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B Nebe

Publications and source records attributed to B Nebe.

At least 19 recordsLinked to original sources
PubMed2003

The in vitro effects of H-89, a specific inhibitor of protein kinase A, in the human colonic carcinoma cell line Caco-2.

SUMMARY: H-89 is a compound characterized in vitro as a potent and selective inhibitor of protein kinase A. In the present study, we observed that H-89 induced morphological transformation and caused growth inhibition of the human colon cancer cell line Caco-2 in a dose-dependent manner. However, another protein kinase A inhibitor, H-8, had no effect on Caco-2 cells. To evaluate the possible molecular mechanism of H-89-evoked effects in Caco-2 cells, we analysed the capacity of H-89 to regulate the protein kinase B (Akt/PKB) signalling pathway. H-89 treatment led to an activation of Akt/PKB in Caco-2 cells. This activation was phosphatidylinositol 3 (PI3)-kinase-dependent and promoted survival of Caco-2 cells because the PI3 kinase inhibitor LY294002 inhibited the Akt/PKB activation and induced apoptosis of Caco-2 cells. To test whether Akt/PKB activity promoted resistance to H-89-induced effects, LY294002 was added in combination with H-89. LY294002 greatly potentiated the H-89-induced growth inhibition and apoptosis of Caco-2 cells. These results suggest that the H-89-induced growth inhibition of Caco-2 cells is associated with phosphorylation of Akt/PKB protein and that the cells become more sensitive to H-89 and die by apoptosis upon inhibition of the PI3K/Akt pathway.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material.

The interaction of cells with the extracellular matrix at the interface of an implant determines the biology of cells and tissues. We analysed components of cell adhesion and measured physico-chemical characteristics of structural modifications of titanium surfaces: polished, machined, glass particle-blasted, corundum-blasted, vacuum plasma-sprayed. Scanning electron microscopy and profilometry revealed a differentiated topography from smooth to rough surfaces, respectively. Osteoblastic MG-63 cells showed an increased spreading on surfaces with low roughness, although without a straight correlation with the surface topography. Integrin expression was increased on structured surfaces compared with polished material, and the organization of the actin cytoskeleton and fibronectin was impaired on extremely rough surfaces. Electrochemical methods, especially the electrochemical impedance spectroscopy (EIS) was used to evaluate physico-chemical characteristics, and the impedance curves revealed a dependence on the roughness of the material surfaces. Further analyses of the EIS results were performed using equivalent circuits which model the electrical flow through the interface. First indications for a correlation between parameters from the equivalent circuits with surface properties were obtained which promise a relevance for the biological response of the cells.

Actins↗
PubMed2002

[Fibronectin-chemotaxis and collagen-gel contraction of the palmar aponeurosis in morbus dupuytren].

Causes for Morbus Dupuytren (MD) on the cellular level are largely unknown. We have studied chemotaxis and collagen-gel contraction of cultivated cells from nodules and cords of Morbus Dupuytren patients and normal palmar aponeurosis. The cells of the seized tissue showed an increased chemotaxis in gradients of the chemo-attractant fibronectin. Furthermore embedded into collagen-gels cells from MD patients especially from the region of cords to the skin had an enhanced ability to contract a three-dimensional collagen network compared to those originating from the center of the nodules and control palmar aponeurosis. One reason for the increase of chemotactic motion and the ability to contract gels could be a surplus of receptors for extracellular matrix proteins (integrins) on the surface of cells from seized tissues. Flow cytometry of cells fluorimetrically stained for beta 1 -, alpha 2 - and alpha 3 -integrins displayed no differences in the quantity of these main cell surface receptors.

Cell Movement↗
PubMed2001

Inhibition of lens epithelial cell adhesion by the calcium antagonist Mibefradil correlates with impaired integrin distribution and organization of the cytoskeleton.

BACKGROUND: Posterior capsule opacification is the most common complication of primary cataract surgery and is caused by migration and proliferation of residual lens epithelial cells onto the posterior capsule. Interfering with the mechanisms involved in cell adhesion is a suitable approach to prevent posterior capsule opacification. METHODS: Mibefradil, a T-type calcium-channel blocker, was used to examine the influence on adhesion-mediating mechanisms in human lens epithelial cells derived from cataract surgery. Adhesion was evaluated by light microscopy on the anterior capsules. Expression of integrin receptors was studied by flow cytometry. The influence on the distribution of integrin receptors on the cell surface and the organization of the cytoskeleton was examined by immunofluorescence using a confocal microscope. RESULTS: The calcium-channel blocker Mibefradil inhibited cell adhesion on the anterior capsule wall at concentrations between 10 and 100 pNM. The cells expressed the integrin subunits beta1 and alpha3. Mibefradil distinctly impaired the distribution of these integrins on the cell surface in culture. The cells express the cytoskeletal components actin, vimentin and, very weakly, cytokeratin. The structural organization of the actin filaments and vimentin was strongly disrupted with pronounced fragmentation of the actin filaments in the presence of the calcium-channel blocker. CONCLUSION: The results suggest that the inhibition of cell adhesion by the calcium-channel blocker Mibefradil involves the impairment of integrin-mediated mechanisms. The use of this calcium antagonist appears to be a suitable therapeutic approach to prevent posterior capsule opacification.

Calcium Channel Blockers↗
PubMed2001

Structural alterations of adhesion mediating components in cells cultured on poly-beta-hydroxy butyric acid.

Polymers may serve as a biodegradable material in tissue engineering. To assess the biocompatibility of poly-beta-hydroxy butyric acid (PHB), we studied the structural organization of cellular molecules involved in adhesion using osteoblastic and epithelial cell lines. On PHB, both cell lines revealed a rounded cell shape due to reduced spreading. The filamentous organization of the actin cytoskeleton was impaired. In double immunofluorescence analyses we demostrated that the colocalization of the fibronectin fibrils with the actin filaments was lost in cultures on PHB. Similarly, collagen II distribution was altered, whereas the organization of collagen I was not obviously affected. Further evidence for impaired structural organization was obtained for the beta1-integrin receptor and vinculin which mediate the interaction of the cytoskeleton with the extracellular matrix. In confluent epithelial cells, the tight junction protein ZO-1 showed a larger lateral extension in the cell-cell contacts when cells were grown on PHB. Because structural organization of components which mediate cell-matrix and cell-cell adhesion controls cell physiology these parameters could be a sensitive indicator for the biocompatibility of implant materials.

Actins↗
PubMed2001

Use of human preconditioned phagocytes for extracorporeal immune support: introduction of a concept.

Neutrophils are critical effector cells in humoral and innate immunity and play a vital role in phagocytosis and bacterial killing. If they and/or their specific functions are lacking, then immunoparalysis may occur, and severe diseases like systemic inflammatory response syndrome (SIRS) or sepsis can take a fatal course. In this paper, we discuss the possibility of using preconditioned cells in an extracorporeal biohybrid immune support system. A human promyelocytic cell line was stimulated for different times with all-trans retinoic acid. The resulting cells displayed major signs and functions of mature neutrophilic granulocytes including oxygen radical production, phagocytosis of living and dead Escherichia coli, Staphylococcus aureus, Candida albicans, intracellular killing, and interleukin production. The cells can be expanded to yield a sufficient cell mass, and subsequent prestimulation results in an expression of specific neutrophil functions. Extracorporeal bioreactor experiments seem to be feasible to test the benefit in immunoparalysis-associated diseases like SIRS or sepsis.

Bioreactors↗
PubMed2001

Effects of lectins on CCK-8-stimulated enzyme secretion and differentiation of the rat pancreatic cell line AR42J.

INTRODUCTION: The peptide hormone cholecystokinin (CCK) plays an important role in the gastrointestinal tract. The rat pancreatic CCK receptor is a highly glycosylated membrane receptor that is able to bind to plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIMS AND METHODOLOGY: We used both lectins to block this receptor for studying the pathophysiologic relevance of its oligosaccharide side chains. In the present study we investigated the influence of WGA and UEA-I on CCK-8-induced alpha-amylase secretion of the rat pancreatic tumor cell line AR42J, which expresses both CCK-A and CCK-B receptors. RESULTS: Under the influence of WGA (25 microg/mL), the alpha-amylase release was reduced by 25% after 30 minutes compared with the hormone-stimulated controls. UEA-I (25 microg/mL) caused a reduction of 20%. The simultaneous application of the lectins with CCK antagonists L 364,718 or L 365,260 led to a reduction of secretion, but the assignment to CCK-A or CCK-B receptors was not possible. CONCLUSION: In long-term studies, both lectins revealed no toxic or apoptosis-inducing effects. On the contrary, WGA showed an inhibitory effect on cell proliferation and led to improved differentiation of cells.

Animals↗
PubMed2000

Dielectric changes in membrane properties and cell interiors of human mesothelial cells in vitro after crocidolite asbestos exposure.

Asbestos induces cytogenetic and genotoxic effects in cultured cell lines in vitro. For further investigations of the fiber-induced cellular changes, electrorotation (ROT) measurements can be used to determine early changes of surface properties and dielectric cellular changes. In the present study, human mesothelial cells (HMC) were exposed to nontoxic concentrations of crocidolite asbestos (1 microg/cm(2)) for 12, 24, 30, 50, and 72 hr, and were investigated for changes in dielectric properties, morphologic and biochemical changes using ROT measurements, electron microscopy, and flow cytometry, respectively. The results of ROT measurements revealed slightly increased internal conductivity and decreased membrane conductance of HMC during the first 12 hr of exposure to crocidolite. This may be due to functional changes of ion channels of the cellular membrane. However, after exposures of >= 30 hr, reduced internal conductivity and increased membrane conductance of HMC occurred. These effects may be caused by permeabilization of the cell membrane and the leakage of ions into the surrounding medium. The membrane capacitance of HMC is always decreased during exposure of cells to crocidolite fibers. This decreased membrane capacitance may result from the observed reduction in the number of microvilli and from the shrinkage of cells as observed by electron microscopy and flow cytometry. Changes in composition of the plasma membrane were also observed after the labeling of phosphatidylserines (PS) on the cell surface. These observed changes can be related to apoptotic events. Whereas during the first 50 hr of exposure only a small number of HMC with increased exposure of PS on the cell surface was detected by flow cytometry, the dielectric properties of HMC showed marked changes during this time. Our results show that surface property changes of the cellular membrane of HMC as well as interior dielectric changes occur after the exposure of cells to crocidolite fibers. The observed changes are discussed in terms of complex combined cellular effects after amphibole asbestos exposure.

Apoptosis↗
PubMed1998

Hepatocyte growth factor enables enhanced integrin-cytoskeleton linkage by affecting integrin expression in subconfluent epithelial cells.

Hepatocyte growth factor (HGF) exerts mitogenic and motogenic effects in different cell types. In the epithelial cell line mHepR1 we found that HGF induced pronounced alterations in cell morphology and promoted cell adhesion and spreading. To analyze the mechanisms how HGF affects these integrin mediated functions we studied the physical linkage of integrins with the cytoskeleton. First we found that HGF increased the expression of different integrin subunits in subconfluent cells and influenced the distribution of integrins on the cell surface. To address the physical association of integrins with the cytoskeleton we analyzed Triton X-100-extracted cell fractions using flow cytometry. Here we show that cultivation of the cells with HGF for 24 h prior to integrin cross-linking significantly enhanced the cytoskeletal anchorage of integrins. To further find out whether HGF directly induces an integrin-cytoskeleton link without subsequent cross-linking we added HGF to suspended cells but failed to detect cytoskeletally immobilized integrins in the detergent-insoluble cell fraction which could be related to the absence of a calcium response induced by HGF. Overall, the results indicate that HGF promotes the physical linkage of integrins to the cytoskeleton which requires additional stimulation of integrins.

Animals↗
PubMed1998

Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins.

Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the beta1 and the alpha2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.

Antigens, CD↗
PubMed1998

Analysis of spatial distributions of cellular molecules during mechanical stressing of cell surface receptors using confocal microscopy.

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.

Humans↗
PubMed1998

Targeted chemotherapy by intratumour injection of encapsulated cells engineered to produce CYP2B1, an ifosfamide activating cytochrome P450.

The prognosis of pancreatic adenocarcinoma is poor and current treatment ineffective. A novel treatment strategy is described here using a mouse model system for pancreatic cancer. Cells that have been genetically modified to express the cytochrome P450 2B1 enzyme are encapsulated in cellulose sulphate and implanted into pre-established tumours derived from human pancreatic cells. Cytochrome P450 2B1 converts the chemotherapeutic agent ifosfamide to toxic metabolites. Administration of ifosfamide to tumour-bearing mice that were recipients of implanted encapsulated cells results in partial or even complete tumour ablation. These results suggest that in situ chemotherapy with genetically modified cells in an immunoprotected environment may prove useful for application in man.

Adenocarcinoma↗
PubMed1998

Increased cytosolic Ca2+ amplifies oxygen radical-induced alterations of the ultrastructure and the energy metabolism of isolated rat pancreatic acinar cells.

BACKGROUND: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca2+ concentration and energy metabolism. METHODS: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca2+ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. RESULTS: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca2+ was found, a change which could be inhibited by BAPTA, reducing cellular damage. CONCLUSION: Cytosolic Ca2+ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.

Acute Disease↗
PubMed1997

Flow cytometric detection of the association between cell surface receptors and the cytoskeleton.

The cytoskeleton can serve as a structure at which receptors and signaling molecules can be immobilized to react with each other and induce signal transduction, which, consequently, leads to functional responses of the cell. Furthermore, transduction of mechanical forces into the cell can be realized by a physical linkage between receptor and cytoskeleton. We present a flow cytometric approach to analyze integrin receptors that are physically linked to the cytoskeleton. Epithelial cells were suspended and extracted with Triton X-100 containing lysis buffer to obtain the detergent-insoluble cytoskeletal fraction. To detect immobilized receptors, the fractions were incubated with antibodies against the receptors. We were able to measure these cytoskeletons as single particles in flow cytometry. The extracted fractions revealed distinct lower forward and side light scatter intensities compared with normal cells. Our results demonstrated that integrin receptor cross linking induced their association to the cytoskeleton. Incubation of cells with a receptor antibody alone had no effect. We conclude that flow cytometry enables the evaluation of the receptor-cytoskeleton linkage on the basis of objective fluorescence data and on a single cell level.

Animals↗
PubMed1997

Adhesiveness of the free surface of a human endometrial monolayer for trophoblast as related to actin cytoskeleton.

Adhesiveness of the apical (free) plasma membrane of uterine epithelial cells for trophoblast is essential for the process of human embryo implantation. As epithelial cells are normally repellent, i.e. apically non-adhesive, we argue that a remodelling of the epithelial organization from a polarized to a non-polarized phenotype might prepare the apical pole for cell-cell adhesion during the so-called receptive phase. To identify details of apical adhesiveness we examined human epithelial RL95-2 cells (RL cells) which, in contrast to other cell lines, allow trophoblast to adhere to their apical plasma membrane. To determine whether the cytoskeletal structure is functionally critical for adhesiveness for trophoblast, RL cells were treated with actin depolymerizing cytochalasin D, i.e. 0.4 microM for 120 min. Changes in adhesiveness for trophoblast were monitored with a centrifugal force-based adhesion assay. Moreover, ultrastructural features, organization of the actin network and expression of integrins, i.e. alpha 6, beta 1, beta 4, were studied using electron microscopy, confocal laser scanning microscopy and cell surface immunogold-labelling techniques. Changes in transmission of mechanical signals via integrins into uterine cells were examined using a magnetic drag force device, thereby monitoring intracellular calcium responses. The results suggest that adhesiveness of the free surface of RL cells for human trophoblast requires an intact but non-polarized actin cytoskeleton, apically localized integrins linked to actin, and calcium signalling originating at the free surface.

Actins↗
PubMed1996

Induction of a physical linkage between integrins and the cytoskeleton depends on intracellular calcium in an epithelial cell line.

In most cases epithelial cells reveal a polarized distribution of integrin receptors in basolateral domains of the plasma membrane. To evaluate the functional state of integrin receptors in these restricted sites we were interested in the physical association of integrins with the cytoskeleton. Basically, we extracted cells with Triton X-100 to obtain detergent insoluble cytoskeleton fractions and used monoclonal antibodies for the detection of integrins linked to the cytoskeleton. We found that no permanent physical integrin-cytoskeleton associations exist in a confluent culture of the hepatocyte cell line mHepR1. However, incubation with anti-integrin antibodies and cross linking with a secondary antibody induced a physical linkage of beta1 as well as of different alpha subunits to the cytoskeleton. The association of integrins with the cytoskeleton was also inducible in suspended cells, which was detected in flow cytometric analyses and indicates that the formation of a physical integrin-cytoskeleton connection is independent of the localization of integrins, cell shape, and adhesion on a substrate. Using the Ca2+ chelators BAPTA-AM and EGTA, we found that intracellular calcium is a necessary prerequisite to induce a connection of integrins to the cytoskeleton. ATP or tauroursodeoxycholic acid, which provoke an intracellular calcium elevation, partly induced the formation of an integrin-cytoskeleton linkage. These results indicate the obvious role of intracellular calcium in integrin-dependent outside-in as well as inside-out signaling.

Adenosine Triphosphate↗
PubMed1996

Assessment of chronic rejection in permanent accepted renal allografts in anti-CD4 treated rats.

In rats, transient prophylactic anti-CD4 therapy with the nondepleting mAB RIB5/2 prevents acute rejection of MHC-mismatched allografted kidneys and induces long-lasting unresponsiveness. However, little is known about long-term benefits of this prophylactic anti-CD4 regimen. Here we report experimental results of permanently accepted rat renal allografts after prophylactic anti-CD4 treatment in regard to signs of chronic rejection. Kidneys from Wistar Furth donors were orthotopically grafted into bilateral nephrectomized BDIX recipients under the cover of anti-CD4 treatment (20 mg/kg b.w). Kidney function was serially monitored by measurement of serum creatinine and urine protein excretion. After 100 or 300 days respectively renal allografts were harvested, histologically and immunohistologically assessed and intragraft cytokine gene expression determined. Serum creatinine increased in few allografted rats. 30% of the 300-day-old grafts had an increased proteinuria and higher degrees of glomerular sclerosis. In these grafts cellular infiltration was more pronounced. However, no activated leukocytes (IL-2 receptor positive) were detected. Correspondingly, intragraft gene expression of CD3, IL-10 and IFN gamma was low. The results of our study indicate that a prophylactic anti-CD4 regimen diminishes chronic rejection to a level comparable to isografted or naive mass-reduced or ischemic kidneys. Thus, the signs of chronic rejection observed seem to be mainly caused by alloantigen-independent processes.

Animals↗