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M Schweizer

Publications and source records attributed to M Schweizer.

At least 55 records · Page 3Linked to original sources
PubMed1999

FIRE3 in the promoter of the rat fatty acid synthase (FAS) gene binds the ubiquitous transcription factors CBF and USF but does not mediate an insulin response in a rat hepatoma cell line.

Several putative insulin-responsive elements (IRE) in the fatty acid synthase (FAS) promoter have been identified and shown to be functional in adipocytes and hepatocytes. Here we report on the insulin-responsiveness in the rat hepatoma cell line H4IIE of four cis-elements in the FAS promoter: the FAS insulin-responsive elements, FIRE2 and FIRE3; the inverted CCAAT element, ICE; and the insulin/glucose-binding element, designated hepatic FIRE element, hFIRE, originally identified in rat hepatocytes. Using electrophoretic mobility shift assay (EMSA) competition experiments together with supershifts and in vitro transcription/translation we show that FIRE3 (-68/-58) binds not only the upstream stimulatory factors USF-1/USF-2 but also the CCAAT-binding factor CBF, also known as the nuclear factor Y, NF-Y. The putative IRE FIRE2, which shows sequence similarity to FIRE3, is located between -267 and -249. Gel retardation experiments indicate that USF-1 and USF-2 also bind to this element, which contains an imperfect E-box motif. Using the same approach we have shown that hFIRE binds the stimulatory proteins Sp1 and Sp3 in addition to CBF. Transient transfection experiments using FAS promoter constructs deleted for FIRE2 and FIRE3 demonstrate that neither of these elements mediates the insulin response of the FAS promoter in the rat hepatoma cell line H4IIE, however, ICE at -103/-87 is responsible for mediating the effect of the insulin antagonist cAMP. The hFIRE element located at -57/-34, in spite of its role in the glucose/insulin response in primary rat hepatocytes, is apparently not involved in the insulin regulation of the rat FAS promoter in H4IIE cells. The fact that the topology of the promoters of the FAS genes in rat, human, goose and chicken is conserved regarding CBF-binding sites and USF-binding sites implies an important role for these ubiquitously expressed transcription factors in the regulation of the FAS promoter.

Animals↗
PubMed1999

Activity-dependent formation of perforated synapses in cultured hippocampal neurons.

The study investigated the formation of perforated synapses in rat hippocampal cell cultures. Perforated synapses are defined by their discontinuous postsynaptic densities (PSDs) and are believed to occur in parallel with changes in synaptic activity and possibly also synaptic efficacy. Several in vivo studies have demonstrated an increase in the frequency of perforated synapses induced by development and environmental stimulation as well as long-term potentiation (LTP). Also in in vitro brain slices, LTP was associated with an elevated number of perforated spine synapses. Our study demonstrated for the first time that the formation of perforated synapses can be induced by a short-term increase in spontaneous neural activity in a hippocampal cell culture model. Stimulation with the GABAA-antagonist picrotoxin (PTX) induced a significant increase in the percentage of perforated synapses. This strong increase was blocked when APV was added together with PTX, indicating that the formation of perforated synapses depended on the activation of NMDA receptors. We also showed that inhibition of the tissue type plasminogen activator (tPA-stop/PAI-1) significantly interfered with the activity-induced increase in perforated synapses. This implies that the proteolytic activities of tPA might be involved in steps which are downstream from the NMDA receptor-mediated synaptic plasticity leading to structural changes at synaptic contacts. In contrast, even long-term inhibition of electrical network activity by tetrodotoxin had no effect on the number of perforated synapses, but almost completely abolished the formation of spine synapses. These results indicate that a short-term increase in neural activity via NMDA receptors and a proteolytic cascade involving tPA lead to the formation of perforated synapses.

2-Amino-5-phosphonovalerate↗
PubMed1999

Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis.

Bovine viral diarrhoea virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae. Both a noncytopathic (ncp) and an antigenically related cytopathic (cp) BVDV can be isolated from persistently infected animals suffering from mucosal disease. In every case studied so far, the genomic changes leading to the cp biotype correlate with the production of the NS3 nonstructural protein, which, in the ncp biotype, is present in its uncleaved form, NS23. This report shows that, in contrast to ncp BVDV, the cp biotype induces apoptosis in cultured embryonic bovine turbinate cells. Early in the process of apoptosis, cells show a rise in the intracellular level of reactive oxygen species, which is indicative of oxidative stress. This precedes two hallmarks of apoptosis, caspase activation as shown by cleavage of the caspase substrate poly(ADP-ribose) polymerase, and DNA fragmentation. Cells were protected from apoptosis only by certain antioxidants (butylated hydroxyanisole and ebselen), whereas others (N-acetylcysteine, pyrrolidine dithiocarbamate, lipoic acid, dihydrolipoic acid and tiron) turned out to be ineffective. Antioxidants that protected cells from apoptosis prevented oxidative stress but failed to block virus growth. These observations suggest that oxidative stress, which occurs early in the interaction between cp BVDV and its host cell, may be a crucial event in the sequence leading to apoptotic cell death. Hence, apoptosis is not required for the multiplication of the cp biotype of BVDV.

Animals↗
PubMed1999

An active foamy virus integrase is required for virus replication.

Foamy viruses (FVs) make use of a replication strategy which is unique among retroviruses and shows analogies to hepadnaviruses. The presence of an integrase (IN) and obligate provirus integration distinguish retroviruses from hepadnaviruses. To clarify whether a functional IN is required for FV replication, a mutant in the highly conserved DD35E motif of the active centre was analysed. This mutant was found to be able to express Gag and Pol protein precursors and cleavage products and to generate and deliver cDNA. However, this mutant was replication-deficient. The junctions of individual foamy proviruses with cellular DNA were sequenced. The findings suggest that FV integration is asymmetrical, because the proviruses started with what is believed to be the U3 end of the free linear DNA to generate the conventional TG dinucleotide, while apparently two nucleotides from the U5 end were cleaved to create the complementary CA dinucleotide. Alignment of known FV genome sequences indicated that this mechanism of integration is not restricted to the two FV isolates from which integrates were studied, but appears to be a common feature of this retrovirus subfamily. In conclusion, with respect to the necessity of a functionally active IN for virus replication FVs behave like other retroviruses; their mechanism of integration, however, is probably unique.

Animals↗
PubMed1999

Genetic stability of foamy viruses: long-term study in an African green monkey population.

The genetic variability of the envelope surface domain (SU) of simian foamy virus (FV) of African green monkeys was studied. To assess the interindividual diversity of FV, isolates were obtained from 19 animals living together in a monkey house. The monkeys had been imported from Kenya prior to being placed in long-term housing in the research institute. In addition, a simian FV isolate and proviral DNA were obtained from an animal caretaker infected in this setting. DNA of the complete SU (1779 to 1793 bp) was analyzed by PCR and sequencing. The sequences revealed four clusters with high homologies (>95%). Between the clusters, divergencies ranged from 3 to 25%. Obviously, the clusters reflect four different strains or subtypes of simian FV type 3 that were prevalent in the colony. In contrast to lentiviruses, hypervariable regions could not be detected in the FV SU. Furthermore, to analyze the intraindividual diversity of FV, we investigated the virus population within an individual monkey at a given time point and its evolution over 13 years. For this purpose, 22 proviral SU clones generated by PCR from one oral swab and seven isolates obtained from the same animal between 1982 and 1995 were examined. These sequences revealed exceptionally high homology rates (99.5 to 100%), and only a minimal genetic drift was recognized within the series of isolates. In conclusion, the low in vivo divergency of FV SU suggests that genetic variability is not important for the maintenance of FV persistence.

Amino Acid Sequence↗
PubMed1999

Gamma interferon is a major suppressive factor produced by activated human peripheral blood lymphocytes that is able to inhibit foamy virus-induced cytopathic effects.

The activation of human peripheral blood lymphocytes by mitogens or by triggering the T-cell receptor with anti-CD3 antibodies leads to the production of a potent soluble inhibitory activity against foamy virus-induced cytopathic effects in vitro. The inhibitory activity acts in a species-specific manner. As a consequence, the isolation of foamy viruses from blood lymphocytes of infected humans is accelerated in a heterologous coculture system. Antibodies against gamma interferon (IFN-gamma) are able to suppress most of the inhibitory activity, suggesting that IFN-gamma is the dominant component.

3T3 Cells↗
PubMed1999

Supernatants of virus-infected macrophages prime uninfected macrophages for lipopolysaccharide-induced apoptosis by both an interferon-dependent and an independent mechanism.

Virus-infected macrophages (M phi) release factors priming uninfected M phi for LPS-induced apoptosis. This was shown for bovine M phi infected with cytopathogenic bovine virus diarrhea (BVD) virus or with bovine herpes virus-1 (BHV-1) but not for M phi infected with noncytopathogenic BVD virus. The former two viruses also induced interferon type I in M phi, which also primes macrophages for LPS-induced apoptosis. However, several lines of evidence suggested that virus-infected M phi produce IFN-independent factors priming for apoptosis. For example, a soluble IFN type I receptor abrogated the antiviral activity, but not the ability of supernatants to prime uninfected M phi for LPS-induced apoptosis. It is suggested that the production of factors priming uninfected cells for apoptosis plays a part in virus-induced pathogenesis, e.g. in cattle afflicted with mucosal disease, or with infectious bovine rhinotracheitis, or in viral diseases inducing a marked lymphopenia.

Animals↗
PubMed1999

A high affinity binding site for the polypyrimidine tract binding protein (PTB) is located in the 5'-untranslated region of the rat proteinase alpha1-inhibitor 3 variant I gene.

As a first step towards understanding the mechanism underlying the differential gene expression of the two variants of the rat proteinase-inhibitor alpha1-inhibitor 3 (alpha1-I3) corresponding genomic clones were isolated. The 100% similarity between the sequence of one genomic clone and that of the alpha1-13 variant I cDNA strongly suggested that its 5'-sequence represented the upstream region of the corresponding gene. Several putative cis-regulatory elements were identified as well as a polypyrimidine tract located between the transcription start site of the alpha1-I3 variant I mRNA and the AUG codon. The polypyrimidine tract functions as a positive cis-element in a heterologous promoter. By electrophoretic mobility shift assays (EMSA) we have shown that a GST (glutathione S-transferase) fusion of the rat polypyrimidine tract binding protein (PTB) has a high affinity for the pyrimidine-rich sense strand but not for the complementary sequence of the 5'-untranslated region of the alpha1-I3 variant I gene.

5' Untranslated Regions↗
PubMed1998

Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation.

Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

Acetylene↗
PubMed1998

Construction of PCR-ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV.

Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homology method (Wach et al., 1994). Transformants were selected as Geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418-resistant marker. We have developed a widely applicable method for the construction of gap repair plasmids to obtain the cognate clones for each of the disrupted ORFs. The 5'- and 3'-flanks of the ORF in question are linked by a unique restriction endonuclease. When the plasmid is cut at this site it can be used to obtain, by selection for the appropriate antibiotic resistance, long flanking homology (LFH) cassettes containing the cognate clone or the disrupted allele. The LFH cassette containing the cognate clone or the disrupted allele can be released from the gap-repaired plasmid by cutting at the inserted flanking restriction sites. One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex. Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non-essential ORFs were subjected to growth test on different media at 15 degrees C, 30 degrees C and 37 degrees C. Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37 degrees C and the product of YOL091w appears to play a role in sporulation since the homozygous diploid disruptant has lost the ability to sporulate.

Anti-Bacterial Agents↗
PubMed1998

Heterologously expressed acyl carrier protein domain of rat fatty acid synthase functions in Escherichia coli fatty acid synthase and Streptomyces coelicolor polyketide synthase systems.

INTRODUCTION: Fatty acid synthases (FASs) catalyze the de novo biosynthesis of long-chain saturated fatty acids by a process common to eubacteria and eukaryotes, using either a set of monofunctional proteins (Type II FAS) or a polypeptide containing several catalytic functions (Type I FAS). To compare the features of a Type I domain with its Type II counterpart we expressed and characterized an acyl carrier protein (ACP) domain of the Type I rat FAS. RESULTS: An ACP domain of rat FAS was defined that allows expression of a small percentage of active holo-ACP both in Escherichia coli, increasing fivefold upon co-expression with an E. coli holo-ACP synthase, and in Streptomyces coelicolor. The rat ACP domain functions with some components of the E. coli FAS, and can replace the actinorhodin polyketide synthase (PKS) ACP in S. coelicolorA3(2). Purification of the rat ACP domain from E. coli resulted in loss of its functionality. Purified apo-ACP could be converted to its holo-form upon incubation with purified E. coli holo-ACP synthase in vitro, however, suggesting that the loss of functionality was not due to a conformational change. CONCLUSIONS: Functionality of the recombinant rat ACP was shown in distantly related and diverse enzyme systems, suggesting that Type I and Type II ACPs have a similar conformation. A procedure was described that might permit the production of rat FAS holo-ACP for structural and further biochemical characterization.

Acyl Carrier Protein↗
PubMed1998

Identification of a human population infected with simian foamy viruses.

Studying the transmission of simian retroviruses to humans can help define the importance of these infections to public health. We identified a substantial prevalence (4/231, 1.8%) of infection with simian foamy viruses (SFV) among humans occupationally exposed to nonhuman primates. Evidence of SFV infection included seropositivity, proviral DNA detection and isolation of foamy virus. The infecting SFV originated from an African green monkey (one person) and baboons (three people). These infections have not as yet resulted in either disease or sexual transmission, and may represent benign endpoint infections.

Animals↗
PubMed1998

PRS5, the fifth member of the phosphoribosyl pyrophosphate synthetase gene family in Saccharomyces cerevisiae, is essential for cell viability in the absence of either PRS1 or PRS3.

In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a phosphoribosyl pyrophosphate binding site. We regard YOL061w as the fifth member of the PRS gene family, PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Deltaprs5 strains are not affected in growth or in the size of their nucleotide pools. However, simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 rendered the strains inviable, which implies that PRS5 plays an important role in the maintenance of PRS function in S. cerevisiae.

Base Sequence↗
PubMed1997

Cooperative binding of NF-Y and Sp1 at the DNase I-hypersensitive site, fatty acid synthase insulin-responsive element 1, located at -500 in the rat fatty acid synthase promoter.

In vitro DNase I footprint analysis of the rat fatty acid synthase (FAS) promoter from -568 to -468 revealed four protein binding sites: A, B, and C boxes and the FAS insulin-responsive element 1 (FIRE1). As demonstrated by gel mobility shift analysis and supershift experiments, FIRE1, located between -516 and -498, is responsible for binding NF-Y. The C box located downstream of FIRE1 was shown by in vitro footprinting to be a Sp1 binding site, and furthermore, competition with Sp1 also abolished FIRE1 binding. Since the half-life of the Sp1.NF-Y.DNA complex is significantly longer than the half-lives of the Sp1.DNA or NF-Y.DNA complexes, the two transcription factors are deemed to bind cooperatively in the FAS promoter at -500. It is unusual that NF-Y binds at this distance from the start site of transcription. NF-Y binding sites are found in the promoters of at least three other FAS genes, viz. goose, chicken, and man. A second NF-Y binding site is located in the FAS promoter at the more usual position of -103 to -87, and it too has a neighboring Sp1 site. CTF/NF-1 competes for proteins binding to the B box. The A box binds Sp1 and contains a 12/13 match of the inverted repeat sequence responsible for binding the nuclear factor EF-C/RFX-1 in the enhancer regions of hepatitis B virus and the major histocompatibility complex class II antigen promoter. The same relative positions of NF-Y and Sp1 binding sites in the promoters of FAS genes of goose, rat, chicken, and man emphasize the involvement of these transcription factors in the diet and hormonal regulation of FAS.

Animals↗
PubMed1997

Afferent innervation influences the development of dendritic branches and spines via both activity-dependent and non-activity-dependent mechanisms.

The present investigation uses an in vitro co-culture system to study the role of afferent innervation in early development and differentiation of hippocampal neurons. Our experiments indicate that the formation of two key morphological features, dendritic branches and dendritic spines, is induced by afferent innervation. Hippocampal neurons develop multiple dendritic branches and spines only when extensively innervated by living axonal afferents. No morphological changes occurred when hippocampal neurons were plated on other cell surfaces such as fixed axons or astrocytes. Furthermore, afferents exerted their effect locally on individual dendrites that they contacted. When one portion of the dendritic arbor of a neuron was contacted by afferents and the other portion was not, morphological effects were restricted to the innervated dendrites. Innervation of some of the dendrites on a neuron did not produce global effects throughout the neuron. Afferent-induced dendritic branching is independent of activity, since branch induction was unaffected by chronic application of TTX or glutamate receptor blockers. In contrast, the formation of dendritic spines is influenced by activity. The number of developing spines was reduced when TTX or a cocktail of three glutamate receptor blockers was applied. Blockade of individual AMPA, NMDA, or metabotropic glutamate receptors did not affect the number of spines. These results, taken together, demonstrate that afferents can have a prominent influence on the development of postsynaptic target cells via both activity-dependent and non-activity-dependent mechanisms, indicating the presence of multiple signals. Accordingly, this suggests an important interplay between pre- and postsynaptic elements early in development.

Animals↗
PubMed1997

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell.

Amino Acid Sequence↗
PubMed1997

NF-Y binds to the inverted CCAAT box, an essential element for cAMP-dependent regulation of the rat fatty acid synthase (FAS) gene.

Using EMSA competition experiments together with supershifts and in vitro transcription/translation we show that the basal transcription factor NF-Y or a related factor binds to the cAMP-responsive inverted CCAAT box recently identified in the rat fatty acid synthase (FAS) gene from nucleotide -99 to -92 relative to the transcription start site of the FAS mRNA. This result indicates a putative novel role for NF-Y in the cAMP-dependent gene regulation in a small class of genes such as FAS and tryptophan hydroxylase. Since NF-Y is a constitutively produced factor, not surprisingly, no differences in the specific DNA/protein complex with the CCAAT(FAS) box and nuclear proteins from H4IIE cells treated with cAMP and/or insulin or not could be observed. This implies that NF-Y might be modified in response to cAMP or might interact with another factor whose properties are altered by cAMP.

Animals↗