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Biomedical subjects

P Wagner

Publications and source records attributed to P Wagner.

At least 73 records · Page 4Linked to original sources
PubMed1997

Reversible, site-specific immobilization of polyarginine-tagged fusion proteins on mica surfaces.

A large variety of genes is expressed as fusion proteins for the purpose of characterization and purification in molecular biology. We have used this strategy to append polyarginine peptides in order to achieve specific binding of the Arg-tag to atomically flat, negatively charged mica surfaces. We show that the model protein, hexaarginine-tagged green fluorescent protein (GFP), binds to mica via its Arg-tag based on ion exchange of naturally occurring potassium cations. Only non-specific binding was observed with the control protein that is free of the Arg-tag. This novel technology will be widely applicable to orient functional proteins on flat surfaces.

Adsorption↗
PubMed1997

Phosphorylation mutants of p53 show differential complex formation with putative dehydrogenase Tms1 of fission yeast.

The yeast tms1 gene was originally identified as a multi-copy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast. The tms1 gene product (Tms1) was found to form stable complexes with p53 in yeast and in vitro; using purified recombinant proteins, the interaction was mapped to the C-terminal region of p53. This part is known to be modified by several protein kinases resulting in a transition of p53 from a latent to an activated state capable of transactivating various cellular genes involved in growth suppression or apoptosis. Since there is evidence for an evolutionary conservation of a Tms1-related protein in mammals, the effect of the phosphorylation status of the C-terminus of p53 on Tms1/p53 complex formation in vitro has been investigated. Whereas mutants changing the cdc2 phosphorylation site at position 315 of human p53 had only little effect on Tms1/p53 complex formation, we found that mutants involving the protein kinase CK2 site at position 392 showed a significantly decreased relative affinity for the Tms1 protein. The same result was obtained by using a C-terminal fragment of p53 which was phosphorylated by purified protein kinase CK2, suggesting that the complex formation of p53 with cellular C-terminal binding proteins like Tms1 impairs regulation by phosphorylation.

Binding Sites↗
PubMed1997

Differential pathophysiological mechanisms of reduced P300 amplitude in schizophrenia and depression: a single trial analysis.

In order to address basic mechanisms behind a reduced averaged P300 wave in schizophrenia and depression, 17 unmedicated schizophrenic and 11 unmedicated depressive subjects were tested in an 'oddball paradigm' against healthy controls matched for gender and age. The amplitude distributions of single trials' maximum positive deflections after stimulation (P300) for both target and nontarget stimuli were determined, which served as a basis for calculating the discrimination index d'. This index characterizes differences in the electrophysiological responses to target and nontarget stimuli of a subject being engaged in a discrimination task. As a main result d' was significantly lower for schizophrenics than for their controls. Directly compared to depressive subjects, schizophrenics also depicted a statistically significant decrease of the discrimination index, which could not be explained by differences in age. Although the averaged P300 signals did not show any significant differences between the two diagnostic groups, the approach of calculating d' on the basis of single trial analysis differentiated between schizophrenics and depressives. In conclusion, schizophrenic patients revealed different functional features when generating event-related potentials in an 'oddball paradigm' compared to healthy controls and depressives.

Adult↗
PubMed1997

The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation.

The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5-1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (alpha-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained with the holoenzyme. Polylysine stimulated MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the alpha-subunit-catalyzed MDM2 phosphorylation which was reduced by about 66% when polylysine was added. Full length p53, but also a peptide representing a C-terminal fragment of the tumor suppressor gene product p53 (amino acids 264-393 which also harbors the CK2beta interaction site at amino acids 287-340) mimicked the polylysine effect in all respects, ie. stimulation of phosphate incorporation by CK2 holoenzyme and inhibition in the presence of the catalytic CK2 alpha-subunit. Stimulation by p53(264-393) was on the average close to twofold and inhibition in the case of the alpha-subunit-catalyzed MDM2 phosphorylation was about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21(WAF1/CIP1), known to be a potent inhibitor of cyclin-dependent protein kinases, also led to a significant reduction of phosphate incorporation into MDM2 indicating that p21(WAF1/CIP1) does not exclusively inhibit cell cycle kinases. Furthermore, these data add new insight into the autoregulatory loop which include p21(WAF1/CIP1), MDM2 protein, CK2 and p53.

Animals↗
PubMed1997

Reactive nitrogen intermediates promote low density lipoprotein oxidation in human atherosclerotic intima.

Oxidized low density lipoprotein (LDL) may be of central importance in triggering atherosclerosis. One potential pathway involves the production of nitric oxide (NO) by vascular wall endothelial cells and macrophages. NO reacts with superoxide to form peroxynitrite (ONOO-), a potent agent of LDL oxidation in vitro. ONOO- nitrates the aromatic ring of free tyrosine to produce 3-nitrotyrosine, a stable product. To explore the role of reactive nitrogen species such as ONOO- in the pathogenesis of vascular disease, we developed a highly sensitive and specific method involving gas chromatography and mass spectrometry to quantify 3-nitrotyrosine levels in proteins. In vitro studies demonstrated that 3-nitrotyrosine was a highly specific marker for LDL oxidized by ONOO-. LDL isolated from the plasma of healthy subjects had very low levels of 3-nitrotyrosine (9 +/- 7 micromol/mol of tyrosine). In striking contrast, LDL isolated from aortic atherosclerotic intima had 90-fold higher levels (840 +/- 140 micromol/mol of tyrosine). These observations strongly support the hypothesis that reactive nitrogen species such as ONOO- form in the human artery wall and provide direct evidence for a specific reaction pathway that promotes LDL oxidation in vivo. The detection of 3-nitrotyrosine in LDL isolated from vascular lesions raises the possibility that NO, by virtue of its ability to form reactive nitrogen intermediates, may promote atherogenesis, counteracting the well-established anti-atherogenic effects of NO.

Arteries↗
PubMed1997

Bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as a new class of atomically flat substrates for biological scanning probe microscopy.

This is the first report of bioreactive self-assembled monolayers, covalently bound to atomically flat silicon surfaces and capable of binding biomolecules for investigation by scanning probe microscopy and other surface-related assays and sensing devices. These monolayers are stable under a wide range of conditions and allow tailor-made functionalization for many purposes. We describe the substrate preparation and present an STM and SFM characterization, partly performed with multiwalled carbon nanotubes as tapping-mode supertips. Furthermore, we present two strategies of introducing in situ reactive headgroup functionalities. One method entails a free radical chlorosulfonation process with subsequent sulfonamide formation. A second method employs singlet carbenemediated hydrogen-carbon insertion of a heterobifunctional, amino-reactive trifluoromethyl-diazirinyl crosslinker. We believe that this new substrate is advantageous to others, because it (i) is atomically flat over large areas and can be prepared in a few hours with standard equipment, (ii) is stable under most conditions, (iii) can be modified to adjust a certain degree of reactivity and hydrophobicity, which allows physical adsorption or covalent crosslinking of the biological specimen, (iv) builds the bridge between semiconductor microfabrication and organic/biological molecular systems, and (v) is accessible to nanopatterning and applications requiring conductive substrates.

Alkanes↗
PubMed1997

Analysis of sleep EEG microstructure in subchronic paroxetine treatment of healthy subjects.

Paroxetine is a selective and potent serotonin reuptake inhibitor and its efficacy for the treatment of depression has been proven. Under acute and subchronical treatment regimens, disturbances of the regular sleep pattern are a reported side effect of the drug. The present study was therefore performed to investigate the impact of subchronic treatment with the selective serotonin reuptake inhibitor paroxetine on the microstructure of the sleep EEG. The study especially addressed the question of subchronic effects of paroxetine medication (30 mg/day) in eight healthy male volunteers in a double blind, placebo-controlled crossover design. Conventional sleep EEG parameters and a spectral power analysis for different sleep stages after 4 weeks of treatment were computed. Additionally, the correlation of certain EEG rhythms across the night was calculated in order to detect subtle dynamical EEG alterations, not necessarily obvious when regarding conventional EEG analysis. Although we could not detect any alterations of the spectral power values in certain frequency bands either during NREM nor during REM sleep following subchronic paroxetine medication, the dynamical EEG attributes across the night revealed a significant enhancement of the correlation between certain EEG rhythms mainly during NREM sleep.

Adult↗
PubMed1997

The influence of lorazepam upon pre- and poststimulus EEG during sleep in man.

Enhancement of beta power in the spontaneous EEG under the influence of benzodiazepine medication is a well known phenomenon under waking conditions as well as during sleep. On the other hand, in certain frequency bands EEG activity following external stimulation is also enhanced. Therefore, the question arises what relationship exists between pre- and poststimulus EEG under the influence of benzodiazepines. Is an augmentation of beta activity during benzodiazepine medication further enhanced when external stimulation is applied? In order to address this question we compared the spectral power of pre- and poststimulus EEG under 2.5 mg lorazepam during sleep with placebo conditions. The hypothesis tested was that of Basar (1980): if the beta power is high prior to the stimulation, no further enhancement following stimulation is observed. Our results confirm the hypothesis during sleep stages II and III. At the present time it is unclear whether this effect might be dose-dependent. In any case, the investigation presented here has furnished additional information on the mechanisms by which benzodiazepines alter the brain's electrical activity.

Acoustic Stimulation↗
PubMed1997

[Results after combined phacoemulsification and trabeculectomy by scleral tunnel incision and goniotrepanation alone].

PURPOSE: We retrospectively evaluated the results of combined phacoemulsification and trabeculectomy through scleral tunnel incision in comparison with those of goniotrephination alone. PATIENTS AND METHODS: 27 eyes of 20 patients (78.3 +/- 7.45 years) underwent a combined trabeculectomy and phacoemulsification with IOL-implantation (PT-group). The trabeculectomy was performed by excision of 2 mm middle part of the lower tunnel lip. In comparison with the TP-group 29 eyes of 28 patients (69.3 +/- 8.1 years) underwent a goniotrephination (Fronimopoulos), (G-group). The median of follow up was 8 weeks. RESULTS: In both groups a significant reduction of the IOP was achieved; in the PT-group the IOP was reduced from 31.0 +/- 5.42 to 15.0 +/- 4.5 mm Hg, in the G-group from 31.4 +/- 6.3 to 11.6 +/- 4.12 mmHg. In the PT-group the visual acuity increased significantly from 0.25 +/- 0.14 to 0.37 +/- 0.27 and decreased significantly from 0.46 +/- 0.28 to 0.32 +/- 0.25 in the G-group. 3 patients (= 11%) needed topical medication in the PT-group and 6 (= 24%) in the G-group. Four patients (= 15%) of the PT-group underwent a secondary surgery and 7 (= 24%) of the G-group. CONCLUSIONS: The combined phacoemulsification and trabeculectomy is a successful method of IOP control and visual rehabilitation. Due to the decrease of visual acuity and a higher frequency of secondary surgery after goniotrephination alone the combined procedure should be preferred even in presence of a moderately visual acuity reducing cataract.

Aged↗
PubMed1997

On the role of myosin-II in cytokinesis: division of Dictyostelium cells under adhesive and nonadhesive conditions.

We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (DeltaBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing DeltaBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, DeltaBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a DeltaBLCBS-myosin is similar to that in wild-type cells.

Animals↗
PubMed1997

Copper ions promote peroxidation of low density lipoprotein lipid by binding to histidine residues of apolipoprotein B100, but they are reduced at other sites on LDL.

Oxidized LDL is implicated in the pathogenesis of atherosclerosis. A widely studied model for oxidation of the lipid in LDL involves Cu2+. Recent studies suggest that Cu2+ may be reduced to Cu1+ by alpha-tocopherol to initiate LDL lipid peroxidation. LDL demonstrates binding sites for Cu2-, but the nature of these binding sites, as well their role in promoting Cu2+ reduction and lipid peroxidation, has not been established. In the current studies, we used diethylpyrocarbonate (DEPC) to modify the histidine residues of apolipoprotein B100, the major protein in LDL. First, we demonstrated that histidine residues were preferentially modified by DEPC under our experimental conditions. Then we monitored the kinetics of Cu(2+)-promoted oxidation of LDL and DEPC-modified LDL. In both cases, the progress curve of lipid peroxidation exhibited a lag phase and a propagation phase. However, when LDL was modified with DEPC, the length of the lag phase was prolonged whereas the rate of lipid peroxidation during the propagation phase was lower. Studies with LDL oxidized by 2,2'-azobis (2-amidinopropane) hydrochloride and phosphatidylcholine liposomes oxidized with hydroxyl radical established that DEPC was not acting simply as a nonspecific inhibitor of lipid peroxidation. DEPC treatment of LDL almost completely inhibited its ability to bind Cu2+. These observations suggest that peroxidation of the lipids in LDL can proceed with normal kinetics only when Cu2+ binds preferentially to sites on apolipoprotein B100 that contain histidine residues. We also compared the kinetics of Cu2+ reduction in the absence and presence of DEPC. There was no effect of DEPC modification on either the rate or extent of Cu2+ reduction by LDL. Therefore LDL is likely to contain a second class of binding sites for Cu2+ that does not involve histidine residues. Thus, LDL appears to contain at least two classes of Cu(2+)-binding sites: histidine containing sites, which are responsible in part for promoting lipid peroxidation during the propagation phase, and sites at which Cu2+ is reduced without binding to histidine.

Antioxidants↗
PubMed1997

Human p53 restores DNA synthesis control in fission yeast.

The p53 gene is the most common target for genetic alterations in human cancers. As a transcriptional regulator p53 enhances the expression of proteins that control cellular proliferation. Although there is no evidence of a p53 homologous gene in yeast, the p53 protein was found to be functional in terms of growth repression and transactivation in yeast, suggesting that some features of p53 function are conserved. Here we report the construction and characterization of a p53 wild type expression strain of fission yeast. Upon induction of wild type p53 expression a dosage dependent growth arrest was observed rendering recipient yeast cells sensitive to UV irradiation in a p53 dosage dependent fashion. The observed growth arrest was efficiently suppressed by coexpression of human CDC25C phosphatase, which restored a normal resistance to UV irradiation in p53 and CDC25C coexpressing yeast cells. Furthermore, expression of CDC25C alone inactivated the DNA synthesis control whereas p53 and CDC25C coexpressing yeast cells showed an intact checkpoint control. Upon moderate expression of wild type p53 a restoration of the DNA synthesis control was also observed in a cdc2.3w mutant background, whereas a tumor mutant of p53 failed to restore this important checkpoint in fission yeast.

Cell Cycle Proteins↗
PubMed1996

Single trial analysis of event related potentials: a comparison between schizophrenics and depressives.

The aim of the present paper was to perform a single trial analysis of event-related potentials in order to elucidate the mechanisms behind a reduced P300 amplitude occurring in unmedicated depressives (n = 11) and schizophrenics (n = 18). For this purpose, tools from linear system theory were applied to single trials in an oddball paradigm. This analysis provides estimates of the magnitude of the positive deflection occurring around the latency of P300 following target and nontarget simuli. According to the density functions of these amplitude distributions, we operationally defined "false-negative" (P300 amplitude lower than an individual threshold under target conditions) as well as "false-positive" responses (P300 amplitude higher than the threshold following nontarget stimuli). Our investigations revealed a reduction of the single-trial P300 amplitude in depression and a combination of amplitude reduction along with fewer elicited single-trial P300 waves in schizophrenics.

Adult↗
PubMed1996

Amplitude frequency characteristics of evoked potentials during sleep: an analysis of the brain's transfer properties in depression.

Classical analysis of the spontaneous sleep EEG in depressive disorder commonly reveals alterations of sleep continuity, number of awakenings, slow-wave sleep, and REM sleep compared to healthy controls; however, conventional analysis can not help understand dynamic differences of the sleep EEG during different sleep stages. In order to elicit qualitative alterations of information processing between depressives and healthy controls, we measured late components of auditory and visual evoked potentials (AEPs and VEPs) during different sleep stages of 15 depressive inpatients and in a sex- and age-matched control group from scalp positions Fz, Cz and Pz. According to linear system theory, we then computed the amplitude frequency characteristic (AFC) from averaged AEPs and VEPs in different sleep stages. These AFCs describe the input/output relation of the system under study leading to a characterization of the transfer properties of the brain during sleep in depression. Our investigations showed that information processing appears characteristically altered in depression during non-REM sleep for both auditory and visual stimulation compared to healthy controls. The transfer properties for processing auditory as well as visual information during REM sleep do not appear dynamically impaired in depressive disorder.

Adult↗
PubMed1996

[Acute kidney insufficiency in an alcoholic patient with liver cirrhosis].

We report on a 67-year-old patient who was hospitalized because of worsening of general condition, generalized edema and ascites with decompensated ethylic cirrhosis of the liver. On hospital admission renal failure was present which had developed within 5 months. In spite of intensive treatment with diuretics and repeated ascites punctures, fluid repletion reappeared repeatedly. Renal function remained stable for the first 10 days but worsened thereafter rapidly.

Aged↗
PubMed1996

p21WAF1/CIP1 interacts with protein kinase CK2.

p21WAF1/CIP1 which belongs to a class of regulatory proteins that interact with cyclin dependent kinases is a potent inhibitor of these kinases. The inhibition of the cyclin dependent kinases induces an arrest of cells in the G phase of the cell cycle. In addition p21WAF1/CIP1 associates with PCNA and inhibits DNA replication. Here, we show that p21WAF1/CIP1 binds to the regulatory beta-subunit of protein kinase CK2 but not to the catalytic alpha-subunit. Binding of p21WAF1/CIP1 down regulates the kinase activity of CK2 with respect to the phosphorylation of the beta-subunit of CK2, casein and the C-terminus of p53. This study demonstrates a new binding partner for the regulatory beta-subunit of protein kinase CK2 which regulates the activity of the holoenzyme.

Amino Acid Sequence↗