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N Fischer

Publications and source records attributed to N Fischer.

At least 55 records · Page 3Linked to original sources
PubMed1997

Axial (HNF3beta) and retinoic acid receptors are regulators of the zebrafish sonic hedgehog promoter.

The signalling molecule Sonic hedgehog is involved in a multitude of distinct patterning processes during vertebrate embryogenesis. In the nascent body axis of the zebrafish embryo, sonic hedgehog is co-expressed with axial (HNF3beta in mammals), a transcription regulator of the winged helix family. We show here that misexpression of axial leads to ectopic activation of sonic hedgehog expression in the zebrafish, suggesting that axial is a regulator of sonic hedgehog transcription. The sonic hedgehog gene was cloned from zebrafish and its promoter was characterized with respect to activation by axial. Expression of axial or rat HNF3beta in HeLa cells results in activation of co-transfected sonic hedgehog promoter-CAT fusion genes. This effect is mediated by two Axial (HNF3beta) recognition sequences. We furthermore identified a retinoic acid response element (RARE) in the sonic hedgehog upstream region which can be bound by retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers in vitro and confers retinoic acid inducibility to the sonic hedgehog promoter in the HeLa cell system. Our results suggest that both Axial (HNF3beta) and retinoic acid receptors are direct regulators of the sonic hedgehog gene.

Animals↗
PubMed1997

Characterization of human foamy virus proteins expressed by recombinant vaccinia viruses.

We report the generation of recombinant vaccinia viruses (VVs) expressing the gag, pol, bel-1, and bet open reading frames of human foamy virus (HFV), and the establishment of a transient, VV-T7 RNA polymerase-directed expression system for the HFV env gene. The correct expression of the HFV proteins was demonstrated by radioimmunoprecipitation using monospecific rabbit antisera, by analysis of the subcellular distribution (for VVgag, VVpol, VVbel-1, and VVbet), and by the ability to induce syncytium formation (for the env expression system). The HFV pol gene was successfully expressed using its own ATG start codon. Foamy viruses are regarded as retroviruses with intracytoplasmatic capsid assembly. However, when VVgag and VVpol were used to study the HFV Gag-Pol protein interaction and particle formation, no HFV capsid structures were observed in singly or doubly infected cells. In addition, no cleavage of the Pr74gag precursor molecule by the pol-encoded protease was detected in doubly infected cells. Our results indicate that foamy virus particle assembly is fundamentally different from that of other retroviruses.

Animals↗
PubMed1997

Identification of sites that act together to direct dimerization of human foamy virus RNA in vitro.

Retroviral particles contain two molecules of genomic RNA, which are noncovalently linked near their 5' ends in a region called the dimer linkage structure (DLS). By using complementary DNA oligonucleotides and deletion mutants to impair RNA dimerization of the human foamy virus (HFV), three sites, designated SI, SII, and SIII, were found within a 159-nucleotide RNA fragment of HFV that are involved in dimerization in vitro. SI overlaps the primer-binding site; and SII contains the palindromic sequence, UCCCUAGGGA, the disruption of which impairs dimer formation; and SIII extends into the gag gene. The first two sites are highly conserved in the other primate foamy viruses, SFV-1, SFV-3, and SFVcpz, whereas the third appears to be shared only by HFV and SFVcpz. RNA of HFV and SFV-3 could form heterodimers, indicating that both viruses dimerize by similar mechanisms. On testing thermal stability, dimers of the 159-nucleotide fragment dissociated between 40 and 70 degrees, with half of the dimers dissociating at 55 degrees. Since the splice donor site of HFV is located at position 51 of viral RNA, the DLS is part of the genomic RNA exclusively.

Binding Sites↗
PubMed1997

Epstein-Barr virus nuclear antigen 1 forms a complex with the nuclear transporter karyopherin alpha2.

The Epstein-Barr virus (EBV) is implicated in the induction of several malignancies. The nuclear antigen 1 (EBNA1) is the only viral protein that is expressed consistently in all EBV-associated tumors. EBNA1 is involved in the replication and maintenance of the viral episome in the infected cell and exhibits oncogenic activity in transgenic mice. Here we report the identification of the nuclear transporter karyopherin alpha2 as a cellular partner of EBNA1 using the yeast "two-hybrid system." Karyopherin alpha2 is also called importin alpha or Rch1. The binding to karyopherin alpha2 was mediated through a C-terminal region of EBNA1 encompassing the nuclear localization signal, whereas clones of EBNA1 devoid of the nuclear localization signal failed to bind to karyopherin alpha2. The interaction was biochemically confirmed by far-Western analysis using bacterially expressed karyopherin alpha2 and karyopherin alpha2-specific monoclonal antibodies. The nuclear transport of EBNA1 was impaired by expression of N-terminally truncated karyopherin alpha2. Zone velocity sedimentation in a sucrose gradient indicated that: (i) EBNA1 and Rch1 colocalize; and (ii) the association of karyopherin alpha2 with high molecular weight protein complexes might be impeded by the presence of EBNA1.

Animals↗
PubMed1997

Cloning of human 2H9 heterogeneous nuclear ribonucleoproteins. Relation with splicing and early heat shock-induced splicing arrest.

Using antibody 2H9 from our heterogeneous nuclear ribonucleoproteins (anti-hnRNP) monoclonal antibody library, we previously showed in HeLa cells that a 35-37-kDa protein doublet switches from the hnRNP complexes to the nuclear matrix following a 10-min heat shock at 45 degrees C (1 Lutz, Y., Jacob, M., and Fuchs, J. P. (1988) Exp. Cell Res. 175, 109-124). cDNA cloning and sequencing revealed an hnRNP protein (2H9) which is a new member of the hnRNP F, H/H' family. Protein 2H9 displays two consensus sequence-type RNA binding domains (CS-RBD) showing 80-90% homology with two of the three CS-RBDs of hnRNP F and H/H'. Another common feature is the presence of two glycine/tyrosine-rich auxiliary domains located at the C terminus and between the two CS-RBDs. At the functional level we show that specific anti-2H9 peptide antibodies can directly inhibit an in vitro splicing system. Moreover, the 2H9 protein doublet is no more present in nuclear extracts from such briefly stressed cells, which interestingly correlates with the inability of these extracts to catalyze in vitro splicing reactions. Taken together, our data suggest that these proteins are involved in the splicing process and also participate in early heat shock-induced splicing arrest by transiently leaving the hnRNP complexes. These 2H9 proteins, which are encoded by a single gene located on human chromosome 10, were also found to be associated with nuclear bodies in situ.

Amino Acid Sequence↗
PubMed1997

Targeted mutations in the psaC gene of Chlamydomonas reinhardtii: preferential reduction of FB at low temperature is not accompanied by altered electron flow from photosystem I to ferredoxin.

The terminal part of the electron pathway within the photosystem I (PSI) complex includes two [4Fe-4S] centers, FA and FB, which are coordinated by the PsaC subunit. To gain new insights into the electron transfer mechanisms through PsaC, we have generated three mutant strains of the alga Chlamydomonas reinhardtii in which two positively charged residues, K52 and R53, near the FA center have been altered in different ways. The mutations K52S/R53D and K52P/R53D lead to a strong destabilization of PSI. The third mutant K52S/R53A accumulates PSI to 30% of wild-type levels and shares the same residues between two of the cysteine ligands of FA as the PsaC homologue in the green sulfur bacterium Chlorobium limicola, in which FB has a higher redox potential than FA [Nitschke, W., Feiler, U., & Rutherford, A. W. (1990) Biochemistry 29, 3834-3842]. Low-temperature electron paramagnetic resonance (EPR) studies reveal that, in contrast to wild type, FB is preferentially photoreduced in this mutant, as was also observed for C. limicola. The preferential photoreduction of FB could be due to changes in the redox potential of FA and/or to slight structural modifications of the PsaC subunit. However, room temperature optical measurements show that stable charge separation still occurs and, surprisingly, that electron transfer from PSI to ferredoxin proceeds at normal rates in the mutant. As C. limicola, the K52S/R53A and K52S/R53D C. reinhardtii mutants are photosensitive when grown aerobically, but can grow photoautotrophically under anaerobic conditions.

Anaerobiosis↗
PubMed1997

Expression and regulation of a netrin homologue in the zebrafish embryo.

Proteins of the Netrin family have been implicated in axon guidance in both C. elegans and vertebrates. Here, we report the cloning and expression analysis of a zebrafish netrin homologue (net1). net1 is expressed in the floor plate and the anterior ventral neural tube. Its expression is ectopically induced by misexpression of sonic hedgehog (shh) and a dominant negative mutant of the regulatory subunit of protein kinase A (dnReg). Ectopic activation of net1, however, is restricted to distinct regions in the brain. Upon overexpression of shh or dnReg in cyclops mutants, which have strongly impaired net1 expression in the ventral neural tube, rescue of net1 expression was observed in the brain but not in the spinal cord. Ectopic expression of dnReg and Shh protein can be detected at high levels throughout injected embryos from pre-gastrula stages onwards suggesting that the competence of the neural plate to respond to Shh signalling activity differs regionally. Similar to net1, axial, the zebrafish homologue of mammalian HNF3beta, which is also expressed along the ventral neural tube, is ectopically induced in the brain of embryos injected with dnReg mRNA. Neurons differentiate normally within domains of ectopic net1 and axial expression. Thus, dorsal neuronal differentiation appears to be unaffected despite co-expression of a gene program specific for the ventral neural tube. This also suggests that these ectopically expressing regions have not differentiated into floor plate.

Amino Acid Sequence↗
PubMed1997

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked.

Amino Acid Sequence↗
PubMed1997

The activity of neurogenin1 is controlled by local cues in the zebrafish embryo.

Zebrafish neurogenin1 encodes a basic helix-loop-helix protein which shares structural and functional characteristics with proneural genes of Drosophila melanogaster. neurogenin1 is expressed in the early neural plate in domains comprising more cells than the primary neurons known to develop from these regions and its expression is modulated by Delta/Notch signalling, suggesting that it is a target of lateral inhibition. Misexpression of neurogenin1 in the embryo results in development of ectopic neurons. Markers for different neuronal subtypes are not ectopically expressed in the same patterns in neurogenin1-injected embryos suggesting that the final identity of the ectopically induced neurons is modulated by local cues. Induction of ectopic motor neurons by neurogeninl requires coexpression of a dominant negative regulatory subunit of protein kinase A, an intracellular transducer of hedgehog signals. Moreover, the pattern of endogenous neurogenin1 expression in the neural plate is expanded in response to elevated levels of Hedgehog (Hh) signalling or abolished as a result of inhibition of Hh signalling. Together these data suggest that Hh signals regulate neurogenin1 expression and subsequently modulate the type of neurons produced by Neurogenin1 activity.

Amino Acid Sequence↗
PubMed1996

Intranigral or intrastriatal injections of GDNF: effects on monoamine levels and behavior in rats.

The present studies were designed to determine whether administration of recombinant human glial cell line-derived neurotrophic factor (rhGDNF) into either the substantia nigra or striatum is capable of augmenting dopamine function of the nigrostriatal pathway in normal rats. Single bolus intracranial injections of rhGDNF at either site increased locomotor activity and decreased food and water consumption and body weight in a dose-dependent manner when compared to vehicle-treated animals. These behavioral responses returned to pre-control levels within 3 weeks post rhGDNF administration. Administration of rhGDNF intranigrally increased dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels of the ipsilateral substantia nigra at 2 and 6 weeks post injection but had no augmenting effects on dopamine or its metabolites in the striatum. Administration of rhGDNF intrastriatally increased DOPAC and HVA levels of the ipsilateral striatum, although striatal dopamine levels were unchanged. Ipsilateral nigral dopamine levels were increased after intrastriatal injection of rhGDNF. The effects of intracranial rhGDNF were not specific to the nigrostriatal dopamine system, since nigrostriatal serotonin, 5-hydroxyindoleacetic acid (5-HIAA), epinephrine and norepinephrine transmitter levels were altered depending on administration route for rhGDNF and dose. Taken together, these data demonstrate long-lasting neurochemical and behavioral changes which suggest that rhGDNF can augment function in adult rat dopamine neurons. Therefore, rhGDNF may have therapeutic potential for Parkinson's disease.

Animals↗
PubMed1996

The human hnRNP-M proteins: structure and relation with early heat shock-induced splicing arrest and chromosome mapping.

With anti-hnRNP monoclonal antibody 6D12 we previously showed in HeLa cells that as early as 10 min after the onset of a heat shock at 45 degrees C, a 72.5-74 kDa antigen doublet leaves the hnRNPs and strongly associates with the nuclear matrix, the effect being reversed after a 6 h recovery at 37 degrees C. cDNA cloning and sequencing enabled us to identify these antigens as hnRNP-M proteins and further to show that the correct sequence differs by an 11 amino acid stretch from the originally published sequence. We also show that monoclonal antibodies raised against synthetic hnRNP-M peptides can directly inhibit in vitro splicing. Furthermore, stressing cells at 45 degrees C for 10 min is sufficient to abolish the splicing capacity of subsequently prepared nuclear extracts which, interestingly, do not contain the hnRNP-M proteins any more. Taken together, our data suggest that these proteins are involved in splicing as well as in early stress-induced splicing arrest. Further in situ hybridization assays located the hnRNP-M encoding gene on human chromosome 19.

Amino Acid Sequence↗
PubMed1996

Selectable marker recycling in the chloroplast.

The bacterial gene aadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of the aadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking the aadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which the aadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas the aadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse the aadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.

Animals↗
PubMed1996

Anisometropia and binocularity.

PURPOSE: To determine the effects of experimentally induced anisometropia on binocular function in healthy adults as a means of assessing the potentially detrimental effects of uncorrected anisometropia on binocular development in childhood. METHODS: Nineteen adults with normal binocularity, ranging in age from 26 to 59 years, were studied. Unilateral myopia, hyperopia, or astigmatism (at 90 degrees or 45 degrees) was induced in each subject using trial lenses. Sensory status then was assessed by measuring stereoacuity, Worth four-dot fusion, and Bagolini lens response. RESULTS: All subjects showed a decline in binocular function with increasing levels of anisometropia. Foveal suppression was evident on the Worth four-dot test, and increased in proportion to the anisometropia. Stereoacuity was similarly degraded by the induced anisometropia, with some subjects showing significant loss of stereoacuity with as little as 1 diopter of spherical anisometropia. Bagolini lens responses were binocular in almost all patients, although occasional abnormalities were found. CONCLUSIONS: Relatively low degrees of anisometropia may cause significant abnormalities in high-grade binocular visual functions in adults. The potential effects of uncorrected anisometropia on binocularity in children require further investigation, but should be considered in developing guidelines for the empiric correction of refractive errors.

Adult↗
PubMed1996

Glial cell line-derived neurotrophic factor: the lateral cerebral ventricle as a site of administration for stimulation of the substantia nigra dopamine system in rats.

The direct application of recombinant human glial cell line-derived neurotrophic factor (rhGDNF) to the deep structures of the nigrostriatum has been shown previously to augment dopamine function and inhibit loss of substantia nigra neurons in rodent models of Parkinson's disease. The present studies were designed to determine whether administration of rhGDNF into the lateral ventricle, a more clinically accessible intracranial target, is capable of augmenting dopamine function of the nigrostriatal pathway in normal rats. Single bolus intracerebroventricular (i.c.v) injections of rhGDNF increased locomotor activity and decreased food and water consumption and body weight gain in a dose-dependent manner. rhGDNF increased concentrations of dopamine and dopamine metabolites in the substantia nigra, ventral tegmental area and hypothalamus, but had no significant effects in the striatum. rhGDNF had no effect on striatal or substantia nigral serotonin (5-HT) and 5-hydroxyindoleacetic acid levels, but these levels were significantly increased in the ventral tegmental area and hypothalamus respectively. The augmentation of the dopamine and 5-HT systems was detected 2 weeks but not 3 days or 6 weeks after rhGDNF administration. After a repeat injection of i.c.v rhGDNF (6 weeks after the initial injection), substantia nigral dopamine, 5-HIAA and noradrenalin levels were increased. These results indicate that i.c. v administration of rhGDNF has an influence on adult rat dopamine neurons. This route of administration may be useful for stimulating dopamine neurons in Parkinson's disease.

Animals↗
PubMed1995

[Which dosage concept for adrenaline is correct in cardiopulmonary resuscitation? A data analysis of preclinical resuscitations].

AIM: Epinephrine is the drug of choice in cardiopulmonary resuscitation. Its dosage, however, is controversially discussed. The American Heart Association recommends for standard use in adults 1 mg epinephrine every 3-5 minutes, but classifies a medium dose, a high dose and a step-by-step escalating dosage concept as potentially useful alternatives. Aim of this study was to develop a rationale for the escalating dosage concept using an analysis of preclinical resuscitation data. METHODS: Bonn city (141 km2, 310,000 residents, 52% female, 13.9% > 65 years) was served by a double-response system of two ALS-units (staffed by physicians) and four BLS-units (staffed by paramedics). All patients were included in this data analysis, which were cardiopulmonary resuscitated according to the AHA guidelines by the ALS-unit Bonn-North (66% of area and 240,000 residents) from 1989 to 1994. All relevant data were documented by the emergency physicians using preformed treatment sheets. Discharge rates were determined by reviewing the hospital records, and one-year survival data were collected by mail contact with the primary physicians. The correlations between duration of cardiac arrest, dosage of epinephrine and outcome were determined by regression analysis in patients older than 17 years suffering from unwitnessed and bystander-witnessed cardiac arrest. Statistical significance was assumed for p < 0.05. RESULTS: Within 1989 to 1994 the ALS-team of Bonn-North resuscitated 685 cardiac arrest patients with presumed cardiac aetiology in 383 (56%) return of spontaneous circulation (ROSC) could be achieved. The epinephrine dosage required to achieve ROSC increased with prolongation of cardiac arrest interval (1989-1994; patients with ROSC selected, n = 263; r = 0.2276; p = 0.0002). Resuscitation success, however, decreased with increasing, dosage of epinephrine (1989-1992; n = 345; ROSC: r = -0.2643; p < 0.001; survival > 24 h: r = -0.3393; p < 0.001; discharge: r = -0.1677; p = 0.0018; survival > 1 year: r = -0.2685; p < 0.001). CONCLUSION: Based on these data, we recommend an escalating epinephrine dosage concept, which facilitates titration of the drug to an effective level and meets the needs of the individual patient. This concept avoids overdosage in patients who had just collapsed shortly before initiation of CPR, attains higher levels of epinephrine in patients suffering from prolonged cardiac arrest, and takes into consideration that the effective epinephrine dose varies individually and increases with prolongation of the cardiac arrest interval.

Adolescent↗
PubMed1994

In situ extracorporeal shockwave lithotripsy of distal ureteral stones: parameters for therapeutic success.

Between January and December 1989, 123 patients with distal ureteral stones were treated with in situ extracorporeal shockwave lithotripsy (ESWL) using a modified HM3 Dornier lithotriptor. One hundred and three patients (83.7%) were stone-free, 20 patients (16.3%) had small residual stones (< 1 mm2) that passed spontaneously. In situ ESWL treatment alone was successful in 88 of the 103 stone-free patients (85.4%). Fifty-six (54.4%) needed 1 ESWL session, 26 (25.2%) needed 2 ESWL sessions. Nineteen of the 123 patients (15.4%) needed auxiliary endourological measures. The results of ESWL treatment were correlated to pretherapeutically identified parameters such as stone size, radiopacity, outer contour, shape, inner structure, biochemical analysis of the stones and grade of dilatation of the upper urinary tract. The results prove that size (> or = 75 mm2), radiopacity and grade of dilatation have a direct correlation to the difficulty to disintegrate ureteral stones.

Female↗
PubMed1993

Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues.

Heparin, among the best studied glycosaminoglycans, is well known for its involvement in a variety of physiological processes. Many proteins, whose activities are modulated via heparin binding, were identified, and the consequences of their interaction with heparin were characterized. However, in the absence of solid structural information regarding heparin-protein complexes, the mechanism by which heparin operates at the molecular level is still obscure. The structure of such a complex is hereby explored via the identification of a common motif in heparin binding sequences. To avoid ambiguity we included in our data base only sequences that have been shown experimentally to be directly involved in heparin binding. Then, a comparison of the spatial distribution of basic residues was conducted among those peptides for which three-dimensional structures were defined. Using computer graphics techniques we were able to identify a unique distribution shared by all of these segments. Two basic amino acids (most frequently arginine) are located at about 20 A apart, facing opposite directions of an alpha-helix. Other basic amino acids are dispersed between these two residues, facing one side, while nonpolar residues face the opposite side, forming an amphipathic structure. The distribution of basic amino acids in other heparin binding sequences that preserves the same spatial arrangement seems to be compatible with a beta-strand structure. The 20-A interval accommodates a glycosaminoglycan pentasaccharide, and the spatial distribution of the basic residues suggests an intertwinement of the heparin-protein complex. The dynamics of such an interaction may provide a clue regarding the ensuing change in protein activity.

Amino Acid Sequence↗