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Biomedical subjects

M Schmidt

Publications and source records attributed to M Schmidt.

At least 361 records · Page 20Linked to original sources
PubMed1998

Lack of interferon consensus sequence binding protein (ICSBP) transcripts in human myeloid leukemias.

Interferon consensus sequence binding protein (ICSBP) was first identified as a transcription factor of the interferon (IFN) regulatory factor family (IRF) which regulates expression of IFN-dependent genes by binding to DNA at specific sites, IFN-stimulated responsive elements. Analysis of ICSBP-deficient mice showed hematologic alterations similar to chronic myelogenous leukemia (CML) in humans and suggested a novel role for ICSBP in regulating proliferation and differentiation of hematopoietic progenitor cells. Here we show that ICSBP-mRNA expression is impaired in human myeloid leukemias: 27 of 34 CML patients (79%) and 21 of 32 patients with acute myeloid leukemia (AML) (66%) showed very low or absent transcript numbers of ICSBP. In contrast, only 2 of 33 normal volunteers (6%) showed low transcription of ICSBP (P < . 0001 both for CML and AML values). The lack of expression was not associated with lack of lymphatic cells, which normally have been shown to express ICSBP at the highest level. More detailed analysis showed an absence of ICSBP-mRNA also in sorted B cells derived from CML patients. To analyze whether ICSBP may be induced in leukemic cells, ex vivo experiments using a known inducer of ICSBP, IFN-gamma, were performed. Ex vivo treatment of primary CML cells using IFN-gamma resulted in induction of ICSBP transcripts. Furthermore, samples of CML patients during IFN-alpha treatment were analyzed. In 11 of 12 CML patients ICSBP-mRNA was inducible upon in vivo treatment with IFN-alpha, but decreased with progression of CML. Stable transfection of K-562 cell line with ICSBP led to no difference in bcr-abl expression in vitro, although two patients showed an inverse correlation between bcr-abl and ICSBP in vivo. These data suggest that lack of ICSBP may have an important role also in human myeloid leukemogenesis.

Chronic Disease↗
PubMed1998

Identification and kinetic analysis of a functional homolog of elongation factor 3, YEF3 in Saccharomyces cerevisiae.

Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nM). K(m) values for ATP were similar, but the Kcat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/K(m) values for EF-3A were about two-fold higher; however, the difference in Kcat/K(m) values between the two factors was small for basal ATPase activity.

Base Sequence↗
PubMed1998

Comparative investigations of algorithms for the detection of breaths in newborns with disturbed respiratory signals.

The correct detection of the beginning of inspiration and expiration in the respiratory signals is an essential prerequisite for accurate lung function testing in newborns. Five algorithms for breath detection using pneumotachographically measured flow and volume signals were investigated with regard to the error rate. To compare and to evaluate the reliability of these algorithms 12 minimally and 12 severely disturbed flow and volume signals from spontaneously breathing newborns were used. With the exception of an algorithm based on Walsh-transformed signals, all algorithms work reliably (error rate <1.1%) if disturbances are minimal. In severely disturbed signals there is a great difference between the algorithms. The most robust algorithm tested (trigger of the flow signal with an additional plausibility check of the recognized breath) resulted in an error rate of <3.4%. Not all algorithms tested are suitable for real-time applications because they differ considerably in delay time for breath detection.

Algorithms↗
PubMed1998

Baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein.

The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies.

Amino Acid Sequence↗
PubMed1998

Correlation of electrophysiology, morphology, and functions in corticotectal and corticopretectal projection neurons in rat visual cortex.

In most mammals the superior colliculus (SC) and the pretectal nucleus of the optic tract (NOT) receive direct input from the ipsilateral visual cortex via projection neurons from infragranular layer V. We examined whether these projection neurons belong to different populations and, if so, whether it is possible to correlate the electrophysiological features with the suggested function of these neurons. Projection cells were retrogradely labeled in vivo by rhodamine-coupled latex beads or fast blue injections into the SC or the NOT 2-5 days prior to the electrophysiological experiment. Intracellular recordings of prelabeled neurons were made from standard slice preparations and cells were filled with biocytin in order to reveal their morphology. Both cell populations consist of layer V pyramids with long apical dendrites that form terminal tufts in layer I. In electrophysiological terms, 12 of the corticotectal cells could be classified as intrinsically bursting (IB), while two neurons showed a doublet firing characteristic and one neuron was classified as regular-spiking (RS). Intracortical microstimulation of cortical layer II/III revealed that SC-projecting neurons responded optimally to stimulation sites up to a distance of 1000 microm from the recorded cell. The morphological features of the SC-projecting cells reveal an apical dendritic tuft in layer I with a lateral extension of 300 microm, a mean spine density of 65 spines per 40 microm on the apical dendrites located in layer II/III, and a bouton density of 13 boutons per 100 microm on the intracortical axons. Sixteen NOT-projecting neurons exhibited an IB and five cells an RS characteristic. Intracortical microstimulation of cortical layer II/III showed that NOT-projecting neurons responded optimally to stimulation sites up to a distance of 1500 microm. Their morphological features consist of an apical dendritic tuft with a lateral extension of 500 microm, a mean spine density of 25 spines per 40 microm on the apical dendrites located in layer II/III, and a bouton density of 6 boutons per 100 microm on the intracortical axons. When the passive membrane parameters, responses to intracortical microstimulation in layer V, the extension of the basal dendritic field, and spine densities in layers I or V were compared between SC- and NOT-projecting cells, no differences were revealed. Differences were only consistently found in the supragranular layers, either for morphological parameters or for intracortical microstimulation. The results suggest that NOT-projecting and SC-projecting neurons, although biophysically similar, could integrate and transmit different spatial aspects of cortical visual information to their target structures.

Action Potentials↗
PubMed1998

Somatostatin receptor imaging in intracranial tumours.

The somatostatin analogue [111In-DTPA-d-Phe1]-octreotide (111In-octreotide) allows scintigraphic visualization of somatostatin receptor-expressing tissue. While it is well known that a large variety of tissues express somatostatin receptors and 111In-octreotide scintigraphy has a clearly defined role in various neuroendocrine diseases, the clinical value of 111In-octreotide scintigraphy in brain tumours is still under clinical investigation. In 124 patients with 141 brain lesions (63 meningiomas, 24 pituitary adenomas, 10 gliomas WHO class I and II, 12 gliomas WHO class III and IV, 11 neurinomas and 2 neurofibromas, 7 metastases and 12 other varieties: three non-Hodgkin B-cell lymphomas, two epidermoids, one abscess, one angioleiomyoma, one chordoma, one haemangiopericytoma, one osteosarcoma, one plasmacytoma and one pseudocyst), 111In-octreotide scintigraphy was performed 4-6 and 24 h after i.v. injection of 110-220 MBq 111In-octreotide. Planar images of the head in four views with a 128x128 matrix and single-photon emission tomographic images (64x64 matrix) were acquired, and lesions were graded according to qualitative tracer uptake. Fifty-nine of the 63 meningiomas showed moderate to intense tracer uptake. Nine of 24 pituitary adenomas were visible; the remaining 15 did not show any tracer uptake. None of the class I and II gliomas with an intact blood-brain barrier were detected whereas 11/12 class III and IV gliomas showed 111In-octreotide uptake. None of the neurinomas or neurofibromas were positive. Five of seven metastases were classified as positive, as were the osteosarcoma, two of three non-Hodgkin B-cell lymphomas, one abscess, one angioleiomyoma, one chordoma and one haemangiopericytoma. The other varieties (one non-Hodgkin B-cell lymphoma, two epidermoids, one plasmacytoma and one pseudocyst) did not show 111In-octreotide uptake. The results demonstrate that a large variety of intracranial lesions express somatostatin receptors and therefore can be visualized by [111In-DTPA-d-Phe1]-octreotide scintigraphy. This technique can be valuable in the differentiation between meningiomas and pituitary adenomas, based on qualitative tracer uptake. [111In-DTPA-d-Phe1]-octreotide scintigraphy allows differentiation between meningiomas and neurinomas or neurofibromas and therefore provides complementary information to computed tomography or magnetic resonance imaging. Furthermore, this technique allows differentiation between scar tissue and recurrent meningiomas postoperatively and can help in non-invasive tumour differentiation of multiple intracranial lesions, which can be of value in defining the most adequate therapeutic strategy.

Adolescent↗
PubMed1998

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-alpha.

Tumor necrosis factor (TNF)-alpha has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-alpha. Among the cell lines tested those resistant to TNF-alpha were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin beta1 resulted in a significant increase in the number of cells surviving TNF-alpha treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-alpha. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-alpha retained TNF-alpha activity. This scFv(14E1)-TNF-alpha molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-kappaB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays.

Animals↗
PubMed1998

Low glutathione pools in the original pso3 mutant of Saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype.

The original pso3-1 mutant isolate of the yeast Saccharomyces cerevisiae exhibits a pleiotropic mutagen-sensitivity phenotype that includes sensitivity to UVA-activated 3-carbethoxypsoralen, to UVC-light, to mono- and bi-functional nitrogen mustard, to paraquat, and to cadmium; on the other hand, it shows hyper-resistance (HYR) to nitrosoguanidine when compared to established wild-type strains. Also, the original pso3-1 mutant exhibits a low UVC-induced mutability and mitotic gene conversion and a high rate of spontaneous and UVC-induced petite mutations. Since the HYR to the nitrosoguanidine (MNNG) phenotype resembles that of low glutathione-containing yeast cells, the original pso3-1 mutant was crossed to a gsh1 knock-out mutant that lacks the enzyme for the first step in glutathione biosynthesis and the resulting diploid was tested for complementation. While there was none for HYR to nitrosoguanidine, and other low glutathione-related phenotypes, some other phenotypic characteristics of pso3-1, e.g. UVC sensitivity and UVC-induced mutability were restored to a wild-type level. Tetrad analysis of a diploid derived from a cross of the original haploid pso3-1 isolate with a repair-proficient, normal glutathione-containing, PSO3 GSH1 wild-type led to the separation of a leaky gsh1 mutation phenotype from that of the repair-deficient pso3-1 phenotype. Linkage studies by tetrad and random spore analyses indicated no linkage of the two genes. This shows that the low glutathione content in the original pso3-1 isolate is due to a second, additional, mutation in the GSH1 locus and is unrelated to the pso3-1 mutation. Thus, the original pso3-1 isolate is a pso3-1 gsh1 double mutant with most of the particular characteristics of the pleiotropic sensitivity phenotype contributed by either the pso3-1 or the gsh1-leaky mutant allele. The expression of a few phenotypic characteristics of pso3, however, were most pronounced in pso3-1 mutants with a low glutathione pool.

Fungal Proteins↗
PubMed1998

Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli.

BACKGROUND: Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described. OBJECTIVE: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity. METHODS: In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells. RESULTS: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli. CONCLUSIONS: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.

Allergens↗
PubMed1998

Optimization of contrast dosage for gadolinium-enhanced 3D MRA of the pulmonary and renal arteries.

To determine the minimal contrast dosage required for diagnostic contrast-enhanced three-dimensional (3D) magnetic resonance angiography (MRA) image quality of the pulmonary (PAs) or renal arteries (RAs). In 12 volunteers (10 females, 2 males; mean age 24 years) imaging was performed with 4 different dosages: 0.05, 0.1, 0.2 and 0.3 mmol/kg of body weight (BW) 0.5 M gadolinium (Gd) contrast agent. The PAs and RAs were evaluated separately each in groups of six volunteers. Qualitative and quantitative signal-to-noise ratio (SNR) image analysis was performed. For the PAs, the increases in signal-to-noise ratio were paralleled by increases in image quality ratings. For the PAs, with the use of 0.05 mmol/kg, only 50.3% of all segments were rated diagnostic, whereas with higher dosages the percentage rose to 89.2% for 0.1 mmol/kg, 98.2% for 0.2 mmol/kg. and 99.1% for 0.3 mmol/kg. For the RAs, 0.3 mmol/kg provided no significant increase in singal-to-noise ratio compared to 0.2 mmol/kg (p = 0.4). Only by a dosage of 0.2 and 0.3 mmol/kg, all evaluated segments were diagnostic evaluable. A dose of 0.2 mmol/kg is required for proper assessment of the RAs or PAs.

Adult↗
PubMed1998

Computer simulation of the measured respiratory impedance in newborn infants and the effect of the measurement equipment.

The forced oscillation technique (FOT) is a non-invasive method to investigate lung mechanics. FOT does not require active cooperation and therefore it seems to be useful for lung function measurements in newborn infants. The aims of this simulation study were to investigate the effects of development and growth of the lung, pulmonary inhomogeneities and the measurement equipment on the respiratory impedance (Zrs). The respiratory impedance was simulated by using four lung models with lumped parameters in the frequency range of 3-50 Hz considering resistive and elastic resistances of the respiratory system and the inertance of breathing air and tissue. The simulation has shown that the maturation of lungs produces only a parallel shifting of the real and imaginary part of the impedance curves whereas respiratory diseases change the course of the curves. Furthermore, a high influence of the measurement equipment (e.g. compliance of the face mask, endotracheal tube leaks) on Zrs was found. In conclusion, the simulation has shown that FOT offers a deeper insight in the structure of the respiratory system. However, the technical requirements for accurate measurements in newborns are very high.

Airway Resistance↗
PubMed1998

Computer-based quality control in high-dose chemotherapy and bone marrow transplantation.

High-dose chemotherapy with autologous and allogeneic blood stem cell transplantation has become a standard therapy for hematological malignancies and solid tumors. To ensure quality of treatment and adherence to internationally approved protocols, the use of computer systems has been introduced in some oncology centers. Most software packages cannot be used automatically by other institutions because of different treatment strategies and medications. To overcome this problem, we designed a software system based on an Oracle database, which allows the user to develop his own therapy blocks and assemble them to generate specific therapy plans. The system supports the daily work of the physicians by suggesting medications based on changes in the vital parameters or the clinical chemistry. Parameters can be given upper and lower limits whose surpassing leads to treatment suggestions by the system. All quantitative variables may be depicted in a graphical way. The system is connected to external laboratory devices. Export of data to international registries is possible. Until now, 43 patients have been transplanted with the help of this system. There is a high degree of acceptance among the nursing staff and the physicians. The system can be transferred to other centers, if the local hardware and network capacities meet the minimal requirements.

Adult↗
PubMed1998

Screening for mutations in the neuropeptide Y Y5 receptor gene in cohorts belonging to different weight extremes.

OBJECTIVE: The neuropeptide Y (NPY) Y5 receptor is presumed to be involved in the regulation of food intake. DESIGN: To investigate the possible role of this receptor in weight regulation, the whole coding region of the NPY Y5 receptor gene was screened for mutations using temperature gradient gel electrophoresis (TGGE). Detected mutations were screened in extended cohorts. STUDY COHORTS AND METHODS: Cohorts of 87 extremely obese children and adolescents, 15 underweight subjects and 25 patients with anorexia nervosa (AN) were initially screened by TGGE. Extended samples of these cohorts (160 obese children and adolescents; mean body mass index (BMI) 33.5 +/- 6.4 kg/m2, 128 underweight subjects; mean BMI 18.4 +/- 1.0 kg/m2 and 58 patients with AN; mean BMI 14.6 +/- 1.7 kg/m2) were screened to determine the frequencies of a detected mutation and a detected polymorphism in the NPY Y5 receptor gene. In addition, a previously described polymorphism in the first intron of the NPY Y1 receptor gene was analysed. RESULTS: The coding region of the NPY Y5 receptor gene encompasses one exon. A single mutation, which results in a non-conservative amino acid substitution in the first extracellular domain of the receptor (Glu-4-Ala), and one silent polymorphism (Gly-426-Gly-Gly) at nucleotide position 1278 (G-->A) were detected by TGGE. Both tests for association and linkage to the NPY Y1 and NPY Y5 receptor polymorphisms were negative among all cohorts. The Glu-4-Ala mutation was found only in a single patient with AN and her mother. CONCLUSION: The results do not support a major role of the NPY Y5 receptor gene in the variability of body weight in children and adolescents.

Adolescent↗
PubMed1998

Expression of two isoforms of Lep d 2, the major allergen of Lepidoglyphus destructor, in both prokaryotic and eukaryotic systems.

BACKGROUND: The dust mite Lepidoglyphus destructor is a major cause of allergic diseases among farmers. We have previously cloned and sequenced two isoforms of the major allergen Lep d 2 (formerly designated Lep d 1) and found significant homology to group 2 allergens of the house dust mite species Dermatophagoides. We now report on the production and characterization of recombinant Lep d 2. OBJECTIVE: We have expressed both isoforms in two different expression systems; a eukaryotic system, baculovirus in insect cells and a prokaryotic system, E. coli. We have compared the two systems in regard to production yields and immunoreactivity of the recombinant allergens. METHODS: The complete cDNA including the natural leader sequence was cloned into the pBlueBacIII transfer vector, and the rLep d 2 was produced as a secreted protein in baculovirus. For the expression in E. coli, the cDNA was cloned into the pET vector, and the rLep d 2 was produced with six C-terminal histidine residues. The purified recombinant allergens were tested for immunoreactivity with 10 sera from subjects allergic to Lepidoglyphus destructor and were compared with native Lep d 2 using inhibition immunoblotting. The ability of the recombinant allergens to release histamine from basophils was evaluated using a histamine release assay. RESULTS: Both expression systems produced immunoreactive recombinant allergens. They inhibited the binding of human sera to native Lep d 2 confirming their retained IgE binding properties. The yield of pure recombinant protein from the prokaryotic system was approximately 1 mg/L compared to the eukaryotic system which produced up to 4 mg/L in an adherent cell culture system. CONCLUSIONS: We have produced recombinant Lep d 2 in prokaryotic and eukaryotic expression systems which are comparable to the native allergen. Recombinant Lep d 2 might now be included in more extensive clinical studies to confirm its usefulness in the in vitro and the in vivo diagnosis of Lepidoglyphus destructor.

Allergens↗
PubMed1998

Characterization of a directional selective inhibitory input from the medial terminal nucleus to the pretectal nuclear complex in the rat.

The receptive field properties of neurons in the medial terminal nucleus of the accessory optic system (MTN) that project to the ipsilateral nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN), as identified by antidromic electrical activation, were analysed in the anaesthetized rat. The great majority (88%) of MTN neurons that were antidromically activated from NOT and DTN preferred downward directed movement of large visual stimuli while the remaining cells preferred upward directed stimulus movement. Distinct retrograde tracer injections into the NOT/DTN and the ipsilateral inferior olive (IO) revealed that no MTN neurons project to both targets. MTN neurons projecting to the ipsilateral NOT/DTN were predominantly found in the ventral part of the MTN, whereas those projecting to the IO were found in the dorsal part of the MTN. In situ hybridization for glutamic acid decarboxylase (GAD) mRNA was used as a marker for GABAergic neurons. Up to 98% of MTN neurons retrogradely labelled from the ipsilateral NOT/DTN also expressed GAD mRNA. Earlier studies have shown that MTN neurons that prefer upward directed stimulus movements are segregated from MTN neurons that prefer downward directed stimulus movements. It also has been demonstrated that directionally selective neurons in the NOT/DTN prefer horizontal stimulus movements and receive an inhibitory input from ipsilateral MTN. Our results indicate that this input is mediated by GABAergic cells in the ventral part of MTN, which to a large extent prefer downward directed stimulus movements, and that the great majority of MTN neurons that prefer upward directed stimulus movements project to other targets one of which possibly is the IO.

Animals↗
PubMed1998

ET(A) and ET(B) specific ligands synergistically antagonize endothelin-1 binding to an atypical endothelin receptor in primary rat astrocytes.

Using a whole-cell binding procedure with long incubations at low temperature and subsequent acid stripping, we have characterized an atypical endothelin (ET) receptor in primary rat cortical astrocyte cultures. We found the following: (a) no competition for 125I-ET-1 binding by the ET(A) antagonists BQ-123 and LU 135252 or the ET(B) agonist IRL 1620; (b) weak competition by the ET(B) antagonist BQ-788 and by the predominant ET(B) ligand ET-3; (c) potent synergistic competition of ET(A) and ET(B) ligands in combination for 125I-ET-1 binding; (d) potent competition of ET-1 with any of the radioligands used, 125I-ET-1, 125I-IRL 1620, and [3H]BQ-123; (e) lack of competition of IRL 1620 and BQ-123 with the respective other radioligand; (f) shifting of the amount of acid-strippable 125I-ET-1 binding from 20 to 80% by ET(B) ligands and to 4% by ET(A) ligands; and (g) as a control, typical ET(A) and ET(B) binding characteristics of the RAT-1 fibroblast and the U373MG astrocytoma cell line, respectively, under our assay conditions. The unusual binding properties of astrocytic ET receptors described in this study appear to be the result of several binding sites in the receptor for different ET ligands or ligand epitopes.

Animals↗