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Biomedical subjects

R Hofbauer

Publications and source records attributed to R Hofbauer.

70 records · Page 4Linked to original sources
PubMed1996

Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells.

Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.

Blotting, Northern↗
PubMed1994

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase.

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.

Amino Acid Sequence↗
PubMed1994

Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions.

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.

Journal Article↗
PubMed1994

[Emergency intubation with the Combitube in a patient with persistent vomiting].

Prompt establishment of a patient airway and effective ventilation are the major goals during initiation of cardiopulmonary resuscitation in patients with cardiac arrest. Endotracheal intubation is the definitive method to maintain an optimal airway. However, endotracheal intubation is not always possible, even for the skilled physician. The Combitube has been developed to overcome this disadvantage. Studies have proved the effectivity of ventilation with this device. A case is reported where a patient suffered from acute respiratory failure and attempts at endotracheal intubation failed due to continued vomiting rendering fibre-optical visualisation of the vocal cords impossible. Blind insertion of the Combitube led to successful ventilation, and hence replacement by an endotracheal airway could be performed without danger of aspiration.

Adult↗
PubMed1991

A computer program for molecular weight determination of DNA fragments (HOWBIG).

The computer program HOWBIG has been developed based on the reciprocal correlation of size to migration distance of DNA in high voltage gradient gel systems (Southern, 1979). For calculation the program automatically chooses three marker bands migrating closest to the calculated band, reducing the error down to approximately 0.5% or less (reciprocal method, local form of calculation). The big advantages of the completely menu-driven program are high accuracy, error detectability, speed and ease of data handling. Management of marker-DNA molecular weights, data and analyses as files included in the menu driven program speeds up every-day lab work.

DNA↗
PubMed1991

Cell cycle-regulated and proliferation stimulus-responsive genes.

We have reviewed here genes whose expression may vary during the "cell cycle" and we discuss the underlying regulatory mechanisms. Given a correlation between the cell cycle and expression of a particular gene, the question arises whether that gene regulates the cycle, whether the cycle regulates that gene, or whether the correlation is simply the consequence that both the cell cycle and that gene respond to the same signal(s). Gene expression is regulated at diverse levels, and the relative importance of regulation at these different levels depends on which version of the cell cycle one has in mind; depending upon the context, the concept of the (higher eukaryote) cell cycle has a number of different operational meanings. Thus the first few divisions of the fertilized egg consist of successive S and M phases, with insignificant G1 and G2 phases, regulated entirely at the translational and post-translational level by the phosphorylation/dephosphorylation of p34cdc2 and the synthesis/degradation of one or more cyclins-keyed perhaps to the cytoplasm/nucleoplasm ratio and the completion of DNA replication. In contrast, cells stimulated to exit quiescence, (G0), require new gene transcription and changes in the post-transcriptional control of gene expression. Cells proliferating in a constant environment proceed directly from mitosis into G1 and are less dependent on (but not independent of) new transcription; here controls at the post-transcriptional and post-translational levels are more pronounced. In addition to regulation by p34cdc2, input from cell-specific growth factors or other extracellular signals is essential for most untransformed cells to continue through the cycle. Many transformed cells in contrast do not require exogenous signals and are altered in the way that key regulatory genes (e.g., p53, RB) are controlled. While cells of many lower eukaryotes appear capable of an indefinite number of cell cycles, the typical higher eukaryotic cell appears to have a limit on this number--untransformed, nonestablished vertebrate cells are usually mortal. For unknown reasons, established cell lines and certain embryonic or stem cells under the right conditions, are immortal and capable of indefinite proliferation. Apparently, the price paid to construct a differentiated multicellular organism is a limit on the number of cell divisions that the constituent somatic cells are capable of undergoing.

Amino Acid Sequence↗
PubMed1989

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs.

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.

Amino Acid Sequence↗
PubMed1987

Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA.

The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA.

Animals↗
PubMed1983

Increased levels of dihydrofolate reductase mRNA can be measured in normal, growth-stimulated mouse fibroblasts.

Levels of mRNA for the enzyme dihydrofolate reductase (EC 1.5.1.3) were determined in growth-stimulated 3T6 cells which contained wild-type dosage of the gene coding for this enzyme. As in the case of methotrexate-resistant cells having highly amplified levels of genes for dihydrofolate reductase, an increase in dihydrofolate reductase mRNA by a factor of 2-4 can be determined when cells enter the S phase. This increase is inhibited by sodium butyrate (which inhibits growth-stimulated 3T6 cells in mid G1 phase) but not by hydroxyurea (which inhibits in early S phase). We conclude that with the available methods it is possible to study the regulation of S phase-specific enzymes after growth stimulation at the level of the mRNA, even if gene amplification is not possible or desirable.

Animals↗
PubMed1982

Translational control of catalase synthesis by hemin in the yeast Saccharomyces cerevisiae.

mRNA-dependent cell-free protein synthesis systems were prepared from a heme-deficient ole3 mutant of the yeast Saccharomyces cerevisiae grown either in the absence or in the presence of the heme precursor delta-aminolevulinate. When supplemented with total yeast mRNA, the two systems-from heme-deficient and from heme-containing cells-translate most mRNAs with comparable efficiencies. mRNAs coding for the hemoproteins catalase T and catalase A, however, are translated at a low rate by the system from heme-deficient cells whereas their translation in the system from heme-containing cells is comparable to that observed in a heterologous in vitro system from wheat germ. Addition of 10 muM hemin to the system from heme-deficient cells stimulates translation of catalase mRNAs significantly. Control experiments showed that the results obtained cannot be explained by specific proteinase or nuclease action. Together with previous findings indicating lack of translation of catalase T mRNA in heme-deficient cells in vivo, the results demonstrate that specific control of yeast catalase formation occurs at the level of translation.

Catalase↗
PubMed1982

Preparation of a mRNA-dependent cell-free translation system from whole cells of Saccharomyces cerevisiae.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by mechanical breakage of cells, isolation of a 30000 x g supernatant fraction and removal of endogenous mRNA by treatment with micrococcal nuclease. The system thus isolated is dependent on added mRNA and translates yeast mRNA to discrete products, many of then identical with yeast proteins synthesized in vivo. Activity and properties of this system are comparable to those of other eukaryotic cell-free translation systems. It offers the following advantages, compared to yeast translation systems described previously. (a) Its isolation is simple and fast. (b) Since it is not isolated from spheroplasts there is no danger of its inactivation by contaminants in enzymes used for spheroplast preparation. (c) Isolation appears to be less strain-dependent and can be carried out starting from cells in various physiological states.

Cell-Free System↗
PubMed1981

[The hereditary factor as a criterion for the classification of stationary congenital night blindness (author's transl)].

The night-blind subject examined in 1952 by Schubert and Bornschein was reexamined electrophysiologically. All findings were unchanged. Normal a, d and g-waves in the ERG indicated undisturbed receptor function. Fast and slow oscillations of the EOG were normal. The subjective darkness-adaptation curve revealed minimal scotopic function after the 14th min of adaptation. The significance of clinical and electrophysiological findings for the classification of stationary congenital night blindness is discussed. The hereditary factor appears to be the most preferable criterion for the classification of stationary congenital night blindness.

Dark Adaptation↗
PubMedDate not supplied

Effects of garlic extract (Allium sativum) on neutrophil migration at the cellular level.

Studies of the effects of garlic extract on oxidative and lipoprotein levels have yielded widely different findings. Leukocytes play an important role during many processes, including inflammation. They migrate from intravascular spaces into tissues and attack microorganisms. In a recent study, the inhibitory effects of the nonsteroidal antiinflammatory drug, ibuprofen, on leukocyte transmigration were demonstrated using an in vitro assay. Little is known about the cellular effects of garlic extracts (Allium sativum). The aim of the current study was to investigate the influence of garlic extract on leukocyte migration through endothelial cell monolayers and thereby evaluate a possible role in inflammatory processes. Human umbilical endothelial cells were cultured on microporous membranes to make an endothelial cell monolayer (ECM). Freshly isolated neutrophils were used in a recently described migration assay. The amount of untreated neutrophils migrating through the untreated ECM was used as control and set at 100%. Neutrophils and/or ECM were pretreated with garlic extract using moderate, higher, and lower concentrations. Moderate plasma concentrations of garlic extract inhibited neutrophil migration through ECM significantly (64 +/- 5.8% standard deviation [SD]; P < 0.05) when both cell types were treated, (a situation that may have clinical relevance). Treating either neutrophils or ECM alone showed significant reductions in migratory rate (neutrophils treated alone: 81 +/- 7.7% SD, P < 0.05; ECM alone: 70 +/- 6.7%, P < 0.05). Thus, garlic extract is identified as a potent inhibitor of leukocyte migration through endothelial cell monolayers. Treatment of both cell types has an additive effect. Endothelial cells seem to be more affected than neutrophils. Further investigations are necessary to understand the potential clinical consequences.

Cell Adhesion↗
PubMedDate not supplied

Quantification of leukocyte migration: improvement of a method.

Eighteen different permeable membrane supports with and without confluent endothelial cell monolayers were incubated with normal donor derived neutrophils in the upper chambers of a 24 multiwell double chamber system. In order to study transmembrane or transendothelial leukocyte migration leukocytes were stimulated by chemoattractants, or endothelial cells were activated by IL-1. After coincubation the membrane supports building the upper chambers were discarded. Using this technique, leukocytes that had migrated into the lower chamber were exposed to the fluorescent dye calcein AM without additional washing or transfer steps. Absolute cell counts were determined computer assisted using dilution series of calcein AM labeled leukocytes as standards. Serial dilutions of neutrophils exposed to calcein AM showed reproducible linear fluorescence intensity, and relative fluorescence intensity correlated significant with cell counts (r2 = 0.974, p < 0.0001). Out of 18 membrane supports only one was suitable for our assay set up. Best technical and optical performance was achieved with a membrane made of polyethylene terephtalate with a pore size of 3 mm at a pore density of 0.8 x 10(6)/cm2. Stimulation of leukocytes or endothelium by FMLP or IL-1 revealed an increase of transendothelial migration to 7.2 +/- 1.8 x 10(5) PMN and 5.1 +/- 0.7 x 10(5) PMN respectively if compared with medium (0.6 +/- 0.2 x 10(5) PMN). IL-1 induced migration of neutrophils was inhibited by anti IL-1 autoantibodies derived from chronic renal failure patients (IL-1: 100% of PMN migrated, anti IL-1 antibody: 39% of PMN migrated, control antibody: 84% of PMN migrated). In summary, a simple fluorimetric assay was established for the quantification of transmembrane and transendothelial leukocyte migration.

Autoantibodies↗