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Biomedical subjects

F Bühling

Publications and source records attributed to F Bühling.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cathepsin K expression in human lung.

Tissue remodeling is crucial in different lung diseases, in the embryonal development as well as in bronchial carcinoma. Cathepsins were proposed to be involved in the degradation of matrix proteins. Cathepsin K is one of the most potent matrix-degrading cysteine proteinases known as yet. The elastinolytic and collagenolytic activity of this papain-like protease is comparable with that of neutrophil elastase. We have investigated the cathepsin K expression in normal adult lung tissues, in embryonal lung tissue and in bronchial carcinoma. With help of specific anti-cathepsin K antibodies it could be shown that cathepsin K was expressed in bronchial epithelial cells. These data could be confirmed at mRNA level using a quantitative RT-PCR as well as by visualisation of the specific enzymatic activity in epithelial cell lines. During the embryonal development cathepsin K was expressed in the epithelial cells of the developing bronchi. The expression seemed to be upregulated in parallel with the development of the bronchial and alveolar lumen. In the later phase of lung development the cathepsin K expression was restricted to bronchial epithelial cells. Furthermore, using quantitative RT-PCR it could be shown that cathepsin K-mRNA was upregulated in lung tumor tissues in comparison to normal tissues from the same patients. These data suggest that cathepsin K may play an important role in matrix remodeling of the lung under physiological and pathological conditions.

Bronchi↗
PubMed2000

Expression of cathepsins B and L in human lung epithelial cells is regulated by cytokines.

The cathepsins B, L, and H are expressed ubiquitously and represent the major proportion of lysosomal enzymes. They are involved in bulk proteolysis in the lysosomes, processing of proteins and matrix degradation. Under pathological conditions the participation of cathepsins, especially their secreted forms, was observed in inflammation, tumor progression and metastasis. The enzymatic activity of cathepsins is regulated by posttranslational modification, localization, maturation, changes in pH, and their interaction with inhibitors. Regulation at the level of transcription is not well elucidated. The aim of this study was to investigate the effect of IL-1 beta, IL-6, IL-10, TGF-beta 1, and HGF on mRNA expression and protein level in human lung epithelial cell lines A-549 and BEAS-2B. IL-6 leads to a twofold increase in cathepsin L mRNA expression, whereas TGF-beta 1 decreases the amount of cathepsin L mRNA. At protein level, using enzyme immunoassay, it was shown that IL-6 induced increased amounts of cathepsin L but not cathepsin B. In contrast, after incubation of bronchial epithelial cells with TGF-beta 1 the cathepsin L concentration was decreased. In conclusion, gene expression of cathepsins B and L is variable. The cytokines IL-6 and TGF-beta 1 modulate cathepsin gene expression.

Carcinoma↗
PubMed1999

Increased release of transforming growth factor (TGF)-beta1, TGF-beta2, and chemoattractant mediators in pneumonia.

Transforming growth factor-beta (TGF-beta), interleukin-8 (IL-8), and leukotrienes are potent neutrophil chemoattractants that are released in several lung diseases. There is limited information about the release of TGF-beta in bronchoalveolar lavage fluid (BALF) of patients with pneumonia. Furthermore, it is not clear if TGF-beta is differentially expressed in different lung diseases. The aim of our study was to compare the concentrations of TGF-beta1 and TGF-beta2 in the BALF of patients with pneumonia and other lung diseases. Furthermore, correlation of the TGF-beta levels with the concentration of chemoattractant mediators as well as with indicators of macrophage and granulocyte activation should be investigated. Patients with pneumonia, interstitial lung disease (ILD), or chronic obstructive pulmonary diseases (COPD) were included. Patients with ischemic heart disease without pulmonary involvement served as controls. The concentrations of TGF-beta1 and TGF-beta2, of the chemoattractant cytokine IL-8, of leukotriene B4, and of the leukotrienes C4, D4, and E4 were measured. Neutrophil elastase and granulocyte content (PMN) were used as markers for granulocyte activation, and neopterin was used as a marker for the activation of macrophages. Significantly elevated levels of TGF-beta1 (mean = 0.216 ng/ml, p < 0.01) were found in patients with microbiologically positive pneumonia but not in patients with ILD or COPD. A significant (p < 0.001) correlation was found between the TGF-beta1 concentrations and the IL-8 levels and the percentage of granulocytes (r = 0.76, and r = 0.44, respectively). Elevated TGF-beta2 concentrations were measured in the BALF of patients with pneumonia (mean = 1.4 ng/ml, p < 0.01) and with ILD. Pneumonia was also associated with increased concentrations of leukotrienes C4, D4, and E4 (mean = 91.61 pg/ml, p < 0.05) and leukotriene B4 (mean = 203.9 pg/ml, p < 0.01), significantly elevated levels of PMN elastase (mean = 2958.26 ng/ml, p < 0.01), and neopterin (mean = 0.42 nmol/L). Our results strongly suggest that different lung diseases do differ with regard to the released cytokines. TGF-beta1 probably plays a key role in regulation of pulmonary inflammation, particularly in pneumonia.

Bronchoalveolar Lavage Fluid↗
PubMed1999

Replacement of threonine 394 by alanine facilitates internalization and resensitization of the rat mu opioid receptor.

Signaling of G protein-coupled receptors is terminated by phosphorylation of intracellular serine and threonine residues. Resensitization of these receptors requires internalization and subsequent dephosphorylation. We have recently shown that the resensitization rate of the rat micro opioid receptor (MOR) isoforms MOR1 and MOR1B is mainly determined by the amino acid composition of their alternatively spliced C-terminal tails. Upon agonist stimulation, MOR1B passes through an accelerated cycle of receptor endocytosis and reactivation, which in turn promotes a greater resistance to agonist-induced desensitization, as compared with MOR1. Given the fact that MOR1B lacks only one putative phosphorylation site (T394 of MOR1), we replaced this threonine by an alanine and stably expressed the wild-type MOR1 and its T394A mutant in mouse neuroblastoma Neuro2a cells. We show that during prolonged [D-Ala2, MePhe4, Gly5-ol]enkephalin exposure (5 h), the T394A receptor mutant desensitized at a slower rate than MOR1. In contrast, T394A is more rapidly removed from the cell surface than MOR1, as determined by flow cytometry using epitope-tagged receptors. This fast internalization was followed by immediate resensitization of T394A during 20 min of agonist removal while the wild-type MOR1 remained inactive. Similar to MOR1B, rapid internalization and reactivation of T394A may explain its delayed desensitization. These findings suggest that T394 represents a negative regulatory signal for MOR1 internalization. Furthermore, phosphorylation of this threonine residue may influence the time course of micro opioid receptor resensitization.

Alanine↗
PubMed1999

Expression of cathepsin K in lung epithelial cells.

Alveolar and bronchial epithelial cells have been shown to have regulatory functions in the maintenance of lung structure and function. Recent evidence supports the premise that these cells can synthesize a variety of extracellular matrix components in vitro, suggesting an active participation in connective tissue remodeling. Their possible role in extracellular matrix degradation, however, is less clear. This study addresses the question of whether alveolar and bronchial epithelial cells express the highly collagenolytic and elastinolytic cysteine proteinase cathepsin K, which has recently been newly described. We provide evidence that the epithelial cell lines A549 and BEAS-2B are capable of expressing cathepsin K messenger RNA. Furthermore, we show that cathepsin K is expressed in normal bronchial epithelial cells. Western blot analyses of human lung-tissue lysates revealed specific immunoreactivity at molecular weights of 46 and 27 kD, corresponding to the procathepsin and the mature cathepsin K. Immunohistochemical analyses showed a pronounced staining of bronchial epithelial cells and in single alveolar epithelial cells. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized. These findings demonstrate that bronchial and alveolar epithelial cells are capable of expressing cathepsin K, which could be of considerable importance for remodeling processes of the extracellular matrix in the lung.

Bronchi↗
PubMed1999

[Cathepsin cysteine proteinases in the lung].

Proteolytic enzymes play an important role during remodeling and digestion of extracellular matrix proteins. An overproduction of extracellular matrix or insufficient extracellular matrix digestion may result in fibrosis. Enhanced proteolytic activity or an insufficient inhibitory potential could be followed by emphysema development. Since the first reports showed an emphysema induction in rats after intratracheal application of the cysteine protease papain, a number of proteolytic enzymes involved in the remodeling of the extracellular matrix of the lung were discovered. Most of them are cysteine-, metallo-, serine- or aspartic proteases. In this paper some new findings concerning the expression, function and regulation of the activity of papain-like cysteine proteases in the process of tissue destruction and remodeling in the lung are reviewed. The functional relationship between cathepsins and other proteolytic enzymes are discussed.

Animals↗
PubMed1998

Dipeptidyl peptidase IV (DP IV, CD26) is involved in regulation of DNA synthesis in human keratinocytes.

Various studies have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK, and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. Here, we clearly demonstrate that this enzyme is highly expressed also on human epidermal foreskin and split-skin keratinocytes and that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit the enzymatic activity as well as the DNA synthesis of these cells. These data demonstrate that CD26 plays a role also in regulation of DNA synthesis of epidermal keratinocytes and that the enzymatic activity is required for mediating these effects.

Adult↗
PubMed1997

A detailed protocol for the measurement of TGF-beta1 in human blood samples.

Quantification of the multifunctional cytokine Transforming Growth Factor-beta1 (TGF-beta1) in blood samples has aroused increasing interest in recent years, since an abnormal regulation of this cytokine appears to play a key role in the pathogenesis of different diseases, such as autoimmundiseases or malignant tumors. The measurement of TGF-beta1 is complicated by a lot of problems concerning the collection, preparation and handling of blood samples, the platelet contamination, and the TGF-beta1 activation procedure. Here, we recommend detailed instructions for measurement of TGF-beta1 in blood plasma samples which should be followed to exclude the determination of false positive or negative results.

Humans↗
PubMed1997

The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression.

Recently, it was shown that aminopeptidase N (E.C. 3.4.11.2, CD13) is up-regulated during mitogenic stimulation of peripheral T-cells. In this study, we demonstrate that the half-life of APN mRNA was considerably prolonged in these cells leading to a 2.7-fold increase of APN transcript level. The apparent half-life time of the APN transcript was investigated by the RNA synthesis inhibitor-chase method using actinomycin D. The steady-state APN mRNA levels was determined by a competitive RT-PCR. The half-lives estimated in resting T-cells, natural killer cells and permanently growing tumour cells varied between 3.5 and 6 h. Finally, nuclear run-on assays revealed that the APN gene expression of stimulated T-cells is controlled by increased promoter activity as well. These studies suggest a control of APN gene expression at the post-transcriptional level in addition to promoter-mediated regulation.

Binding, Competitive↗
PubMed1997

CD26/dipeptidyl peptidase IV in lymphocyte growth regulation.

DP IV/CD26 is involved in regulation of DNA synthesis and proliferation as well as production of cytokines of hematopoietic cells under various conditions. Inhibition of DNA synthesis in T lymphocytes, B lymphocytes, NK cells and myelomonocytic cells as well as of the production of IL-2, IL-6 TNF alpha, IL-1, IL-10, IL-12, IL-13, IFN-gamma, GM-CSF are not due to apoptosis of these cells. DP IV/CD26 inhibitors induce TGF-beta 1 mRNA synthesis and latent protein release demonstrating a crucial role of TGF-beta 1 in mediating CD26 function. X-X-Pro peptides as HIV-Tat protein strongly inhibit DP IV enzymatic activity and suppress DNA synthesis. This group of peptides may represent a class of natural DP IV/CD26 ligands and effectors, respectively. Hyperphosphorylation of p56lck as well as protein tyrosine phosphorylation of a number of proteins in T lymphocytes can be modulated by DP IV inhibitors. These data suggest that enzymatic activity or, at least in part, the active site of DP IV are both essential for its regulatory function in lymphocytes. Further work is required to determine the natural ligands, i.e. substrates and effectors, which are play the central role in DP IV/CD26 action in T cell growth and to understand the molecular mechanism of the early steps of this fundamental process.

Animals↗
PubMed1997

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-beta 1 (TGF-beta 1) in stimulated mouse splenocytes and thymocytes.

Various studies have shown that the ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. The DP IV/CD26 was found also on mouse splenocytes and thymocytes. Here, we show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of IL-2, IL-6 and IL-10 of PHA-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes. Most importantly, these inhibitors induce a three to fourfold increased secretion of latent transforming growth factor beta 1 (TGF-beta 1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-beta 1 enzyme-linked immunosorbent assay (ELISA). These data demonstrate that CD26 plays a role also in regulation of DNA synthesis and cytokine production by murine immune cells, that the enzymatic activity is required for mediating these effects, and that TGF-beta 1 might have key functions in these processes.

Animals↗
PubMed1997

Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor-beta 1 in PWM-stimulated PBMC and T cells.

Various studies have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV; CD26), expressed on T, natural killer (NK) and B cells in the immune system, is involved in the regulation of DNA synthesis and cytokine production. We show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-piperidide, and Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of interleukin-2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-gamma) of pokeweed mitogen (PWM)-stimulated purified T cells. Most importantly, these inhibitors induce a three- to fourfold increased secretion of latent transforming growth factor-beta 1 (TGF-beta 1) by PWM-stimulated peripheral blood mononuclear cells (PBMC) and T cells, as measured with a specific TGF-beta 1 enzyme-linked immunosorbent assay and in the Mv1Lu bioassay. As we could demonstrate previously, TGF-beta 1 exhibits the same inhibitory effects as DP IV inhibitors on DNA synthesis and cytokine production (Cytokine 1994, 6, 382-8; J Interferon Cytokine Res 1995, 15, 685-90). A neutralizing chicken anti-TGF-beta 1 antibody was capable of abolishing the DP IV inhibitor-induced suppression of DNA synthesis of PWM-stimulated PBMC and T cells. These data suggest that TGF-beta 1 might have key functions in the molecular action of DP IV/CD26 in regulation of DNA synthesis and cytokine production.

Cells, Cultured↗