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Biomedical subjects

R Gebhardt

Publications and source records attributed to R Gebhardt.

At least 19 recordsLinked to original sources
PubMed2006

Thin casein films as prepared by spin-coating: influence of film thickness and of pH.

Casein films were successfully prepared with the spin-coating technique of aqueous casein solutions on base-treated glass surfaces. The film structure is investigated in real space with optical microscopy and atomic force microscopy and for the first time in reciprocal space with grazing incidence small-angle X-ray scattering (GISAXS). The size of the substructures detected in the film increases with pH from 170 nm (pH 5.1) up to 490 nm (pH 9.4). Dynamic light scattering experiments reveal that the average diameters of casein micelles in solution exhibit the same quantitative increase. This result suggests that the substructures detected in the bulklike films with GISAXS reflect intact casein micelles. However, with thin homogeneous casein films, the micelle size diminishes with decreasing film thickness. This indicates that the moderate pressures introduced by spin-coating force the micelles to rearrange into a more compact structure.

Caseins↗
PubMed2006

Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

Animals↗
PubMed2005

Pressure-induced dissociation of casein micelles: size distribution and effect of temperature.

Pressure-induced dissociation of a turbid solution of casein micelles was studied in situ in static and dynamic light scattering experiments. We show that at high pressure casein micelles decompose into small fragments comparable in size to casein monomers. At intermediate pressure we observe particles measuring 15 to 20 nm in diameter. The stability against pressure dissociation increased with temperature, suggesting enhanced hydrophobic contacts. The pressure transition curves are biphasic, compatible with a temperature (but not pressure)-dependent conformational equilibrium of two micelle species. Our thermodynamic model predicts an increase in structural entropy with temperature.

Caseins↗
PubMed2005

High pressure near infrared study of the mutated light-harvesting complex LH2.

The pressure sensitivities of the near infrared spectra of the light-harvesting (LH2) complex and a mutant complex with a simplified BChl-B850 binding pocket were compared. In the mutant an abrupt change in the spectral properties occurred at 250 MPa, which was not observed with the native sample. Increased disorder due to collapse of the chromophore pocket is suggested.

Escherichia coli↗
PubMed2005

A novel model of fibroblast-mediated cartilage destruction.

In rheumatoid arthritis (RA), fibroblasts have been shown to be crucial for disease progression as well as joint destruction. In the model of human/murine SCID arthritis, synovial explants as well as fibroblasts from human rheumatoid synovial membrane induce destructive arthritis in immunodeficient mice. Hereby, the underlying cartilage destruction is accomplished by murine fibroblasts. Therefore, murine destructive fibroblasts represent a promising tool to investigate destruction of articular cartilage and bone. In this context, a novel destructive murine fibroblast line (LS48) was examined for morphological, ultrastructural, immunological and functional cellular parameters. These cells were injected into knees of SCID mice. Subsequently, the animals were monitored for joint swelling and serological parameters of arthritis by radiological methods. Finally, cartilage destruction was assessed morphologically. Cultured LS48 cells exhibit characteristic features that resemble those of activated synovial fibroblasts in human RA. Expression levels of inducible nitric oxide synthase, interleukin-6, tumour necrosis factor-alpha and matrix metalloproteinases were comparable to those detected in invasive human fibroblasts. The instillation of 5 x 10(5) LS48 cells into the knee joints of SCID mice initiated a rapid progressive process, that caused cartilage destruction within 10 days, and morphological examinations revealed that articular cartilage was infiltrated by the fibroblasts injected previously. In summary, the intra-articular application of LS48 cells represents a rapid and highly reproducible model to investigate the initiation and progression of cartilage destruction in connection with RA therapy and represents an easy-to-handle animal model.

Animals↗
PubMed2005

Enhanced matrix degradation after withdrawal of TGF-beta1 triggers hepatocytes from apoptosis to proliferation and regeneration.

TGF-beta1 is a profibrogenic cytokine participating in deposition of extracellular matrix in fibrotic disorders. In liver, its anti-proliferative/apoptotic effect on hepatocytes promotes fibrosis. The tetracycline-controlled double-transgenic TA(LAP-2)/p(tet)TGF-beta1 mouse provides a model for reversible liver fibrosis. In livers of TGF-beta1-expressing mice, hepatocytes showed synchronous apoptosis detected by DNA laddering and active caspase-3 staining that disappeared when expression of transgenic TGF-beta1 was switched off. In these 'off' mice, perisinusoidal liver fibrosis resolved within 21 days accompanied by elevated proliferation of hepatocytes. Here, we have specified the intermediary stages (2-3 days off and 6 days off) in terms of (i) proliferation (by immunohistochemical staining of proliferating cell nuclear antigen and expression of cyclin D1 mRNA) and (ii) extracellular matrix remodelling processes (by measuring mRNA expression of matrix metalloproteinases-2 and -13 (mmp-2 and mmp-13) and tissue inhibitor of matrix metalloproteinases 1 (timp-1) and quantitative morphometric analysis. In summary, we show a rapidly declining timp-1 mRNA level together with lastingly high mmp-2 and mmp-13 mRNA levels after 2-3 days, suggesting that high matrix-degrading potential represents a prerequisite for the markedly enhanced proliferation of hepatocytes in the early stages after switching off transgenic TGF-beta1.

Animals↗
PubMed2004

Influence of organosulphur compounds from garlic on the secretion of matrix metalloproteinases and their inhibitor TIMP-1 by cultured HUVEC cells.

Organosulphur compounds from garlic, especially diallyl disulphide (DADS) at non-toxic concentrations, affected production and secretion of some matrix metalloproteinases (MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of their inhibitors, by human umbilical vein endothelial cells. Addition of DADS to the culture medium resulted in a concentration-dependent reduction of secreted MMP-2 protein and activity as well as TIMP-1 protein. In the presence of inducers (phorbol 12-myristate 13-acetate, forskolin and tumor necrosis factor alpha) addition of DADS caused a distinct concentration-dependent decrease of MMP-9 and TIMP-1 secretion, while not affecting MMP-9 mRNA levels. Intracellular protein levels remained low and were not affected. Other organosulphur compounds like allyl mercaptan and S-allylcysteine showed no or less clear effects on MMP-secretion or TIMP-1-secretion. These results suggest that DADS may mediate some of the biological effects ascribed to garlic preparations through affecting MMP-TIMP balance.

Allyl Compounds↗
PubMed2003

Variable influence of kaempferol and myricetin on in vitro hepatocellular cholesterol biosynthesis.

Flavonoids, e. g., quercetin, luteolin, and taxifolin, are known to inhibit hepatocellular cholesterol biosynthesis. Surprisingly, we found that some flavonoids, namely kaempferol and myricetin, are able to considerably stimulate cholesterol biosynthesis, particularly within a low concentration range (between 0.1 and 10 microM), while they show a variable degree of inhibition at higher concentrations. Kaempferol 3-glucoside even stimulated cholesterol synthesis up to 100 microM. In contrast, kaempferol 7-neohesperidoside was ineffective. These effects were more pronounced in rat hepatocytes than in HepG2 cells except for myricetin which acted stimulatory even at high concentrations in HepG2 cells. These opposing effects on cholesterol biosynthesis are exerted in an indirect manner and seem possible though differential modulation of the complex regulation of HMGCoA reductase.

Animals↗
PubMed2002

Oxidative stress, plant-derived antioxidants and liver fibrosis.

There is accumulating evidence that oxidative stress plays a considerable role in the development of liver fibrosis by acting in different cell types and in different signaling pathways. Consequently, antioxidants, particularly those of plant origin, have emerged as potent antifibrotic agents. This review briefly summarizes current views of the mechanisms of fibrogenesis and recent findings on the antifibrotic potential of plant-derived antioxidants.

Animals↗
PubMed2002

Prevention of taurolithocholate-induced hepatic bile canalicular distortions by HPLC-characterized extracts of artichoke (Cynara scolymus) leaves.

The effects of water-soluble extracts of artichoke (Cynara scolymus L.) leaves on taurolithocholate-induced cholestatic bile canalicular membrane distortions were studied in primary cultured rat hepatocytes using electron microscopy. Artichoke extracts at concentrations between 0.08 and 0.5 mg/ml were able to prevent the formation of bizarre canalicular membrane transformations in a dose-dependent manner when added simultaneously with the bile acid. However, prevention also occurred when the hepatocytes were preincubated with the extracts, indicating that absorption of the bile acid to components of the extracts was not involved. These results demonstrate that artichoke leaf extracts exert a potent anticholestatic action at least in the case of taurolithocholate. This effect may contribute to the overall hepatoprotective influence of this herbal formulation.

Animals↗
PubMed2002

Comparison of antioxidative capacities and inhibitory effects on cholesterol biosynthesis of quercetin and potential metabolites.

The flavonol quercetin is known to be rapidly metabolized after ingestion by enterocytes and bacteria in the intestinal tract which may influence the biological, e.g. antioxidative potency of this compound. Therefore, quercetin and several of its possible metabolites were compared with regard to their antioxidant activity and their capacity to inhibit hepatocellular cholesterol biosynthesis. Using the 2,2,-diphenylpicrylhydrazyl radical scavenger assay, all compounds with an ortho diphenolic structure acted as strong antioxidants. In contrast, in a cellular assay focusing on lipid peroxidation in cultured rat hepatocytes challenged with tert.-butylhydroperoxide only the lipophilic compounds quercetin and 3,4-dihydroxytoluene were active. Concerning the inhibition of cholesterol biosynthesis, 3,4-dihydroxytoluene surprisingly mimicked the effect of quercetin in primary rat hepatocytes, but much less so in HepG2 cells. All other metabolites were almost ineffective in both cell types. These results suggest that some of the biological functions of flavonoids detectable by in vitro assays may persist in vivo as long as comparably potent metabolites are systemically present.

Animals↗
PubMed2001

Identification of a cis-acting element and a novel trans-acting factor of the glutamine synthetase gene in liver cells.

In the mammalian liver the expression of the enzyme glutamine synthetase (GS) is restricted to a small population of hepatocytes. In cells expressing the enzyme up to 3.5% of total cellular protein is GS. In order to identify enhancer elements contributing to this extraordinarily high level of expression we focused on a region roughly 2.5 kbp upstream of the GS promoter. Gel mobility shift assays revealed binding of an unknown protein within the most distal part of this region and reportergene assays demonstrated that roughly 60 bp downstream from position -2503 are indispensable for protein binding and the full effect of the enhancer. In UV cross-link analysis a 38 kDa nuclear protein that binds to the sequence was identified in rat hepatocytes. This nuclear protein, designated as upstream binding factor of the GS gene (UFGS) seems to play an important role in high-level expression of GS in liver.

5' Untranslated Regions↗
PubMed2001

Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.

Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.

Cation Exchange Resins↗
PubMed2001

Acquisition of blood--tissue barrier--supporting features by hepatic stellate cells and astrocytes of myofibroblastic phenotype. Inverse dynamics of metallothionein and glial fibrillary acidic protein expression.

A number of similarities between astrocytes and hepatic stellate cells (HSC) rose the question whether or not the protective barrier features of blood-tissue interface may be provided by HSC as well. To test this hypothesis, we investigated the presence of metallothionein (MT), a functional marker of blood--brain barrier, in HSC in situ and in cell culture and compared the results with those obtained with astrocytes. The dynamics of MT expression in cultured astrocytes and HSC was investigated by simultaneous labelling of the cells with a monoclonal antibody (MAb MT) against a lysine-containing epitope of the cadmium-induced monomer of MT-I from rat liver and antiserum against glial fibrillary acidic protein (GFAP). Cell activation was estimated by the presence of smooth muscle alpha-actin (SMAA). In immunoblotting, MAb MT recognized monomeric MT protein and proteins in the 30-kDa range; both bands were pronounced in brain and barely visible in liver homogenates. In situ, MAb MT reacted with very few perivascular cells situated in the parenchyma of the liver. Double immunolabelling of brain slices with MAb MT and antiserum against GFAP showed large areas of brain containing cells expressing both MT and GFAP. However, there were also regions in the brain where the cells produced solely GFAP or MT. In liver cell culture, MT was absent from HSC and hepatocytes in early periods of cultivation, during which the cells maintained their original features; however, MT was expressed strongly in HSC during their activation under prolonged culture conditions. Inversely, in astrocytes MT was expressed during early culturing and disappeared from the cells together with SMAA in late culture when GFAP was upregulated. These results suggest that the acquisition of myofibroblastic features by perivascular cells empowers them to establish a protective blood-tissue permeability barrier. In addition, this study shows that, at least in cell culture, an enrichment of perivascular cells in GFAP results in the disappearance of protective functions.

Animals↗
PubMed2001

Garlic and health.

Garlic is a worldwide cultivated crop species which is well-known for its health beneficial effects. In this paper an overview is given on a number of important plant and health aspects of garlic. Furthermore an outline is presented of a European initiative which is aimed at the development of high quality garlic and the identification of biomarkers in atherosclerosis and cancer in humans for disease prevention.

Animals↗
PubMed2001

Anticholestatic activity of flavonoids from artichoke (Cynara scolymus L.) and of their metabolites.

It is well known that water-soluble extracts of artichoke (Cynara scolymus L.) leaves exert choleresis. When studying this effect in vitro using primary cultured rat hepatocytes and cholephilic fluorescent compounds, it was noticed that the artichoke leaf extracts not only stimulated biliary secretion, but that they also reestablished it when secretion was inhibited by addition of taurolithocholate to the culture medium. Furthermore, taurolithocholate-induced bizarre bile canalicular membrane distortions detectable by electron microscopy could be prevented by artichoke leaf extracts in a dose-dependent manner when added simultaneously with the bile acid. These effects were exerted by the flavonol luteolin and, to a lesser extent, by luteolin-7-O-glucoside, while chlorogenic acid and 1.5-dicaffeoyl quinic acid were almost ineffective. Surprisingly, metabolites produced by the cultured hepatocytes were able to stimulate biliary secretion substantially as well as prevent canalicular membrane deformation. These results demonstrate that artichoke leaf extracts exert a potent anticholestatic action at least in the case of taurolithocholate-induced cholestasis. Flavonoids and their metabolites may contribute significantly to this effect.

Animals↗
PubMed2000

Post-transcriptional inhibition of glutamine synthetase induction in rat liver epithelial cells exerted by conditioned medium from rat hepatocytes.

Recently, a soluble factor produced by primary periportal hepatocytes and different from glutamine was found to completely block induction of glutamine synthetase (GS) by dexamethasone (DEX) in the liver cell line RL-ET-1G. Using Northern and Western blotting we investigated whether this block is regulated on the transcriptional or the post-transcriptional level. Three different species of GS mRNA were detected that all increased (though in different proportions), when the cells were exposed to DEX for 24 h. Further maintenance for another 24 h in normal DEX-containing culture medium, conditioned medium from primary periportal hepatocytes or medium containing glutamine did not result in significant differences in GS mRNA levels. In contrast, GS protein and specific GS-activity remained high only under normal culture conditions, whereas both returned to basal levels in conditioned and glutamine-supplemented culture medium. Throughout, GS protein content and specific GS-activity strongly correlated (r = 0.98) excluding that GS-activity is regulated by chemical modification. These data suggest that the decrease in enzyme activity caused by cultivation in CM is controlled on the post-transcriptional level.

Animals↗