhibit the effects of poly(IC). Additional experiments using transfected poly(IC) showed that ncp BVDV interfered with the intracellular action of dsRNA rather than with its uptake into the cells. Infected cells were not resistant to induction of apoptosis by actinomycin D or staurosporine, which suggests that ncp BVDV may specifically interfere with signaling through dsRNA. Interference with the innate antiviral host responses may explain the successful establishment of persistent infection by ncp BVDV in fetuses early in their development.

Animals↗
PubMed2001

The FIRE3-mediated sterol response of the FAS promoter requires NF-Y/CBF as a coactivator.

The transcription of the fatty acid synthase (FAS) gene is regulated by the sterol status of the cell via cleavage of the sterol regulatory element-binding protein (SREBP). When human HepG2 hepatoma cells were cotransfected with an expression plasmid for mature SREBP-1a together with FAS promoter/reporter constructs significant increases in reporter activity were observed. Deletion analysis of the FAS promoter between -151 and -52 relative to the transcription start site pinpoint two cis-elements important in sterol regulation of the FAS gene. One element, FIRE3, between -71 and -52 can bind in vitro translated and transcribed SREBP-1a whereas the other element, the inverted CCAAT element ICE(-97/-92), binds the trimeric transcription factor NF-Y/CBF as shown with rat liver extract and reconstituted, recombinant NF-Y. The results clearly show that the coactivator for SREBP-1a in this cell line is NF-Y. This finding was confirmed by using a dominant negative form of NF-YA, NF-YAm29, which interferes with the effect of ectopically expressed SREBP-1a on FAS reporter activity.

Base Sequence↗
PubMed2000

Muscarinic acetylcholine receptors induce the expression of the immediate early growth regulatory gene CYR61.

In brain, muscarinic acetylcholine receptors (mAChRs) modulate neuronal functions including long term potentiation and synaptic plasticity in neuronal circuits that are involved in learning and memory formation. To identify mAChR-inducible genes, we used a differential display approach and found that mAChRs rapidly induced transcription of the immediate early gene CYR61 in HEK 293 cells with a maximum expression after 1 h of receptor stimulation. CYR61 is a member of the emerging CCN gene family that includes CYR61/CEF10, CTGF/FISP-12, and NOV; these encode secretory growth regulatory proteins with distinct functions in cell proliferation, migration, adhesion, and survival. We found that CYR61, CTGF, and NOV were expressed throughout the human central nervous system. Stimulation of mAChRs induced CYR61 expression in primary neurons and rat brain where CYR61 mRNA was detected in cortical layers V and VI and in thalamic nuclei. In contrast, CTGF and NOV expression was not altered by mAChRs neither in neuronal tissue culture nor rat brain. Receptor subtype analyses demonstrated that m1 and m3 mAChR subtypes strongly induced CYR61 expression, whereas m2 and m4 mAChRs had only subtle effects. Increased CYR61 expression was coupled to mAChRs by both protein kinase C and elevations of intracellular Ca(2+). Our results establish that CYR61 expression in mammalian brain is under the control of cholinergic neurotransmission; it may thus be involved in cholinergic regulation of synaptic plasticity.

Animals↗
PubMed2000

KCNQ4, a K+ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway.

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, an autosomal dominant form of progressive hearing loss in humans. In the mouse cochlea, the transcript has been found exclusively in the outer hair cells. By using specific antibodies, we now show that KCNQ4 is situated at the basal membrane of these sensory cells. In the vestibular organs, KCNQ4 is restricted to the type I hair cells and the afferent calyx-like nerve endings ensheathing these sensory cells. Several lines of evidence suggest that KCNQ4 underlies the I(K,n) and g(K,L) currents that have been described in the outer and type I hair cells, respectively, and that are already open at resting potentials. KCNQ4 is also expressed in neurons of many, but not all, nuclei of the central auditory pathway, and is absent from most other brain regions. It is present, e.g., in the cochlear nuclei, the nuclei of the lateral lemniscus, and the inferior colliculus. This is the first ion channel shown to be specifically expressed in a sensory pathway. Moreover, the expression pattern of KCNQ4 in the mouse auditory system raises the possibility of a central component in the DFNA2 hearing loss.

Amino Acid Sequence↗
PubMed2000

Prognosis of extrahepatic bile-duct atresia after hepatoportoenterostomy.

Clinical and histologic findings from 206 patients operated upon for extrahepatic biliary atresia (EHBA) are analyzed in order to define the prognosis of patients with EHBA. The prospective study took into consideration both initial fibrosis of the liver and the morphology of the porta hepatis (PH) at surgery. Kaplan-Meier survival estimates and statistical calculations demonstrated a relationship between long-term survival and histologic findings in the liver and porta hepatis. The efficacy of HPE is significantly influenced by the morphology of the PH and to a lesser extent by the initial liver fibrosis. Surgery should thus achieve pattern 1 morphology of the PH, but this is problematic because of the close relationship of the vascular and biliary structures in its two lateral zones.

Biliary Atresia↗
PubMed2000

Bovine viral diarrhoea virus and bovine herpesvirus-1 prime uninfected macrophages for lipopolysaccharide-triggered apoptosis by interferon-dependent and -independent pathways.

The flavivirus bovine viral diarrhoea (BVD) virus exists in two biotypes, cytopathic (cp) and non-cytopathic (ncp), defined by their effect on cultured cells. Cp BVD virus-infected cells undergo apoptosis and may promote apoptosis in uninfected cells by an indirect mechanism. Macrophages (Mφ) infected with cp, but not ncp, BVD virus release a factor(s) in the supernatant capable of priming uninfected Mφ for activation-induced apoptosis in response to lipopolysaccharide. A possible role of interferon (IFN) type I was suggested previously by the observation that this cytokine primed for activation-induced apoptosis and was present in supernatants of Mφ infected with cp, but not ncp, BVD virus. Here, supernatants of both Mφ infected with a wider range of cp BVD virus and Mφ infected with bovine herpesvirus-1 are shown to contain such priming activity. Two lines of evidence indicate that factors in addition to IFN type I prime uninfected Mφ for apoptosis. First, supernatants of Mφ infected with cp BVD virus contained much less IFN than is required for priming for apoptosis. Second, whereas antiviral activity was neutralized by a vaccinia virus-encoded IFN type I receptor, B18R, the capacity of the supernatant to prime for apoptosis was unaffected by this treatment. The apparent molecular mass of the factor(s) priming for apoptosis was between 30 and 100 kDa. Priming of uninfected cells for activation-induced apoptosis may add a new facet to virus pathogenesis and may contribute to the formation of lesions not related directly to virus replication.

Animals↗
PubMed2000

Dependence of rat spot14 promoter activity on NF-Y binding to the inverted CCAAT-element at -100.

Electrophoretic mobility shift assay (EMSA) and in vitro transcription/translation show that NF-Y binds to the inverted CCAAT-element in the promoter of the rodent spot14 gene. The NF-Y-binding sequence has been shown to be responsible for basal activity in H4IIE. Given the similar role found for the inverted CCAAT-element in the promoter of the FAS gene, NF-Y may have an important function in the control of lipogenesis.

Animals↗
PubMed1999

Development of a yeast-based assay system for monitoring microsatellite instability.

Simple sequence repeats (microsatellites) are found in all eukaryotic genomes. Instabilities within these sequences have been associated with several human disorders including Huntington's chorea and myotonic dystrophy. Further studies have identified links between microsatellite instability, faulty mismatch repair and certain human cancers, in particular a form of hereditary colorectal cancer. The assay system described here consists of a congenic set of yeast strains mutated in DNA replication and mismatch repair genes and assay plasmids with which it is possible to measure differences in microsatellite stability in the range of 5-850-fold. The development of this technology will allow monitoring of environmental and dietary influences on the genomic stability in the context of human disease.

Base Pair Mismatch↗
PubMed1999

Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1.

The regulation of the rat fatty acid synthase gene by mediators such as diet, hormones, cAMP, sterols or retinoic acid is controlled by three NF-Y binding sites. All three sites have a neighbouring Sp1-binding GC-box. This NF-Y/Sp1 motif is conserved in the FAS promoters of rat, human, goose and chicken. We have previously shown cooperative binding of NF-Y and Sp1 to the promoter region at -500 coincident with a diet-induced DNAse I-hypersensitive site. Here, we show an in-vivo interaction of NF-YA with Sp1 using the yeast two-hybrid system. The interacting domains are located between amino acids 55 and 139 of the NF-Y subunit NF-YA and between amino acids 139 and 344 of Sp1. In addition, we show by co-immunoprecipitation direct interaction of NF-Y subunit NF-YA with Sp1 in extracts of rat hepatoma cells H4IIE. Furthermore, we demonstrate by the GST pull-down assay that NF-YA interacts physically with Sp1 in-vitro in the absence of DNA. Therefore, NF-Y can be added to the list of transcription factors interacting with Sp1.

Animals↗
PubMed1999

Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP.

In animals and several cellular models of synaptic plasticity, long-lasting changes in synaptic strength are dependent on gene transcription and translation. Here we demonstrate that Pim-1, a serine/threonine kinase closely related to Pim-2 and Pim-3, is induced in hippocampus in response to stimuli that evoke long-term potentiation (LTP). Mice deficient for Pim-1 show normal synaptic transmission and short-term plasticity. However, they fail to consolidate enduring LTP even though Pim-2 and Pim-3 are constitutively expressed in the hippocampus and Pim-3 expression is similarly induced by synaptic activity. Thus, expression of Pim-1 is required for LTP. Its level of expression and, consequently, its capacity to phosphorylate target proteins in dendritic and nuclear compartments of stimulated neurons might be a determining factor for the establishment of long-lasting changes in synaptic strength.

Animals↗
PubMed1999

Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae.

The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).

Gene Deletion↗
PubMed1999

Sites of simian foamy virus persistence in naturally infected African green monkeys: latent provirus is ubiquitous, whereas viral replication is restricted to the oral mucosa.

Foamy viruses (FV), retroviruses of the genus Spumavirus, are able to infect a wide variety of animal species and replicate in nearly all types of cultured cells. To identify the cells targeted by FV in the natural host and define the sites of viral replication, multiple organs of four African green monkeys naturally infected with simian FV type 3 were investigated for the presence of FV proviral DNA and viral transcripts. All organs contained significant amounts of FV proviral DNA. In addition to proviruses containing the complete transactivator gene taf, proviral genomes carrying a specific 295-bp deletion in the taf gene were detected in all monkeys. As in the case of human foamy virus the deletion leads to the formation of the bet gene that is regarded to be instrumental in the regulation of viral persistence. FV RNA was detected by RT-PCR and in situ hybridization only in the oral mucosa of one monkey. No other samples contained detectable levels of viral transcripts. Histopathological changes were not observed in any of the tissue samples analyzed. Our results show that the natural history of FV is characterized by latent infection in all organs of the host and by minimal levels of harmless viral replication in the oral mucosa. The broad host cell range in vivo further encourages the development of FV-derived vectors for therapeutic gene delivery.

Animals↗
PubMed1999

A bioassay for interferon type I based on inhibition of Sendai virus growth.

The expression of type I interferons (IFNs) in eukaryotic cells represents a first line of defense against viral infection. Cells pretreated by IFNs do not support viral replication and are protected from virus-induced cell destruction. A challenge of IFN-pretreated cells with vesicular stomatitis virus (VSV) is frequently used to quantitate this cytokine because, on the one hand, the replication of VSV is highly sensitive to IFNs and, on the other hand, in unprotected cells this virus induces a rapid cytopathic effect that can readily be quantified. However, as VSV may infect humans and is known to cause severe disease in a variety of animal species, this virus must be considered a biohazard. In this paper, we describe a bioassay for bovine IFN using Sendai virus, a paramyxovirus that grows readily in MDBK cells yet is released from these cells in a non-infectious form. The sensitivity and dynamic range of this assay are similar to those of the popular VSV-based IFN assay. We demonstrate that the Sendai-virus-based IFN assay permits rapid quantitation of recombinant bovine type I IFN, and also of native type I IFNs which are present in the supernatants of monocyte-derived macrophages infected with various pathogens. In view of the possible artifacts induced by viruses in samples to be assayed for IFN activity, we evaluated several methods of virus inactivation. Treatment with beta-propiolactone led to virus inactivation without affecting the bioactivity of IFNs as detected in the Sendai-virus-based assay.

Animals↗
PubMed1999

Isotopic ((13)C) fractionation during plant residue decomposition and its implications for soil organic matter studies.

Carbon isotopic fractionations in plant materials and those occurring during decomposition have direct implications in studies of short-and longer-term soil organic matter dynamics. Thus the products of decomposition, the evolved CO(2) and the newly formed soil organic matter, may vary in their (13)C signature from that of the original plant material. To evaluate the importance of such fractionation processes, the variations in (13)C signatures between and within plant parts of a tropical grass (Brachiaria humidicola) and tropical legume (Desmodium ovalifolium) were measured and the changes in (13)C content (signatures) during decomposition were monitored over a period of four months. As expected the grass materials were less depleted in (13)C (-11.4 to -11.9 per thousand) than those of the legume (-27.3 to -25.8 per thousand). Root materials of the legume were less (1.5 per thousand) depleted in (13)C compared with the leaves. Plant lignin-C was strongly depleted in (13)C compared with the bulk material by up to 2.5 per thousand in the legume and up to 4.7 per thousand in the grass. Plant materials were subsequently incubated in a sand/nutrient-solution/microbial inoculum mixture. The respiration product CO(2) was trapped in NaOH and precipitated as CaCO(3), suitable for analysis using an automated C/N analyser coupled to an isotope ratio mass spectrometer. Significant depletion in (13)C of the evolved CO(2) was observed during the initial stages of decomposition probably as a result of microbial fractionation as it was not associated with the (13)C signatures of the measured more decomposable fractions (non-acid detergent fibre and cellulose). While the cumulative CO(2)-(13)C signatures of legume materials became slightly enriched with ongoing decomposition, the CO(2)-C of the grass materials remained depleted in (13)C. Associated isotopic fractionation correction factors for source identification of CO(2-)C varied with time and suggested errors of 2-19% in the estimation of the plant-derived C at 119 days of incubation in a soil of an intermediate (-20.0 per thousand) (13)C signature. Analysis of the residual material after 119 days of incubation showed little or no change in the (13)C signature partly due to the incomplete decomposition at the time of harvesting. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗