Biomedical subjects

B Lüscher

Publications and source records attributed to B Lüscher.

At least 37 records · Page 2Linked to original sources

PubMed2000

Behavioural changes produced by transgenic overexpression of gamma2L and gamma2S subunits of the GABAA receptor.

Transgenic mice overexpressing either the mouse gamma2L or gamma2S subunit of the GABAA receptor were generated in a C57BL/6 J x DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam- and ethanol-enhanced 36Cl- flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]-flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the gamma2L or gamma2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor gamma2 subunit available (regardless of whether it is gamma2L or gamma2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Animals↗

PubMed2000

Transcriptional activity of GLI1 is negatively regulated by protein kinase A.

The transcription factor GLI1 is one of three vertebrate members of the GLI family, which is characterized by a highly conserved DNA-binding domain of five zinc fingers. We have analyzed whether human GLI1 is a target of PKA regulation. It was found that PKA inhibits GLI1 transcriptional activity. However, no evidence for proteolytic processing or for alteration in the subcellular distribution of GLI1 was obtained. The responsive PKA site (aa333-336) was localized to the second zinc finger of GLI1. Mutation of Ser336 revealed that PKA could also stimulate GLI1 transcriptional activity. Thus, our data demonstrate both negative and positive regulation of human GLI1 by PKA.

Animals↗

PubMed1999

Postsynaptic clustering of gamma-aminobutyric acid type A receptors by the gamma3 subunit in vivo.

Synaptic localization of gamma-aminobutyric acid type A (GABA(A)) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The gamma2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABA(A) receptor subtypes. We now show that transgenic overexpression of the gamma3 subunit in gamma2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the gamma3 subunit can substitute for gamma2 in the formation of GABA(A) receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous gamma3 subunit, indicating that the factors present for clustering of gamma2 subunit-containing receptors are sufficient to cluster gamma3 subunit-containing receptors. The GABA(A) receptor and gephyrin-clustering properties of the ectopic gamma3 subunit were also observed for the endogenous gamma3 subunit, but only in the absence of the gamma2 subunit, suggesting that the gamma3 subunit is at a competitive disadvantage with the gamma2 subunit for clustering of postsynaptic GABA(A) receptors in wild-type mice.

Animals↗

PubMed1999

Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation.

The high risk human papillomavirus (HPV) type 16 E7 protein affects cell growth control and promotes transformation by interfering with functions of cellular proteins. A key target of E7 is the tumor suppressor protein p105RB. Although this interaction is required for E7-dependent transformation, other cellular molecules must also be involved, because some E7 mutants that have reduced transforming abilities still bind to p105RB. In order to identify additional proteins that interact with E7 and that may be responsible to mediate its transforming function, we have used the C-terminal half of E7 in a yeast two-hybrid screen. We identified the fork head domain transcription factor M phase phosphoprotein 2 (MPP2) as an interaction partner of E7. Specific interaction of the two proteins both in vitro and in vivo in mammalian cells was detected. The interaction of MPP2 with E7 is functionally relevant since MPP2 enhances the E7/Ha-Ras co-transformation of rat embryo fibroblasts. In addition HPV16 E7, but neither non-transforming mutants of HPV16 E7 nor low risk HPV6 E7, was able to stimulate MPP2-specific transcriptional activity. Thus, MPP2 is a potentially important target for E7-mediated transformation.

Animals↗

PubMed1999

Analysis of the Max-binding protein MNT in human medulloblastomas.

Medulloblastomas (MBs) are the most frequent malignant brain tumors in children. The molecular pathogenesis of these tumors is still poorly understood. Microsatellite and restriction-fragment-length polymorphism studies have revealed allelic loss of genetic material on the short arm of chromosome 17 in the region 17p13 in approximately 50% of MBs, suggesting the presence of a tumor-suppressor gene in this region. A candidate for this putative tumor-suppressor is the MNT gene, located at 17p13.3 and encoding a Max-interacting nuclear protein with transcriptional-repressor activity. In this study, we analyzed MNT mRNA and protein expression in 44 MB samples, including 32 primary tumors, 3 recurrent tumors and 9 MB cell lines. Allelic loss at 17p13.3 was found in 49% of informative cases. RT-PCR showed MNT mRNA expression in all cases analyzed. Endogenous Mnt protein with an apparent molecular weight of 72 to 74 kDa was detected in lysates from MB cell lines. The presence and functional integrity of Mnt in MBs were tested in electrophoretic mobility-shift assays. These experiments demonstrated that Mnt interacts with Max, and that this heterodimer binds DNA specifically, suggesting a functional bHLHZip domain of MB-derived Mnt. In support, single-strand conformation-polymorphism (SSCP) analyses revealed no mutation in the bHLHZip region. Deletion of the Mnt Sin3 interaction domain was shown to convert Mnt from an inhibitor of myc/ras-co-transformation into a molecule capable of cooperating with Ras in transformation. This region therefore was screened for mutation by SSCP: again, no alterations were found. These findings indicate that the MNT gene located at 17p13.3 is not likely to be involved in the molecular pathogenesis of MBs.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed1999

Posttranslational regulation of Myc function in response to phorbol ester/interferon-gamma-induced differentiation of v-Myc-transformed U-937 monoblasts.

The transcription factors of the Myc/Max/Mad network are important regulators of cell growth, differentiation, and apoptosis and are frequently involved in tumor development. Constitutive expression of v-Myc blocks phorbol ester (TPA)-induced differentiation of human U-937 monoblasts. However, costimulation with interferon-gamma (IFN-gamma) and TPA restores terminal differentiation and G1 cell-cycle arrest despite continuous expression of v-Myc. The mechanism by which TPA + IFN-gamma counteract v-Myc activity has not been unravelled. Our results show that TPA + IFN-gamma treatment led to an inhibition of v-Myc- and c-Myc-dependent transcription, and a specific reduction of v-Myc:Max complexes and associated DNA-binding activity, whereas the steady state level of the v-Myc protein was only marginally affected. In contrast, TPA + IFN-gamma costimulation neither increased the expression of Mad1 or other mad/mnt family genes nor altered heterodimerization or DNA-binding activity of Mad1. The reduced amount of v-Myc:Max heterodimers in response to treatment was accompanied by partial dephosphorylation of v-Myc and c-Myc. Phosphatase treatment of Myc:Max complexes lead to their dissociation, thus mimicking the effect of TPA + IFN-gamma. In addition to modulation of the expression of Myc/Max/Mad network proteins, posttranslational negative regulation of Myc by external signals may, therefore, be an alternative biologically important level of control with potential therapeutic relevance for hematopoietic and other tumors with deregulated Myc expression.

Antineoplastic Agents↗

PubMed1999

The basic region/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins: function and regulation.

A large body of evidence has been accumulated that demonstrates dominant effects of Myc proto-oncoproteins on different aspects of cellular growth. Myc is one of the few proteins that is sufficient to drive resting cells into the cell cycle and promote DNA synthesis. In line with this finding is that the constitutive expression of Myc in cells blocks their differentiation. These growth stimulating properties are most likely responsible for Myc's ability to initiate and promote tumor formation. Interestingly Myc can also sensitize cells to apoptosis, suggesting that this protein is part of a life-and-death switch. Molecularly Myc functions as a transcriptional regulator that needs to heterodimerize with Max to exert the biological activities described above and to regulate gene transcription. Myc and Max are just two members of a growing family of proteins referred to as the Myc/Max/Mad network. A hallmark of these proteins is that they possess a C-terminal basic region/helix-loop-helix/leucine zipper domain (bHLHZip). The bHLHZip domain specifies dimerization within the network and determines sequence specific DNA binding. Importantly this domain together with the N-terminal transactivation domain is essential for Myc biology. Here we have summarized the structural, functional, and regulatory aspects of the bHLHZip domain of Myc proteins.

Amino Acid Sequence↗

PubMed1999

The golden hamster aphrodisin gene. Structure, expression in parotid glands of female animals, and comparison with a similar murine gene.

The so-called lipocalins are a family of extracellular proteins that are known to typically fulfill tasks as transport proteins for small hydrophobic molecules. However, in the last decade, a large diversity has been described concerning their functions, for example as enzymes, immunomodulators, or proteins involved in coloration and pheromone action. Aphrodisin belongs to those lipocalins, which are of significant importance for the pheromonal stimulation of copulatory behavior in male hamsters. We recently succeeded in characterizing the corresponding cDNA and demonstrated the expression of the aphrodisin gene in the vagina, uterus, and Bartholin's glands of female hamsters. Here we report the structure of the aphrodisin gene and the functionality of its promoter region. We further compare the aphrodisin gene to the related gene for mouse odorant-binding protein 1a, indicating similar functions of their products. As a novelty, we show that the aphrodisin gene, in addition to the above-mentioned tissues, is also expressed in female hamster parotid glands. In contradiction to the results expected, we finally demonstrate that aphrodisin already occurs in vaginal discharge before the female animals reach fertility. These findings may lead to the identification of as yet unknown aphrodisin functions.

Amino Acid Sequence↗

PubMed1999

Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.

Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.

Animals↗

PubMed1999

DNA binding of Myc/Max/Mad network complexes to oligonucleotides containing two E box elements: c-Myc/Max heterodimers do not bind DNA cooperatively.

Myc proteins function in heterodimeric complexes with Max proteins as transcriptional regulators at least in part by binding to E box sequences with a 5'-CACGTG core. Since such E boxes are found frequently in the human genome and since other proteins besides Myc/Max can bind to similar or identical sequences it is unclear how the specificity of E box-mediated gene transcription is determined. Recent findings were interpreted to suggest that Myc/Max, but not Max/Max or USF complexes, bind cooperatively to DNA sequences that contain two E box elements. This provides a potential mechanism for selective E box-mediated gene transcription. To extend this finding we analyzed DNA binding of c-Myc/Max complexes using a transient COS-7 expression system. In both competition and titration experiments no cooperative binding of c-Myc/Max heterodimers to probes with two E boxes was observed. Furthermore, c-Myc-specific transcription of reporter gene constructs did not reveal cooperativity. Thus ourfindings argue against cooperative DNA binding of Myc proteins as a selection mechanism for E box-dependent transcription.

Animals↗

PubMed1999

Expression and regulation of protein kinase CK2 during the cell cycle.

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2alpha, CK2alpha' and CK2beta on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2alpha and CK2alpha' following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2alpha to CK2alpha' during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2beta relative to CK2alpha in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2beta increase in mitotic cells, that CK2alpha and CK2beta are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.

Amino Acid Sequence↗

PubMed1998

YY1 can inhibit c-Myc function through a mechanism requiring DNA binding of YY1 but neither its transactivation domain nor direct interaction with c-Myc.

The proto-oncoprotein c-Myc and the multifunctional transcriptional regulator YY1 have been shown previously to interact directly in a manner that excludes Max from the complex (Shrivastava et al., 1993). As binding to Max is necessary for all known c-Myc activities we have analysed the influence of YY1 on c-Myc function. We demonstrate that YY1 is a potent inhibitor of c-Myc transforming activity. The region in YY1 required for inhibition corresponds to a functional DNA-binding domain and is distinct from the domains necessary for direct binding to c-Myc. Furthermore the transactivation domain of YY1 was not necessary suggesting that gene regulation by YY1, for example through DNA bending or displacement of regulators from DNA, could be the cause for the negative regulation of c-Myc. This model of indirect regulation of c-Myc by YY1 was supported by the finding that although YY1 did not bind to the c-Myc transactivation domain (TAD) in vitro it was able to inhibit transactivation by Gal4-MycTAD fusion proteins in transient transfections. As for the inhibition of transformation, an intact DNA-binding domain of YY1 was necessary and sufficient for this effect. In addition YY1 did not alter c-Myc/Max DNA binding, further supporting an indirect mode of action. Our findings point to a role of YY1 as a negative regulator of cell growth with a possible involvement in tumor suppression.

Alanine↗

PubMed1998

Identification and characterization of specific DNA-binding complexes containing members of the Myc/Max/Mad network of transcriptional regulators.

In the past, eukaryotic cell-derived complexes of the Myc/Max/Mad network of transcriptional regulators have largely been refractory to DNA binding studies. We have developed electrophoretic mobility shift assay conditions to measure specific DNA binding of Myc/Max/Mad network complexes using a COS7 cell-based overexpression system. With the established protocol, we have measured on- and off-rates of c-Myc/Max, Max/Max, and Mad1/Max complexes and determined relative affinities. All three complexes appeared to bind with comparable affinity to a Myc E-box sequence. Furthermore, our data derived from competition experiments suggested that the Mad3/Max and Mad4/Max complexes also possess comparable DNA binding affinities. The conditions established for COS7 cell-overexpressed proteins were then used to identify c-Myc/Max, Max/Max, and Mnt/Max complexes in HL-60 cells. However, no Mad1/Max could be detected, despite the induction of Mad1 expression during differentiation. Whereas the DNA binding activity of c-Myc/Max complexes was down-regulated, Max/Max binding increased, and Mnt/Max binding remained unchanged. In addition, we have also tested for upstream stimulatory factor (USF) binding and observed that, in agreement with published data, USF comprises a major Myc E-box-binding factor that is more abundant than any of the Myc/Max/Mad network complexes. Similar to the Mnt/Max complex, the binding activity of USF remained constant during HL-60 differentiation. Our findings establish conditions for the analysis of DNA binding of Myc/Max/Mad complexes and indicate posttranslational regulation of the Max/Max complex.

Animals↗

PubMed1998

Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin.

Most fast inhibitory neurotransmission in the brain is mediated by GABAA receptors, which are mainly postsynaptic and consist of diverse alpha and beta subunits together with the gamma 2 subunit. Although the gamma 2 subunit is not necessary for receptor assembly and translocation to the cell surface, we show here that it is required for clustering of major postsynaptic GABAA receptor subtypes. Loss of GABAA receptor clusters in mice deficient in the gamma 2 subunit, and in cultured cortical neurons from these mice, is paralleled by loss of the synaptic clustering molecule gephyrin and synaptic GABAergic function. Conversely, inhibiting gephyrin expression causes loss of GABAA receptor clusters. The gamma 2 subunit and gephyrin are thus interdependent components of the same synaptic complex that is critical for postsynaptic clustering of abundant subtypes of GABAA receptors in vivo.

Animals↗

PubMed1997

Analysis of the DNA-binding activities of Myc/Max/Mad network complexes during induced differentiation of U-937 monoblasts and F9 teratocarcinoma cells.

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Myc:Max or Max:Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.

Animals↗

PubMed1997

Regulatory domains of the A-Myb transcription factor and its interaction with the CBP/p300 adaptor molecules.

The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, we have generated a series of deletion mutants and have investigated their trans-activation potential as well as their DNA-binding activity. Our results have allowed us to delineate the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218-319) are centred on a sequence rich in charged amino acids (residues 259-281). A region (residues 320-482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues markedly inhibits specific DNA binding. Finally the last 110 residues of A-Myb (residues 643-752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. We have also investigated the functional interaction between A-Myb and the nuclear adaptor molecule CBP [cAMP response element-binding protein (CREB)-binding protein]. We demonstrate that CBP synergizes with A-Myb in a dose-dependent fashion, and that this co-operative effect can be inhibited by E1A and can also be observed with the CBP homologue p300. We show that this functional synergism requires the presence of the A-Myb charged sequence and that it involves physical interaction between A-Myb and the CREB-binding domain of CBP.

3T3 Cells↗

PubMed1997

Cell growth inhibition by the Mad/Max complex through recruitment of histone deacetylase activity.

BACKGROUND: The organization of chromatin is crucial for the regulation of gene expression. In particular, both the positioning and properties of nucleosomes influence promoter-specific transcription. The acetylation of core histones has been suggested to alter the properties of nucleosomes and affect the access of DNA-binding transcriptional regulators to promoters. A recently identified mammalian histone deacetylase (HD1) shows homology to the yeast Rpd3 protein, which together with Sin3 affects the transcription of several genes. Mammalian Sin3 proteins interact with the Mad components of the Myc/Max/Mad network of cell growth regulators. Mad/Max complexes may recruit mammalian Rpd3-like enzymes, therefore, directing histone deacetylase activity to promoters and negatively regulating cell growth. RESULTS: We report the identification of a tetrameric complex composed of Max, Mad1, Sin3B and HD1. This complex has histone deacetylase activity which can be blocked by the histone deacetylase inhibitors trichostatin A and sodium butyrate. The inhibition of cell growth by Mad1 is enhanced by Sin3B and HD1, as measured by colony formation assays. Furthermore, a Mad1-induced block of S-phase progression can be overcome by trichostatin A, as shown in microinjection experiments. CONCLUSIONS: The recruitment of a histone deacetylase by sequence-specific DNA-binding proteins provides a mechanism by which the state of acetylation of histones in nucleosomes and hence the activity of specific promoters can be influenced. The finding that Mad/Max complexes interact with Sin3 and HD1 in vivo suggests a model for the role of Mad proteins in antagonizing the function of Myc proteins.

Animals↗

PubMed1997

Characterization of the transcriptional regulator YY1. The bipartite transactivation domain is independent of interaction with the TATA box-binding protein, transcription factor IIB, TAFII55, or cAMP-responsive element-binding protein (CPB)-binding protein.

YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. We show that YY1 is ubiquitously expressed in growing, differentiated, and growth-arrested cells. The protein is phosphorylated and has a half-life of 3.5 h. To define functional domains, we have generated a large panel of YY1 mutant proteins. These were used to define precisely the DNA-binding domain, the region responsible for nuclear localization, and the transactivation domain. The two acidic domains at the N terminus each provide about half of the transcriptional activating activity. Furthermore, the spacer region between the Gly/Ala-rich and zinc finger domains has accessory function in transactivation. YY1 has been shown previously to bind to TAFII55, TATA box-binding protein, transcription factor IIB, and p300. In addition, we identified cAMP-responsive element-binding protein (CBP)-binding protein as a YY1 binding partner. Surprisingly, these proteins did not bind to the domains involved in transactivation, but rather to the zinc finger and Gly/Ala-rich domains of YY1. Thus, these proteins do not explain the transcriptional activating activity of YY1, but rather may be involved in repression or in initiation.

3T3 Cells↗