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Biomedical subjects

K Haupt

Publications and source records attributed to K Haupt.

At least 19 recordsLinked to original sources
PubMed2001

Effect of gluconic acid as a secondary carbon source on non-growing L-lysine producers cells of Corynebacterium glutamicum. Purification and properties of 6-phosphogluconate dehydrogenase.

We studied the production of L-lysine in Corynebacterium glutamicum ATCC 21543 non growing cells obtained by nutrient limitation. Statistical analysis revealed significant differences in the L-lysine titers of glucose, gluconic acid or glucose-gluconic acid cultures. Higher L-lysine titer obtained in batch cultures with mixed carbon sources or gluconic acid alone were found to be associated with a high 6-phosphogluconate dehydrogenase activity (6PGDH, E.C.1.1.1.44). This enzyme is a pivotal enzyme within the hexose monophosphate pathway, and thus of importance for L-lysine production. 6PGDH was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 52.5 kDa. The molecular mass of the native enzyme was estimated to be 120 kDa by molecular exclusion chromatography, thus suggesting a homodimeric structure. The amino terminal sequence shows a strong similarity (a match of 86% of the first 20 amino acid) to the 6PGDH from other microorganisms such as, E. coli and B. subtilis. The pI of the dimeric native enzyme and the optimum pH were 6.2 and 8.0, respectively. For the oxidative decarboxylation of 6-phosphogluconate, K(m) of 71 &mgr;M and 43 &mgr;M were obtained for 6-phosphogluconate and NADP(+), respectively.

Journal Article↗
PubMed2001

Chemiluminescence imaging ELISA using an imprinted polymer as the recognition element instead of an antibody.

An imaging assay analogous to competitive enzyme immunoassays has been developed using a molecularly imprinted polymer instead of an antibody. The antigen 2,4-dichlorophenoxyacetic acid (2,4-D) was labeled with tobacco peroxidase, and the chemiluminescence reaction of luminol was used for detection. Microtiter plates (96 or 384 wells) were coated with polymer microspheres imprinted with 2,4-D, which were fixed in place by using poly(vinyl alcohol) as glue. In a competitive mode, the analyte-peroxidase conjugate was incubated with the free analyte in the microtiter plate, after which the bound fraction of the conjugate was quantified. After addition of the chemiluminescent substrates, light emission was measured in a high-throughput imaging format with a CCD camera. Calibration curves corresponding to analyte concentrations ranging from 0.01 to 100 microg/mL were obtained.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2001

Induction of a cellular and humoral immune response against preprocalcitonin by genetic i: a potential new treatment for medullary thyroid carcinoma.

Currently, no effective therapy exists for patients suffering from progressive medullary thyroid carcinoma (MTC), a calcitonin (CT)-secreting C cell tumor. As CT, which arises from the precursor protein preprocalcitonin (PPCT), is expressed by almost all MTC cases, these molecules may represent target antigens for immunotherapy against MTC. In our study we investigated whether DNA immunization is able to induce cellular and humoral immune responses against human PPCT (hPPCT) in mice. Antigen-encoding expression plasmids were delivered intradermally by gene gun. One group of mice received DNA encoding hPPCT only. Two groups were coinjected with mouse cytokine genes. We observed in lymphocyte proliferative assays substantial proliferation against hPPCT in mice coinjected with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, in contrast to mice vaccinated with hPPCT expression plasmid only. In addition, codelivery of the GM-CSF gene augmented the frequency of anti-hPPCT antibody seroconversions in sera of immunized animals, as shown by enzyme-linked immunosorbent assay. These results illustrate that cellular and humoral immune responses against hPPCT can be generated by DNA immunization and increased by coinjection of the GM-CSF gene. Our findings may have implications for the use of DNA immunization as a potential novel immunotherapeutic treatment for patients suffering from progressive MTC.

Animals↗
PubMed2000

New configurations and applications of molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) are applicable in a variety of different configurations. For example, bulk polymers imprinted with beta-lactam antibiotics are presented to be used as stationary phases for the chromatographic separation of beta-lactam antibiotics with both aqueous and organic mobile phases. However, in some analytical applications, monosized spherical beads are preferred over the currently used ground bulk polymers. A precipitation polymerization technique allows preparation of monosized spherical imprinted beads with diameters down to 200 nm having excellent recognition properties for different target molecules. Nevertheless, with current imprinting protocols a substantial amount of template has to be used to prepare the polymer. This can be problematic if the template is poorly soluble, expensive or difficult to obtain. It is shown that for analytical applications, the functional monomer:template ratio can be drastically increased without jeopardizing the polymer's recognition properties. Furthermore, a substantial reduction of the degree of crosslinking is demonstrated, resulting in much more flexible polymers that are useful for example the preparation of thin imprinted films and membranes for sensors. Apart from analysis, MIPs also are applicable in chemical or enzymatic synthesis. For example, MIPs using the product of an enzyme reaction as template are utilized for assisting the synthetic reaction by continuously removing the product from the bulk solution by complexation. This results in an equilibrium shift towards product formation.

Anti-Bacterial Agents↗
PubMed1999

Imprinted polymer-based sensor system for herbicides using differential-pulse voltammetry on screen-printed electrodes.

A sensor system for the herbicide 2,4-dichlorophenoxy-acetic acid has been developed based on specific recognition of the analyte by a molecularly imprinted polymer and electrochemical detection using disposable screen-printed electrodes. The method involves a competitive binding step with a nonrelated electrochemically active probe. For batch binding assays, imprinted polymer particles are incubated in suspension with the analyte and the probe, followed by centrifugation and quantification of the unbound probe in the supernatant. Two different compounds, namely 2,4-dichlorophenol and homogentisic acid, were tested as potential electroactive probes. Both compounds could be conveniently detected by differential-pulse voltammetry on screen-printed, solvent-resistant three-electrode systems having carbon working electrodes. Whereas 2,4-dichlorophenol showed very high nonspecific binding to the polymer, homogentisic acid bound specifically to the imprinted sites and thus allowed calibration curves for the analyte in the micromolar range to be recorded. An integrated sensor was developed by coating the imprinted polymer particles directly onto the working electrode. Following incubation of the modified electrode in a solution containing the analyte and the probe, the bound fraction of the probe is quantified. This system provides a cheap, disposable sensor for rapid determination of environmentally relevant and other analytes.

Electrochemistry↗
PubMed1998

Improved capillary electrophoretic separation of glycosylated oligopeptides through addition of poly(vinyl alcohol), and analysis by electrospray mass spectrometry.

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.

Amino Acid Sequence↗
PubMed1998

Some new developments and challenges in non-covalent molecular imprinting technology.

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules via the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include the use of molecularly imprinted polymers as tailor-made separation materials, antibody and receptor binding site mimics in recognition and assay systems, enzyme mimics for catalytic applications and as recognition elements in biosensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial-scale production and application.

Antibodies↗
PubMed1998

Plastic antibodies: developments and applications.

Antibodies are natural receptor molecules produced after contact with an antigen. In an attempt to mimic nature, the technique of molecular imprinting has been developed, which allows specific recognition sites to be formed in synthetic polymers through the use of templates. These recognition sites mimic the binding sites of antibodies and may be substituted for them in applications such as affinity separation, assay systems and biosensors. The stability and low cost of these polymers make them particularly attractive to industry.

Animals↗
PubMed1997

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane.

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG1 (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS of Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m2 histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 l of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.

Adsorption↗
PubMed1996

Immobilized metal ion affinity capillary electrophoresis of proteins--a model for affinity capillary electrophoresis using soluble polymer-supported ligands.

An affinity capillary electrophoresis method has been developed that employs small ligands covalently bound to a replaceable soluble polymer matrix. The metal chelate iminodiacetate-Cu(II) coupled to polyethylene glycol was used as a model system and the interactions of different model proteins, namely ribonucleases, cytochromes c, chymotrypsin, and kallikrein, were investigated. The method allowed for easy determination of dissociation constants using a modified Langmuir adsorption isotherm equation which is applicable to interactions with fast on/off kinetics. It is shown that the general rules for protein interaction with matrix-bound metal chelates established for immobilized metal ion affinity chromatography (IMAC) are maintained in immobilized metal ion affinity capillary electrophoresis (IMACE). In contrast to gel electrophoresis, IMACE allowed for using similar conditions as in IMAC, especially regarding the high salt concentrations, usually employed with the latter technique. The usefulness of the method for quantification of low-affinity interactions and studying protein surface structure and structure/function relationship using the metal affinity is demonstrated.

Animals↗
PubMed1996

Immobilized metal ion affinity gel electrophoresis: quantification of protein affinity to transition metal chelates.

This paper describes some recent advances in the methodology of immobilized metal ion affinity gel electrophoresis. Four different ways to incorporate metal chelate ligands in agarose and polyacrylamide-based electrophoresis gels are evaluated, a new polymerizable metal chelating ligand, allyl-2-hydroxy-3-(N,N-dicarboxymethyl)amino-propyl ether, is introduced, and the determination of affinity constants described. The affinities of model proteins (ribonucleases A and B and cytochromes c from different species) for the transition metal chelate iminodiacetic acid-Cu(II) were studied. The results were found to be in agreement with literature data on immobilized metal ion affinity chromatography, and the polymer nature and the different chemistries used influenced the affinity only quantitatively, keeping the basic mechanisms of interaction unchanged.

Animals↗
PubMed1996

Quantitative EEG in long-term survivors of acute lymphoblastic leukemia.

Conventional and quantitative aspects of electroencephalographic recordings obtained during a follow-up surveillance study in long-term survivors of acute lymphoblastic leukemia in childhood were investigated with respect to differences in central nervous system prophylaxis given during antileukemic therapy and compared with data derived from healthy controls. Central nervous system prophylaxis consisted either of cranial irradiation (18 Gray, group A, n = 8) or intermediate high-dose methotrexate (2000 mg/m2; group B, n = 5), each combined with intrathecal methotrexate. Conventional electroencephalographic analysis revealed comparable results in all three study groups. However, quantitative electroencephalography showed significantly increased absolute power scores for all frequency bands in both long-term survivor groups. Relative power estimates revealed a significant increase in delta/tau activities in both prophylaxis groups compared to healthy controls, which were countered by decreased percentage power scores in the alpha-range. Quantitative electroencephalographic comparisons between both central nervous system prophylaxis groups revealed only small differences in quantity, not quality, of the observed power disturbances with slightly higher deviations in irradiated long-term survivors than in nonirradiated ones. Topographical distributions of spectral band power were comparable between all three study groups without evidence for therapy-related topographical differences.

Adolescent↗
PubMed1996

Activation of human platelet protein kinase C-beta 2 in vivo in response to acute hyperglycemia.

Protein kinase C (PKC) is known to be activated in experimental model systems by elevated glucose and may play an important role in the pathogenesis of diabetic complications. Since there is no information about its role in humans in vivo we investigated the activation of PKC in human thrombocytes during infusion of glucose and insulin in normal controls and in 19 NIDDM patients by determining membrane and cytosol levels of PKC beta 2 using immune blots. In the 27 subjects investigated (8 controls, 19 NIDDM) membrane-associated levels of PKC beta 2 increased significantly after 60 and 150 min (p < 0.005). In controls an increase of membrane and of cytosolic PKC beta 2 occurred upon elevation of glucose by 5.5 mmol/L or more and the membrane association persisted for at least 60 min. In NIDDM glucose was elevated by 7.5-10 mmol/L during infusions. Increases of both membrane and cytosolic PKC beta 2 (< 20%-300%) occurred in 10 NIDDM patients suggesting that both, translocation and increased synthesis of PKC beta 2 were stimulated by glucose. Nine other patients showed no alteration (i.e. < 20%) of PKC beta 2. The 2 groups were similar regarding parameters of diabetes control, baseline glucose and glucose elevation during the test. However, the PKC beta 2 responsive group had lower levels of serum triglycerides (1.39 +/- 0.19 vs. 2.32 +/- 0.34 g/L; p = 0.038). To assess whether absolute levels of PKC were altered in human diabetes, platelet levels of PKC alpha, beta 1 and beta 2 were determined in 22 controls and 25 NIDDM subjects with poorly controlled diabetes (HbA1c = 9.8 +/- 0.36%). Cytosolic levels of PKC alpha were significantly decreased by 27% compared to controls in NIDDM but there was no change of PKC beta 1 or PKC beta 2. We conclude that 1. acute elevation of glucose by 5.5 mmol/L or more can activate PKC beta 2 translocation in controls and NIDDM patients in vivo irrespective of parameters of metabolic control. 2. NIDDM patients differ in their PKC beta 2-responses to glucose and 3. poor metabolic control leads to moderate downregulation of PKC alpha suggesting continued activation.

Adult↗
PubMed1996

P300 abnormalities in long-time survivors of acute lymphoblastic leukemia in childhood--side effects of CNS prophylaxis?

The incidence and the degree of P300 abnormalities in relation to psychometric findings was assessed in two groups of long-term survivors of acute lymphoblastic leukemia (ALL) in childhood. The study was performed as part of a follow-up trial, evaluating CNS late-effects after antileukemic therapy, 7.1 +/- 1.6 years after cessation of antileukemic therapy. Subject groups differed primarily in terms of antileukemic CNS prophylaxis: combined radio- and chemotherapy (n = 8) vs. chemotherapy alone (n = 5). Results were compared with those of a third group of healthy controls (n = 13) matched for age and gender. P300 recordings were obtained during a visual oddball-paradigm using checkerboard reversal stimuli of different pattern sizes as target and background events. Neurophysiological data were correlated with the results of a psychological test battery, which measured general intelligence, non-verbal visual memory functioning and concentration. ERP analysis showed a significant prolongation of the P300 latency in irradiated subjects when compared to healthy controls and non-irradiated long-term survivors. Topographical data comparisons revealed substantial P300 differences in both ALL long-term survivor groups when compared with healthy controls. This demonstrated significantly lower amplitudes over the left frontal derivations, associated with lowered concentration abilities, in the irradiated subject group. Higher amplitudes over the parieto-temporal derivations of the right hemisphere, associated with significantly impaired visual memory capabilities, were seen in both long-term survivor groups.

Adolescent↗
PubMed1995

Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes.

L-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their Fab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in KD with increasing temperature and a positive entropy value between 20 and 35 degrees C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.

Adsorption↗