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Biomedical subjects

R Wagner

Publications and source records attributed to R Wagner.

At least 91 records · Page 5Linked to original sources
PubMed2000

Hemodynamic measurement in liver transplantation. Piggyback versus conventional techniques.

The piggyback technique in venous outflow tract reconstruction has been proposed as an alternative to the conventional technique in liver transplantation. Maintaining caval flow during the anhepatic phase with hemodynamic stability is regarded as one of the main advantages of this method. Between November 1994 and November 1998, the piggyback technique was used in 47 patients in our Center. Hemodynamic measurements during the operation showed hyperdynamic circulation with an increase in cardiac output (9.3+/-3.5 L/min) and the calculated cardiac index (5.0+/-1.9 L/min/m2). There was a statistically significant increase in heart rate and a decrease in systolic arterial pressure, cardiac output (CO) and cardiac index (CI) during inferior vena cava clamping ( simulated conventional technique). Only a non-significant decrease in CO and CI was observed during the partial clamp on the inferior vena cava (piggyback technique). Out of those two techniques, piggyback proved to be a safer approach to venous outflow tract reconstruction from the hemodynamic point of view.

Adolescent↗
PubMed1999

Exploring the open pore of the potassium channel from Streptomyces lividans.

The tetrameric potassium channel from Streptomyces lividans (KcsA) embedded in planar bilayers exhibits the following electrophysiological characteristics: (i) K+ ions can cross the pore in a highly hydrated state (nH2O > or = 6), (ii) the selectivity for K+ exceeds that for Na+ ions by 11 times, and both Ca2+ and Mg2+ are permeant, (iii) the internal side is blocked by Ba2+ ions in a voltage-dependent manner, (iv) intrinsic rectification is due to gating, depending on the direction of the electric field, (v) the internal side is pH-sensitive, and (vi) the open pore has a diameter of approximately 5.8 A. In conclusion, our results show that ion conduction and selectivity of KcsA cannot easily be reconciled with the properties deduced from the rigid crystal structure [Doyle et al., Science 280 (1998) 69-77], which must be concluded to have the pore trapped in its closed state.

Bacterial Proteins↗
PubMed1999

Characterization of transient RNA-RNA interactions important for the facilitated structure formation of bacterial ribosomal 16S RNA.

The co-transcribed leader sequences of bacterial rRNA are known to affect the structure and function of the small ribosomal subunits. Base changes in the leader nut -like sequence elements have been shown to cause misfolded but correctly processed 16S rRNA structures at low growth temperature. Transient interactions of leader sequences with the nascent 16S rRNA are considered to guide rRNA folding and to facilitate correct structure formation. In order to understand this chaperone-like activity of the leader RNA we have analyzed the thermodynamic stabilities of wild-type and mutant leader transcripts. We show here that base changes cause subtle differences in the melting profiles of the corresponding leader transcripts. Furthermore, we show that direct interaction between leader transcripts and the 16S rRNA is limited to the 5'-domain of the 16S rRNA for both wild-type and mutant leaders. Binding studies of mutant and wild-type leader transcripts to 16S rRNA revealed small changes in the affinities and the thermal stabilities as a consequence of the base changes. Different complex stabilities as a function of the Mg(2+) ion concentration indicated that mutant and wild-type leader transcripts interact differently with the 16S rRNA, consistent with a less stable and tightly folded structure of the mutant leader. Employing time-resolved oligonucleotide hybridization assays we could show different folding kinetics for 16S rRNA molecules when linked to wild-type leader, mutant leader or in the absence of leader RNA. The studies help to understand how bacterial rRNA leader transcripts may affect the folding of the small subunit rRNA.

5' Untranslated Regions↗
PubMed1999

Cell lines with reduced UDP-N-acetylhexosamine pool in the presence of ammonium.

The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium. Glucosamine-6-phosphate isomerase (GPI) has been proposed as the key enzyme responsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc) by accepting ammonium from the medium of cultured mammalian cells. As previously reported, the increased UDPGNAc pool then affects the N-glycan complexity in glycoproteins. To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK-21 cells and characterized. Thus, the complete pathway by which ammonium enters the cellular metabolism was elucidated. To reduce the negative effects of ammonium, GPI was inhibited using two different strategies. First, the addition of mannose to the culture media and, second, antisense RNA expression. In both cases, the cellular UDPGNAc pool was suppressed in the presence of high ammonium concentrations in the medium. However, constant suppression of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable. Further studies showed that the main reason for instability was the inducibility of GPI by its substrate ammonium. GPI was induced to a factor of two under ammonium-containing medium conditions. We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the presence of ammonium.

Aldose-Ketose Isomerases↗
PubMed1999

A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea.

Phosphorylated carbohydrates are the main photoassimilated export products from chloroplasts that support the energy household and metabolism of the plant cell. Channels formed by the chloroplastic outer envelope protein OEP21 selectively facilitate the translocation of triosephosphate, 3-phosphoglycerate and phosphate, central intermediates in the source-sink relationship between the chloroplast and the cytosol. The anion selectivity and asymmetric transport properties of OEP21 are modulated by the ratio between ATP and triosephosphates, 3-phosphoglycerate and phosphate in the intermembrane space. Conditions that lead to export of triosephosphate from chloroplasts, i.e. photosynthesis, result in outward-rectifying OEP21 channels, while a high ATP to triosephosphate ratio, e.g. dark metabolism, leads to inward-rectifying OEP21 channels with a less pronounced anion selectivity. We conclude that solute exchange between plastids and cytosol can already be regulated at the level of the organellar outer membrane.

Adenosine Triphosphate↗
PubMed1999

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

Biological Transport↗
PubMed1999

Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins.

The negative effects of ammonia on animal cells, especially in vitro cultures, are well known, but the mechanism of how ammonia inhibits cell growth and influences the glycosylation of proteins is not completely understood. We investigated the ammonium action on the synthesis of the intracellular UDP-N-acetylhexos- amines (UDPGNAc), which are precursors of glycosylation as well as on N-linked oligosaccharides of a recombinant human IL-2 mutant variant model glycoprotein expressed in BHK-21 cells under defined and controlled culture conditions in a continuously perfused bioreactor. The examinations were based on our previous observations that increased ammonia concentrations in the medium lead to the intracellular formation and accumulation of UDPGNAc (Ryll et al., 1994). The kinetics of formation of the UDPGNAc pool after adding ammonia and its reconstitution to normal conditions are shown. To study the pathway leading to the intracellular increase of UDPGNAc, the uptake and incorporation of 15NH4+ was confirmed by the detection of 15N in UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was purified using high pH anion-exchange chromatography with pulsed amperometric detection and analyzed by GC/MS. The proportion of UDP-GlcNAc containing 15N was approximately 60% and corresponds quantitatively to the increased intracellular concentration of UDP-GlcNAc. In order to confirm the direct influence of ammonia on protein glycosylation, the human IL-2 mutant glycoprotein variant IL-Mu6, bearing a novel N-glycosylation site, has been produced under defined protein-free medium conditions in the presence of 15NH4Cl. IL-Mu6 glycoprotein was purified and N-glycans released were analyzed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Maximally 60-80% of N-acetylated sugars in N-glycan structures contained 15N indicating that ammonium is used as a building block during synthesis of the carbohydrate structures expressed from in vitro cultivated mammalian cells.

Animals↗
PubMed1999

Competitive inhibition of human immunodeficiency virus type-1 protease by the Gag-Pol transframe protein.

The human immunodeficiency virus type-1 (HIV-1) transframe protein p6* is located between the structural and enzymatic domains of the Gag-Pol polyprotein, flanked by the nucleocapsid (NC) and the protease (PR) domain at its amino and carboxyl termini, respectively. Here, we report that recombinant highly purified HIV-1 p6* specifically inhibits mature HIV-1 PR activity. Kinetic analyses and cross-linking experiments revealed a competitive mechanism for PR inhibition by p6*. We further demonstrate that the four carboxyl-terminal residues of p6* are essential but not sufficient for p6*-mediated inhibition of PR activity. Based on these results, we suggest a role of the transframe protein p6* in regulating HIV-1 PR activity during viral replication.

Amino Acid Sequence↗
PubMed1999

Correlates of protection, antigen delivery and molecular epidemiology: basics for designing an HIV vaccine.

Major obstacles in the development of HIV vaccines are the high variability of the virus and its complex interaction with the immune system. Recent studies demonstrated, that CTLs recognizing highly conserved epitopes in the group-specific antigen are capable of controlling HIV-replication in long-term nonprogressors. Necessary consequences for novel vaccine concepts are the presentation of a large repertoire of antigenic sites as well as the stimulation of different effectors of the immune system. Accordingly, different types of recombinant HIV-1 virus-like particles (VLPs) have been constructed stimulating the induction of neutralizing antibodies and HIV-specific CD8-positive CTL responses in preclinical studies. With respect to future vaccine trials, HIV vaccine formulations may need to be tailored to the local strains circulating within a geographical region. The expert group of the joint United Nations Programme on AIDS recently identified Yunnan, a southwestern province of China, as a region, in which the HIV epidemic is starting to gain speed, resembling to the situation in Thailand 10 years ago. A molecular clone of a representative virus strain is now available for the development of innovative antigen delivery systems aiming to be evaluated in future clinical vaccine trials throughout this area.

AIDS Vaccines↗
PubMed1999

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.

Animals↗
PubMed1999

Conformational changes of the upstream DNA mediated by H-NS and FIS regulate E. coli RrnB P1 promoter activity.

The two proteins FIS and H-NS had previously been shown to regulate ribosomal RNA (rRNA) transcription by interacting with the promoter upstream DNA. FIS is known as an activator whereas H-NS had been demonstrated to function as a repressor. Details of the antagonistic control mechanisms are not yet solved. Here, we have addressed the question how the two proteins cooperate to exert both, positive and negative control of rRNA transcription. By mobility shift experiments and footprinting studies we show that FIS and H-NS binding sites partially overlap but appear to interact with different sites of a curved DNA helix. Although not mutually exclusive, the two proteins compete each other for binding. Both proteins, by changing the DNA curvature, effect circularization reactions of DNA fragments in different ways. Our results imply that binding of the proteins induces alternate DNA conformations with favourable or unfavourable topology for the formation of active transcription complexes. Together the findings presented here help to answer some of the open questions about the concerted molecular mechanism of transcription factors for the regulation of stable RNA synthesis.

Bacterial Proteins↗
PubMed1999

Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction.

Continuous mammalian cell lines are important hosts for the production of biological pharmaceuticals. However, these cell lines show some severe disorders in primary metabolism, which they have in common with many cancer cells. This leads to a high throughput of substrates giving a low energy yield and ample toxic side products such as lactate and ammonia. Because the enzymatic connection between glycolysis and the tricarboxylic acid cycle (TCA) is very poor, glucose is mainly degraded via oxidative glycolysis. It will be shown that introducing a pyruvate carboxylase gene expressed in the cytoplasma into a continuous BHK-21 cell line, and thus reconstituting the missing link between glycolysis and TCA, can reduce this problem. Thus, glucose consumption could be reduced by a factor of four and glutamine utilization up to a factor of two, compared with control. Moreover, a 1.4-fold-higher adenosine triphosphate (ATP) content was achieved. The flux of labeled [(14)C]-glucose into the TCA is shown to be enhanced, indicating a higher rate of oxidative glucose degradation. Host cell lines with an improved energy metabolism will therefore result in better exploitation of substrates, an increasing yield by the more efficient use of carbon source, and higher product integrity combined with lower production costs.

Adenosine Triphosphate↗
PubMed1999

Cultivation of Tetrahymena thermophila in a 1.5-m3 airlift bioreactor.

A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations.

Animals↗
PubMed1999

Observations on emerging patterns of asthma in our society.

Epidemiologic studies of temporal and geographic variation in asthma morbidity have identified asthma as an important public health concern. Knowledge gained from these studies has resulted in intense focus on this condition by agencies such as the National Institutes of Health and the World Health Organization. In this report, studies of recent asthma trends and patterns are explored. These studies show increases in US prevalence through 1994. Data on measures of morbidity show complex longitudinal patterns but are notable for large differences in emergency department services and hospitalizations by age and race. Very recent trends for US asthma mortality suggest widening of an existing racial gap. Limited comparisons are possible between these US trends and international trends but do suggest that US increases in prevalence and mortality rates may reflect world-wide patterns. Also, within the US, it is clear that geographic variation exists in asthma prevalence, morbidity, and mortality rates. Changes in certain environmental risk factors and exposures may contribute to recent trends, but little information is available relating specific risk factors to either longitudinal asthma trends, geographic variability, or high-risk populations.

Asthma↗
PubMed1999

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

AIDS Vaccines↗
PubMed1999

High level expression of hepatitis B virus surface antigen in stably transfected Drosophila Schneider-2 cells.

Two transfer vector systems have been constructed for the generation of Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably and used to express the small surface antigen of hepatitis B virus (HBsAg). One system is based on the cotransfection of an expression vector for the S gene under the control of an inducible Drosophila metallothionein (Mtn) promotor and a resistance plasmid which carries a selectable marker dihydrofolate reductase (dhfr) gene under the control of a Drosophila actin 5C distal promoter. The second system is based on the transfection of a single plasmid, which includes both expression units. Both vector systems were suitable for the generation of stably transfected DS-2 cell-lines secreting high levels of HBsAg. The quantities of HBsAg expression from polyclonal DS-2 cells correlated strictly with the concentration of the transfected S gene expression vector. Clonal cell-lines selected from the most efficient HBsAg producing polyclonal cell-populations were examined in more detail. All of the transfected S genes were found to be integrated and the copy numbers per genome varied extremely between 10 and 240. Furthermore, the levels of secreted HBsAg varied greatly between different clones and in best they reached up to 7 microg/ml under serum-free cell culture conditions. Thus, DS-2 cells transfected stably provide an alternative source for the production of HBsAg particles for diagnostic purposes and vaccine development.

Animals↗
PubMed1999

Purification and characterization of hepatitis B virus surface antigen particles produced in Drosophila Schneider-2 cells.

The small surface antigen of hepatitis B virus (HBV) was produced in Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably using an inducible Drosophila metallothionein promoter. Selected clonal DS-2 cell-lines expressed and secreted large quantities of HBsAg particles consisting exclusively of non-glycosylated 25 kDa proteins. HBsAg produced by DS-2 cells had physical and biochemical properties very similar to 22 nm particles derived from the human hepatoma cell-line PLC/PRF/5. DS-2 cell-derived HBsAg particles were purified near homogeneity by a strategy based on protein concentration, precipitation and ultracentrifugation. The resulting HBsAg product was < 98% pure. A single immunisation of BALB/c mice with both DS-2 and yeast-cell derived purified HBsAg particles without adjuvants elicited a substantial humoral antibody and class-I restricted cytotoxic T lymphocyte (CTL) response. Adsorption of HBsAg particles to aluminium hydroxide resulted in increased levels of HBsAg-specific antibodies. However, CTLs were not elicited by HBsAg/Alum combinations. Thus, stably transfected DS-2 cells provide a useful source for the production of HBV subviral particles for diagnostic and research purposes as well as for novel vaccine development.

Animals↗