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Biomedical subjects

R Wagner

Publications and source records attributed to R Wagner.

At least 181 records · Page 10Linked to original sources
PubMed1996

Nonlinear Optics in Relativistic Plasmas and Laser Wake Field Acceleration of Electrons

When a terawatt-peak-power laser beam is focused into a gas jet, an electron plasma wave, driven by forward Raman scattering, is observed to accelerate a naturally collimated beam of electrons to relativistic energies (up to 10(9) total electrons, with an energy distribution maximizing at 2 megaelectron volts, a transverse emittance as low as 1 millimeter-milliradian, and a field gradient of up to 2 gigaelectron volts per centimeter). Electron acceleration and the appearance of high-frequency modulations in the transmitted light spectrum were both found to have sharp thresholds in laser power and plasma density. A hole in the center of the electron beam may indicate that plasma electrons were expelled radially.

Journal Article↗
PubMed1996

Construction, expression, and immunogenicity of chimeric HIV-1 virus-like particles.

The group-specific antigens Pr55gag of human immunodeficiency virus type-1 (HIV-1) self-assemble into noninfectious virus-like particles (VLP) that are released from various eucaryotic cells by budding. Deletion analysis of Pr55gag mutants revealed three domains into which sequences of the third variable domain V3 or the CD4-binding domain of the gp120 external glycoprotein can be inserted without destroying the capacity of the chimeric proteins to assemble to VLP. Immunization of rabbits with different types of purified chimeric VLP without adjuvants raised a strong antibody response to the Pr55gag carrier component. The magnitude of the antibody response to the inserted gp 120 epitopes strictly depended on their position within the gag polyprotein. These antisera exhibited only weak neutralizing activity. However, BALB/c mice immunized by different routes with different types of chimeric Pr55gag/V3 VLP without adjuvants developed a strong MHC class I (Dd)-restricted, cytolytic CD8+ T-cell (CTL) reactivity against a known epitope within the V3 domain. When the recombinant antigen was emulsified in mineral oil (incomplete Freund's adjuvant) or adsorbed in aluminium hydroxide, its immunogenicity for CTL was drastically reduced or completely abrogated. The magnitude of the V3-specific CTL response was not influenced by the position of the V3 domain within the Pr55gag-carrier moiety; the flanking residues, hence, did not influence processing of the exogenous antigen for MHC class I-restricted peptide presentation. These results indicate ways for the rational design and optimal delivery of CTL-stimulating HIV candidate vaccines.

Animals↗
PubMed1996

Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6.

The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.

Amino Acid Sequence↗
PubMed1996

Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells.

Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodoptera frugiperda) cell line using radioactive substrates. A high degree of similarity in many aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always close to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucose- or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/or productivity.

Animals↗
PubMed1996

Qualitative [18F]FDG positron emission tomography in primary breast cancer: clinical relevance and practicability.

Positron emission tomography (PET) using fluorine-18 2-deoxy-2-fluoro-d-glucose (FDG) is of potential value for the diagnosis of malignant tumours. The aim of this study was to evaluate the use of FDG PET in patients with breast tumours, appraising its applicability in visualising primary carcinomas and regional metastases in a clinical setting. Results of FDG PET were compared with those of mammography, breast ultrasonography and histology in 30 patients with inconclusive breast findings. For PET, transmission and emission images were taken in one or two scan positions, depending on the available time and the clinical status of patients. PET showed focal FDG uptake with high contrast in 21 of 23 primary carcinomas. In one patient, only PET correctly visualized multifocal disease (three foci, O 0.4-1 cm). The accuracy of PET in the detection of primary breast cancer was 90%, and in the detection of involved axillary lymph nodes, 94%. All metastases (lymph nodes, lungs, bones, soft tissues) covered by the field of view and demonstrated by other methods (X-ray, computed tomography, magnetic resonance imaging, bone scan) showed FDG uptake. In three patients, only PET initiated further diagnostic procedures. The results indicate that FDG PET can provide a rapid diagnostic study (45-60 min) and allows accurate tumour staging of several organ systems for primary tumour and metastases with a single imaging study in a routine clinical setting.

Breast Neoplasms↗
PubMed1996

Pre-emptive dynorphin and N-methyl-D-aspartate glutamate receptor antagonism alters spinal immunocytochemistry but not allodynia following complete peripheral nerve injury.

The development of chronically painful states following peripheral nerve injury may involve different mechanisms depending on the nature and extent of the nerve lesion. The altered spinal neurochemistry of two substances, the excitatory amino acid glutamate operating via the N-methyl-D-aspartate receptor and the endogenous opioid peptide dynorphin, have been implicated in behavioral sequelae that follow partial peripheral nerve injury. In addition, dynorphin has nonopioid functions which may involve the N-methyl-D-aspartate receptor. We investigated two hypotheses: that the development of mechanical allodynia following complete nerve injury is not greatly influenced by the N-methyl-D-aspartate receptor, and that spinal dynorphin and glutamate expression is interdependent. These studies employed sciatic cryoneurolysis, a complete but transient peripheral nerve injury that results in a delayed mechanical allodynia beginning 21-28 days after injury. Rats were administered dizocipline maleate (MK-801) at 0.25 mg/kg twice per day intraperitoneally from days 0-7 or from days 0-21 post-lesion to pre-emptively block the N-methyl-D-aspartate receptor. In a separate group of rats, an antibody to dynorphin was administered intraperitoneally at 16.6 mg/kg twice per day from days 14 to 21 post-lesion. For all groups, the outcome of allodynia behavior was assessed using von Frey filaments at 42 days post-lesion and the resulting dynorphin and glutamate immunoreactivity in the substantia gelatinosa was measured using proportional area stained and relative optical density, respectively. Only the 0-7 day MK-801 treatment increased the resulting mechanical thresholds significantly (mean +/- S.E.M. 7.0 +/- 1.2 g) when compared to saline-injected animals (3.9 +/- 0.6 g). However, this effect did not prevent allodynia since baseline thresholds were 12 or 15 g for each group. With regard to resulting spinal immunoreactivity, anti-dynorphin antibody treatment significantly increased glutamate immunoreactivity when compared to saline-treated animals (mean relative optical density +/- S.E.M. = 807.2 +/- 3.6 versus 779.6 +/- 8.3, respectively; P = 0.01) at 42 days post-lesion. We conclude that the development of allodynia following sciatic cryoneurolysis peripheral nerve injury involved a minimal contribution from N-methyl-D-aspartate receptor activity. In addition, this study demonstrated that decreasing available dynorphin using antiserum had a significant and lasting effect on spinal glutamate expression without altering the outcome of allodynia. These conclusions suggest that etiological mechanisms leading to pain behaviors are not equal for all nerve injuries, and that altering kappa opioid levels can affect glutaminergic pathways at a substantially later time.

Animals↗
PubMed1996

Schwann cells produce tumor necrosis factor alpha: expression in injured and non-injured nerves.

We investigated the production of tumor necrosis factor alpha, a pleotropic cytokine, in the rat sciatic nerve using immunohistochemistry for the protein and in situ hybridization for specific messenger RNA sequences. Results demonstrated that in the uninjured sciatic nerve, few endothelial cells, fibroblasts, and Schwann cells were immunoreactive for tumor necrosis factor. In situ hybridization did not yield visible results in this control tissue. Following a compression injury, the number of immunoreactive cells in the nerve fascicle was increased. In situ hybridization showed positive staining in Schwann cells and endothelial cells at seven days. These observations are the first to demonstrate the production of this proinflammatory cytokine by peripheral nerve glia, and further support other studies from this laboratory suggesting that tumor necrosis factor has a pathogenic role in nerve injury. Because this cytokine is produced by Schwann cells in intimate contact with nerve fibers, tumor necrosis factor may also have a role in the hyperalgesia associated with neuropathic pain.

Animals↗
PubMed1996

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human beta 1,2-N-acetylglucosaminyltransferase I.

Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I.

Animals↗
PubMed1996

Characterization of ouabain-like immunoreactivity in human urine.

OBJECTIVE: To characterize ouabain-like immunoreactivity in human urine. METHODS: Sensitive radioimmunoassay for ouabain characterized by high-performance liquid chromatography. RESULTS: Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86 nmol (62.5 micrograms) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7 nmol/l 8 min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain. CONCLUSION: A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma.

Adult↗
PubMed1996

N-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector.

The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (Sf9) cells and in Estigmene acrea cells by using a baculovirus vector. Structural analysis revealed that the endo-H-resistant N-glycans of HA from Sf9 cells were predominantly trimannosyl core oligosaccharides, whereas in E. acrea cells most of these cores were elongated by at least one terminal N-acetylglucosamine residue. To understand the difference in carbohydrate structures, enzymes involved in N-glycan processing have been analyzed. The results revealed that the different glycosylation patterns observed are due to an N-acetyl-beta-glucosaminidase activity that was found in Sf9 cells but not in E. acrea cells. This enzyme specifically used the GlcNAcMan(3)GlcNAc(2) oligosaccharide as a substrate. When N-acetyl-beta-glucosaminidase or alpha-mannosidase II was inhibited by specific inhibitors, the amount of terminal N-acetylglucosamine in hemagglutinin from Sf9 cells was significantly enhanced. These results demonstrate that N glycosylation in both cell lines follows the classical pathway up to the stage of GlcNAcMan(3)GlcNAc(2) oligosaccharide side chains. Whereas these structures are the end product in E. acrea cells, they are degraded in Sf9 cells to Man(3)GlcNAc(2) cores by N-acetyl-beta-glucosaminidase.

Acetylglucosaminidase↗
PubMed1996

Safety and immunogenicity of recombinant human immunodeficiency virus-like particles in rodents and rhesus macaques.

Data from long-term non-progressing human immunodeficiency virus (HIV)-infected individuals and populations at high risk suggest that an early cytolytic T cell response rather than the humoral immune response might be involved in controlling disease progression. These observations may be used as a guide to the type of response that a vaccine should induce. To clarify the role of different arms of the immune system in conferring protection, the candidate vaccine should allow a regulated, selective induction of different immune responses. Based on a better understanding of the molecular mechanisms regulating the morphogenesis of HIV, we developed an autologous, non-replicating and safe antigen delivery system. This system takes advantage of molecular characteristics of the HIV group-specific antigens (gag) to self-assemble to highly immunogenic virus-like particles (VLP). The immunogenicity of the gag-derived VLP was expanded either by replacing defined domains by selected HIV-1 cytotoxic T lymphocyte (CTL) epitopes (type 1 VLP) or by stable anchoring derivatives of the HIV-1 envelope protein on the surface of the VLP (type 2 VLP). In complete absence of adjuvants, type 1 and type 2 VLP stimulated CD8+ CTL in BALB/c mice, which specifically recognised HIV sequences. In contrast to type 1 VLP, generating an HIV-specific CTL response in the absence of env-specific antibodies, type 2 VLP induced both arms of the immune system including reasonable levels of neutralising antibodies. Initial studies performed in rhesus macaques confirmed these results. Thus, depending on the type and formulation of the VLP, the proposed antigen delivery system allows either the induction of a CTL response (1) in the absence and (2) the presence of an envelope-specific antibody response. A comparison of these approaches in appropriate animal models might contribute to further define the correlates of protection.

Animals↗
PubMed1996

[Computer-assisted multimedia interactive learning program "Primary Open-Angle Glaucoma"].

INTRODUCTION: Advances in the area of information technology have opened up new possibilities for the use of interactive media in the training of medical students. Classical instructional technologies, such as video, slides, audio cassettes and computer programs with a textbook orientation, have been merged into one multimedia computer system. The medical profession has been increasingly integrating computer-based applications which can be used, for example, for record keeping within a medical practice. The goal of this development is to provide access to all modes of information storage and retrieval as well as documentation and training systems within a specific context. MATERIAL AND METHODS: Since the beginning of the winter semester 1995, the Department of Ophthalmology in Giessen has used the learning program "Primary Open Angle Glaucoma" in student instruction. One factor that contributed to the implementation of this project was that actual training using patients within the clinic is difficult to conduct. Media-supported training that can provide a simulation of actual practice offers a suitable substitute. The learning program has been installed on Power PCs (Apple MacIntosh), which make up the technical foundation of our system. The program was developed using Hypercard software, which provides userfriendly graphical work environment. This controls the input and retrieval of data, direct editing of documents, immediate simulation, the creation of on-screen documents and the integration of slides that have been scanned in as well as QuickTime films. All of this can be accomplished without any special knowledge of programming language or operating systems on the part of the user. RESULTS: The glaucoma learning program is structured along the lines of anatomy, including an explanation of the circulation of the aqueous humor, pathology, clinical symptoms and findings, diagnosis and treatment. This structure along with the possibility for creating a list of personal files for the user with a collection of illustrations and text allows for quick access to learning content. The program is designed in such a way that working with and through it is done in a manner conducive to learning. Student response to the learning program as an accompaniment to instruction has been positive. CONCLUSION: Independent, supplemental student learning by means of an interactive learning program has raised the quality of study within the sciences. The use of a pedagogically sound multimedia program, that is oriented toward problem solving and based on actual cases offers students the opportunity to actively work ophthalmological material. An additional benefit is the development of competence in working with computer-support information systems, something that is playing an ever-increasing role within the medical profession.

Computer-Assisted Instruction↗