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M Ueffing

Publications and source records attributed to M Ueffing.

At least 19 recordsLinked to original sources
PubMed2006

Reverse genetics for proteomics: from proteomic discovery to scientific content.

Proteomic strategies generally result in rather descriptive outcomes. Although proteomic patterns may lead to understanding of cellular function or failure, they do not provide insights into cellular mechanisms of normal physiology or molecular mechanisms of disease. The integration of single events or patterns into complex biological processes and signaling networks must be functionally validated by studying these processes in a dynamic physiological context. Effective methodologies, applied at the level of intact cells and model organisms that monitor their function, topology and interaction of proteins are necessary and essential. Reverse genetic strategies can greatly advance the understanding of patterns identified through proteomic analysis. RNAi as an accessible method for routine functional analysis of cells and whole model organisms represents a quantum leap in revolutionizing reverse genetics. This paper aims at describing an integrated workflow linking proteome-based discovery to molecular analysis of single genes and proteins by RNAi, potentially contributing to the advanced understanding of molecular mechanisms in neurodegenerative disease, including Parkinson's disease.

Animals↗
PubMed2002

Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones.

Glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic neurones against toxic and physical damage. In addition, GDNF promotes differentiation and structural integrity of dopaminergic neurones. Here we show that GDNF can support the function of primary dopaminergic neurones by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that GDNF, in addition to its action in structural differentiation, also promotes differentiation regarding expression and enzymatic activity of a crucial component in the dopaminergic biosynthetic pathway.

Animals↗
PubMed2001

Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures.

The feasibility of non-viral gene transfer using liposomes is described for human fetal nigral tissue. Ventral mesencephalic explants from 6 to 12 week old fetuses were grown as free-floating roller tube cultures. For the transfection, a vector coding for beta-galactosidase driven by the Rous Sarcoma Virus promoter was used. The developmental stage of the human tissue, time in vitro and the amount of vector DNA used significantly influenced the transfection efficiency. Optimal transfection results were obtained with tissue from a 10 week old fetus, cultured for 4 days and transfected with mixtures containing 4 microg vector DNA. Histological analysis suggested that a specific population of ventral mesencephalic precursor cells were the target for the gene transfer. This finding might have implications for gene delivery and cell replacement strategies in Parkinson's disease.

Avian Sarcoma Viruses↗
PubMed2000

Nonviral glial cell-derived neurotrophic factor gene transfer enhances survival of cultured dopaminergic neurons and improves their function after transplantation in a rat model of Parkinson's disease.

Transplantation of dopaminergic fetal mesencephalic tissue into the striatum is currently being developed for treatment of patients with advanced Parkinson's disease. Ethical concerns regarding the use of human fetal tissue, and the limited availability as well as poor survival and differentiation of dopaminergic neurons after transplantation have reduced the extent and outcome of this approach so far. With the purpose of finding means to increase the yield of dopaminergic neurons in transplants, and to reduce the amount of fetal tissue needed for each transplanted patient, we transfected rat fetal ventral mesencephalic (VM) tissue grown as organotypic free-floating roller tube (FFRT) cultures with a vector encoding human glial cell-derived neurotrophic factor (hGDNF). For transfer of an episomal expression vector (pRep7-GDNF8) a nonviral, nonliposomal cationic transfection technique was applied and optimized. Recombinant hGDNF expression resulted in a higher number of TH-positive neurons in the cultures as measured 6 days after transfection. Ventral mesencephalic cultures expressing hGDNF were then grafted into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Grafting of genetically modified VM cultures resulted in earlier functional recovery compared with grafting nontransfected cultures. We conclude that organotypic free-floating roller tube cultures can be successfully transfected to produce hGDNF with effects on TH-expressing neurons in vitro and functional effects after grafting in a rat Parkinson's disease model.

Animals↗
PubMed1999

Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.

Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)-receptor superfamily and is absolutely required for B cell immortalization by Epstein-Barr virus. Molecular and biochemical approaches demonstrated that LMP1 usurps cellular signaling pathways resulting in the induction of NF-kappaB and AP-1 via two C-terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1's C-terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphorylation of JAK3 within minutes after crosslinking of a conditional NGF-R:LMP1 chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C-terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.

Amino Acid Sequence↗
PubMed1999

The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase.

Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR). The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTP-binding protein Ran. In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDEdelta) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR392). The interaction of RPGR with PDEdelta was confirmed by pull-down assays and plasmon surface resonance. The binding affinity was determined to be 90 nM. Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system. All missense mutations showed reduced interaction with PDEdelta. A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDEdelta. PDEdelta is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family. These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB.

Reactive oxygen species (ROS) play an important role in cell death induced by many different stimuli. This study shows that hydrogen peroxide-induced apoptosis in T-cells did not require tyrosine kinase p561ck, phosphatase CD45, the CD95 receptor and its associated Caspase-8. H2O2-triggered cell death led to the induced cleavage and activation of Caspase-3. Hydrogen peroxide-treatment of T-cells resulted in the formation of mitochondrial permeability transition pores, a rapid decrease of the mitochondrial transmembrane potential delta psi(m) and the release of Cytochrome C. Inhibition of the mitochondrial permeability transition by bongkrekic acid (BA), or interference with the mitochondrial electron transport system by rotenone or menadione prevented the cytotoxic effect of H2O2. Antimycin A, a mitochondrial inhibitor that increases the release of mitochondrial ROS (MiROS), enhanced apoptosis. Overexpression of Bcl-2 and the viral anti-apoptotic proteins BHRF-1 and E1B 19K counteracted H2O2-induced apoptosis. Pharmacological and genetic inhibition of transcription factor NF-kappaB protected cells from hydrogen peroxide-elicited cell death. This detrimental effect of NF-kappaB mediating hydrogen peroxide-induced cell death presumably relies on the induced expression of death effector genes such as p53, which was NF-kappaB-dependently upregulated in the presence of H2O2.

Adenovirus E1B Proteins↗
PubMed1999

Somatic gene therapy in animal models of Parkinson's disease.

Gene therapy in Parkinson's disease (PD) emerged about 10 years ago but until now, no clinical trials are under way, because most approaches have failed to show long-term therapeutic effects in PD animal models and because safety concerns precluded the use in humans so far. This review tries to give an overview on the development of different strategies in gene therapy in PD animal models and point out new and possibly more successful directions, including the transplantation of neural precursor cells and pig tissue.

Animals↗
PubMed1998

Phosphorylation of GTP cyclohydrolase I and modulation of its activity in rodent mast cells. GTP cyclohydrolase I hyperphosphorylation is coupled to high affinity IgE receptor signaling and involves protein kinase C.

GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hültner, L. , Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rödl, W. (1993) J. Biol. Chem. 268, 12544-12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivo labeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.

Animals↗
PubMed1998

Overexpression of PKCepsilon in R6 fibroblasts causes increased production of active TGFbeta.

In previous studies, our laboratory demonstrated that Rat 6 (R6) fibroblasts which stably overproduce high levels of PKCepsilon display abnormalities in growth control that are characteristic of malignant transformation (Cacace et al., 1993, Oncogene, 8:2095-2104). The R6-PKCepsilon overproducing cell lines also exhibited a decreased growth factor requirement. The present study demonstrates that conditioned medium (CM) from two individual clones, R6-PKCepsilon 10 and 30, stimulates DNA synthesis in control R6-C1 cells. Maximal DNA synthesis and morphologic transformation was achieved in control cells when they were treated with medium from R6-PKCepsilon cells grown in the presence of TPA (TPA-CM). Size fractionation of the TPA-CM from PKCepsilon 30 cells revealed that this activity is due to a factor(s) that has an apparent molecular weight in the range of 10-30 kD and is heat and acid stable. This factor, like TGFbeta1, stimulated anchorage-independent growth of NRK cells. Western blot analysis (under nonreducing conditions) of the TPA-CM from R6-PKCepsilon 30 and R6-PKCepsilon 10 cells revealed the presence of the 25 kD active forms of TGFbeta2 and 3. These active forms of TGFbeta were not found in the CM of control R6 cells, or R6 cells that overexpress PKCalpha or PKCbeta1. The addition of a pan-specific TGFbeta antibody to NRK cells treated with the 10-30 kD fraction of TPA-CM from PKCepsilon 30 cells blocked the ability of this material to stimulate thymidine incorporation. Taken together, these studies suggest that the oncogenic activity of PKCepsilon in R6 cells is due, at least in part, to its ability to induce production of the active forms of TGFbeta2 and 3.

Animals↗
PubMed1998

Analysis of the human GDNF gene reveals an inducible promoter, three exons, a triplet repeat within the 3'-UTR and alternative splice products.

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGF-beta superfamily, is a survival factor for various neurons, making it a potential therapeutic agent for neurodegenerative disorders. Here we present the genomic structure and characterization of the promoter of the human GDNF (hGDNF) gene. It contains three exons coding for a cDNA of 4.6 kb including large 5'- and 3'-untranslated regions (UTRs). The 3'-UTR contains a polymorphic AGG repeat that appears not to be expanded in patients suffering from different neurodegenerative disorders. RT-PCR results in at least three different hGDNF transcripts including one that lacks exon 2. Transient expression experiments reveal that exon 2 is essential for proper cellular processing to yield a secreted form of hGDNF, whereas expression of exon 3 alone is sufficient to code for a mature form of hGDNF retained within the cell. Our data show that the hGDNF gene is driven by a TATA-containing promoter preceding exon 1. A second promoter element has been mapped to a region 5' of exon 2. Both promoters are in close proximity to CpG islands covering exons 1 and 2. Using luciferase as a reporter gene, the TATA-containing hGDNF promoter facilitates a 20- to 40-fold increase in transcription when compared with a corresponding promoterless construct, whereas the second promoter confers only weak activity. Furthermore, fibroblast growth factor 2, tetradecanoyl 12-phorbol acetate, an inflammatory agent, and cAMP increase promoter activity, suggesting that GDNF transcriptional regulation is a target of exogenous signals.

Alternative Splicing↗
PubMed1997

Protein kinase C-epsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity.

Several observations indicate that the Raf-1 kinase is a downstream effector of protein kinase C-epsilon (PKC epsilon). We recently have shown that Raf-1 is constitutively activated in PKC epsilon transformed Rat6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Cacace et al., 1996). Cai et al. (1997) demonstrated that PKC epsilon induced proliferation of NIH3T3 cells is independent of Ras or Src, but depends on Raf-1. These authors further suggested that PKC epsilon activates Raf-1 by direct phosphorylation. Here we have investigated the functional interaction between PKC epsilon and Raf-1. PKC epsilon, but not PKC alpha, was found to bind to the Raf-1 kinase domain. The association appeared to be direct, as it could be reconstituted in vitro with purified proteins. Raf-1 and PKC epsilon could be co-precipitated from Sf-9 insect cells and PKC epsilon transformed NIH313 cells (NIH/epsilon). The association was negatively regulated by ATP in vitro and by TPA treatment in NIH/epsilon cells, but not in Sf-9 insect cells. Raf-1 was constitutively activated in NIH/epsilon cells. However, using coexpression experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PKC epsilon. PKC epsilon did not induce translocation of Raf-1 to the membrane. Furthermore, PKC epsilon did not activate Raf-1 nor enhance the kinase activity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase. In contrast, conditioned media from PKC epsilon transformed cells induced a robust activation of Raf-1. This activation could be partially reproduced by recombinant TGFbeta, a growth factors secreted by PKC epsilon transformed Rat6 cells. In conclusion, our results suggest that PKC epsilon stimulates Raf-1 indirectly by inducing the production of autocrine growth factors.

3T3 Cells↗
PubMed1997

Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade.

The Epstein-Barr virus latent membrane protein-1 (LMP-1) is an integral membrane protein which transforms fibroblasts and is essential for EBV-mediated B-cell immortalization. LMP-1 has been shown to trigger cellular NF-kappa B activity which, however, cannot fully explain the oncogenic potential of LMP-1. Here we show that LMP-1 induces the activity of the AP-1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP-1 effects on AP-1 are mediated through activation of the c-Jun N-terminal kinase (JNK) cascade, but not the extracellular signal-regulated kinase (Erk) pathway. Consequently, LMP-1 triggers the activity of the c-Jun N-terminal transactivation domain which is known to be activated upon JNK-mediated phosphorylation. Deletion analysis indicates that the 55 C-terminal amino acids of the LMP-1 molecule, but not its TRAF interaction domain, are essential for AP-1 activation. JNK-mediated transcriptional activation of AP-1 is the direct output of LMP-1-triggered signaling, as shown by an inducible LMP-1 mutant. Using a tetracycline-regulated LMP-1 allele, we demonstrate that JNK is also an effector of non-cytotoxic LMP-1 signaling in B cells, the physiological target cells of EBV. In summary, our data reveal a novel effector of LMP-1, the SEK/JNK/c-Jun/AP-1 pathway, which contributes to our understanding of the immortalizing and transforming potential of LMP-1.

B-Lymphocytes↗
PubMed1997

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.

B-Lymphocytes↗
PubMed1997

Expression of CD44 isoforms in neuroblastoma cells is regulated by PI 3-kinase and protein kinase C.

With respect to a potential role for CD44 in neuronal tumors, we investigated the regulation of variant CD44 exon containing isoforms (CD44V) in the human neuroblastoma cell line SK-N-SH in response to treatment with differentiation-inducing and mitogenic factors. While the standard form of CD44 was expressed at high levels in both treated and untreated cells, variant isoforms were strongly upregulated in response to treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA), insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor (PDGF) as shown by RT-PCR and immunofluorescence. One of the CD44 isoforms contains sequences encoded by variant exon v6 (CD44V6), which was originally described as a metastasis-associated antigen. Using specific inhibitors, we explored the signal transduction pathways involved in the expression of variant CD44. GF-109203X, a specific inhibitor of protein kinase C effectively blocked TPA- and IGF-1-upregulated expression of CD44v6. Wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI 3-kinase) partly reduced IGF-1 and PDGF induced CD44v6 expression. The induction of CD44V by TPA, IGF-1 or PDGF was correlated with an increased cellular binding to hyaluronic acid, a major counterreceptor for CD44. The increased binding caused by TPA or IGF-1 could specifically be blocked by the above inhibitors. Thus, PKC and PI 3-kinase are likely to transduce growth factor induced signals that upregulate specific CD44 splice variants.

Carcinogens↗
PubMed1996

PKC epsilon functions as an oncogene by enhancing activation of the Raf kinase.

Previous studies have indicated that PKCepsilon behaves as an oncogene when overproduced in rodent fibroblasts (Cacace et al., 1993; Mishak et al., 1993). In the present study, Western blot analysis revealed that the hyperphosphorylated form of Raf kinase was present at a high level in PKCepsilon overproducing R6 rat fibroblasts but not in R6 fibroblasts overproducing PKCalpha or beta1. Extracts from the PKCepsilon overproducing cells also exhibited a marked increase in Raf-1 kinase and MAP-kinase activity. To investigate the significance of these findings, dominant negative mutants of ras (N17) or raf (301-1) were stably expressed in early passage control and PKCepsilon-transformed R6 fibroblasts, by transduction using retrovirus-derived constructs. Dominant negative raf expressing clones exhibited a flat morphology, a decreased saturation density, and decreased growth in soft agar. In addition, these reverted clones exhibited decreased Raf kinase activity. In contrast, dominant negative ras expressing clones remained highly transformed. In addition, PKCepsilon was detected in Raf-1 immunoprecipitates indicating that PKCepsilon forms a complex with Raf-1 in vivo. Taken together, these results suggest that PKCepsilon functions as an oncogene in R6 cells by enhancing activation of the Raf-1 kinase.

Animals↗
PubMed1996

Cloning and functional analysis of the hematopoietic cell-specific phospholipase C(gamma)2 promoter.

Phospholipase C(gamma)2 (PLCgamma2) is a phospholipid-converting enzyme which, upon receptor stimulation, is activated within membrane-bound signalling complexes. In contrast to the highly ubiquitous PLCgamma1, PLCgamma2 is expressed predominantly in B-lymphocytes. Associated with antigen-coupling receptors it is activated by tyrosine phosphorylation after the triggering of B-cell surface immunoglobulin. We have cloned and sequenced the human PLCgamma2 promoter. Primer extension analysis reveals the existence of a major transcriptional start site. The TATA-less promoter contains G+C-rich stretches with a cluster of contiguous SP1 consensus sites, an NF1, and an AP2 site between bp -220 to -70. A construct containing the region from -189 to +78 confers full promoter activity, as shown by fusion to a luciferase reporter gene construct. The distal part of the promoter between bp -662 to -293 containing an SRE, EBF and CACCC box contributed negatively to promoter activity in the B-cell line Raji but not in three adherent cell lines. In Raji cells, PLCgamma2 mRNA is expressed at low levels with a half life greater than 4 h. After treatment with serum, TPA, retinoic acid, or with 5-azacytidine increased levels of PLCgamma2 mRNA were induced in B-cells.

3T3 Cells↗