Fakultät Naturwissenschaften

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Biologie, Ernährungs-wissenschaften und Lebensmittelwissenschaften sind die Schwerpunkte der Fakultät. Die Forschung befasst sich mit Schlüsselthemen der Life Sciences.

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Role of reactive oxygen species in anti-cancer treatment: Investigations in 2-methoxyestradiol chemotherapy and 5-aminolevulinic acid based photodynamic therapy combined with hyperthermia
(2003) Lambert, Christine; Frank, Jürgen
The thesis deals with two different ROS-generating anti-cancer treatments: chemotherapy with the endogenous estrogen metabolite 2-methoxyestradiol and 5-aminolevulinic acid based photodynamic therapy. Both treatments were investigated with the rat DS-sarcoma model, which can be used in vitro and in vivo. It the first part, it could be shown that 2-methoxyestradiol induces apoptosis in DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to the mitochondria was identified as initial apoptotic event, followed by a decrease in mitochondrial transmembrane potential and the release of AIF out of the mitochondria. In addition, upregulation of FasL and TNFalpha by 2-ME, two death receptor ligands, was observed. Although, 2-ME administration resulted in activation of caspases, pan caspase inhibitor Z-VAD-FMK could not block 2-ME induced apoptotic cell death pointing to a caspase-independent mechanism. Furthermore, an increase in formation of reactive oxygen species was observed after 2-ME treatment. However, supplementation with different antioxidants could not decrease the toxic effect of 2-ME. This finding may indicate, that reactive oxygen species are not involved in apoptosis induction, rather they are a consequence of mitochondrial damage. In vitro and in vivo combination of 2-ME with another ROS-generating treatment resulted in a synergistic anti-tumour effect. In the second part of the thesis anti-tumour effects of 5-aminolevulinic acid based photodynamic therapy combined with simultaneous hyperthermia was investigated. Analysis of apoptosis associated nuclear changes clearly demonstrated the high efficiency of this treatment regime. Formation of reactive compounds (e.g. ROS, nitrogen monoxide, peroxynitrite) which is mainly responsible for toxicity of PDT, could be assessed in the shape of massive protein nitrosylation in tumours treated with PDT alone or the combined treatment. Detection of decreased amounts of heat shock proteins (HSP70 and HO-1) which protect tumour cells against damaging influences, lowered glutathione levels and reduced MMP-activity indicate an increase in degradation of proteins. This phenomenon may be caused by excessive generation of ROS. Taken together, the presented studies could demonstrate the high benefit of combining 2-ME resp. ALA-PDT with hyperthermia (or other ROS-generating therapies), which make them interesting candidates for future clinical applications.
Einfluss der Ernährung und von Genussmitteln auf Risikofaktoren für das Auftreten von ischämischem Herzinfarkt und Schlaganfall
(2005) Eckoldt, Joachim; Bode, Christiane
Background and aims of the study: Arteriosclerotic changes of blood vessels which contribute to coronary heart disease (CHD) and ischemic stroke are influenced by risk factors like cigarette smoking, overweight, hypertension, diabetes mellitus, missing physical exercise and nutritional factors, such as alcohol consumption. Beyond this, the concentrations of serum lipids, antioxidants, coagulation factors or other risk factors, such as C-reactive protein, and homocysteine are considered to be additional factors that indicate an enhanced or lowered risk of atherosclerosis. In this study we examined the effect of nutritional factors, in particular alcohol consumption, on various plasma components that are believed to play a role in the pathogenesis of atherosclerosis. Multiple epidemiologic studies suggest that moderate alcohol consumption reduces the mortality from cardiovascular diseases and that this effect is chiefly mediated by elevation of high-density lipoprotein (HDL). This cross-sectional study assessed the effect of moderate alcohol consumption and other life-style factors on the composition of HDL in healthy working males. An additional goal of the present study was to find out whether there is an association between alcohol consumption and the concentration of vitamins and cardio-protective substances. Methods: We included a collective of healthy men (n = 284, age 23-66 years), investigated with respect to cardiovascular risk factors. The average daily alcohol consumption, nutrient intake, smoking and other life-style factors was assessed by a computer based questionnaire. Group 1 (n = 62) comprised subjects with an average daily alcohol consumption of 0-5g, group 2 (n = 175): 5-30g, and group 3 (n = 47): 30-70g. In addition, the study design made it possible to subdivide groups 2 and 3 in a so called ?beer drinker group 2+3? (> 80 % beer), a ?wine drinker group 2+3? (> 80 % wine) and persons without preference of a certain alcoholic beverage. Results: The alcohol groups showed no significant differences in the nutritional profile (nutrients, energy intake, and metabolic rate). The markers for regular, higher alcohol consumption (g-GT and MCV) were positive correlated to the amount of alcohol consumption. There was no correlation between the great number of clinical laboratory parameters and the amount or kind of alcohol consumption. Thereby the groups are comparable in view of these laboratory parameters. Besides, there were no indications for the existence of diseases, which might influence blood lipid and vitamin concentrations. Antioxidative substances in the blood: Dietary assessment: The intake of vitamin B2, B6, B12, folic acid, retinol, ß-carotene and other carotenoids, as assessed by the computer interview was comparable in groups 1-3. The subjects in group 1 had a higher supply of the vitamins C, B1 and a-tocopherol, ?beer drinker Gr. 2+3? had a higher intake of vitamin B2, B6 and folic acid. Blood measurements: Antioxidative vitamins: The vitamin B2 status in erythrocytes (EGRAC) was lower in group 3 (vs. group 1 and 2). The plasma level of ß-carotene and ß-cryptoxanthin was lower in group 3 than in group 1. Vitamins that influence homocysteine metabolism (including homocysteine): Influence of beer and wine: The status of vitamin B6 and the concentration of free plasma pydridoxal phosphate in group 3 was significantly higher than in group 1. These results cannot explain the postulated positive influence of moderate or higher alcohol consumption through improvements of the vitamin status and the concentration of vitamins in the blood. The vitamins in beer improved the vitamin status only in case of vitamin B6, no effect was calculated in case of vitamin B2 and folic acid. Higher alcohol consumption (group 3) made the vitamin status respectively the plasma concentration of vitamin B2, ß-carotene and ß-cryptoxanthin lower compared with group 1 ? in spite of comparable supply. Coagulation factors, markers of inflammation: The coagulation factors prothrombin time and fibrinogen as well as the ?newer? risk factors C-reactive protein and homocysteine were not correlated to the amount or the kind of alcohol. Lipoproteins: The serum concentration of total cholesterol, cholesterol ester, phospholipids, apolipoprotein A-1 and A-2 was higher in group 3. Moderate and higher alcohol consumption raises the concentration of cholesterol in the high-density lipoproteins (HDL) (including subfractions) ? independent of the sort of alcoholic beverage. The concentration of cholesterol in the low- (LDL) (including subfractions), very-low (VLDL) and intermediate-density lipoproteins (IDL) in the blood was not influenced by alcohol consumption. Composition of HDL: The induced increase of HDL cholesterol was lower in the subfraction HDL3 as in the subfractions HDL2b und HDL2a. Besides we found qualitative changes of the HDL-components: the phospholipid component increased more than the other HDL-components. This phenomenon might play a beneficial role in the mechanism of atherosclerosis. Conclusions: Vitamins: The changes of antioxidative vitamins and vitamins influencing homocysteine metabolism observed in persons with moderate and increased alcohol consumption do not explain the antiatherogenic effect of alcohol. On the other hand, our study confirmed a positive association of moderate alcohol consumption with HDL plasma levels ? independent of other nutritional factors. In addition, alcohol might induce qualitative alterations of HDL composition (more pronounced increased of HDL2 relative increase of the phospholipid component). The pathophysiological significance of this phenomenon remains unclear.
Ecological studies of the Lactobacillus biota in the human digestive tract and adaptation of intestinal lactobacilli to the sourdough ecosystem
(2005) Dal Bello, Fabio; Hertel, Christian
Among the bacteria inhabiting the human gut, lactobacilli have received considerable attention, due to their putative health promoting effects (Reid, 1999; Vaughan et al., 1999). Cultivation of lactobacilli is considered to be reliable and numerous studies using plating on selective media have been performed to investigate these bacteria in intestinal ecosystems (Tannock, 1995; Reuter, 2001). Recently, the application of PCR-DGGE in combination with primers specific for lactic acid bacteria (LAB) detected species which are not considered to be intestinal inhabitants but food-associated, such as Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Remarkably, these species could not be recovered by traditional bacteriological culture on Rogosa SL agar (Walter et al., 2001). In Chapter III, different cultivation media, as well as new incubation conditions were applied to overcome these difficulties. Human faecal samples were plated on selective and non-selective media and incubated under standard condition (37°C, anaerobiosis) for faecal LAB as well as alternative condition (30°C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected stronger by the incubation condition than by the used medium. It was observed that food-associated LAB such as L. sakei and Lc. mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition on Rogosa SL agar showed that L. sakei is one of the predominant food-associated LAB species in faecal samples, reaching counts of up to 106 CFU per gram faeces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in faeces. Examination of the faecal Lactobacillus populations over longer periods has revealed marked variation in the complexity and stability of these populations among human subjects (Vanhoutte et al., 2004, Walter et al., 2001). Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract (GIT) are likely to be transient (allochthonous), originating from either the oral cavity or food (reviewed in Bibiloni et al., 2004). In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a three months interval (Chapter IV). The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. The results of Chapter IV, together with recently published data (reviewed in Bibiloni et al., 2004), give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity. Lactobacilli have been detected in diverse environments and have been the subject of considerable research due to their commercial use in the food industry (reviewed in Hammes and Hertel, 2003). Several Lactobacillus species are commonly detected in both fermented food and the human GIT, but the genetic background for this ecological versatility is poorly understood. Lactobacillus reuteri is a dominant member of the microbiota of type II sourdough fermentations (Meroth et al., 2003) and is considered one of the truly autochthonous Lactobacillus species in humans (Reuter, 2001). The in vivo expression technology (IVET) developed by Walter et al. (2003) was used to identify genes (so-called ivi genes) of the sourdough isolate L. reuteri LTH5531 that show elevated levels of expression during growth of this organism in a type II sourdough fermentation (Chapter V) and during passage through the GIT of mice (Chapter VI). Thirty-eight induced fusions were found to be highly expressed during the sourdough fermentation (Chapter V), and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g. acid and general stress response) reflecting the harsh conditions prevailing during sourdough fermentation. Further eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway are of technological interest, as they are potentially involved in the formation of aroma precursors. Remarkably, IVET with the genomic library that was successfully used in the sourdough study (Chapter V) did not detect ivi promoters when LTH5531 inhabited the GIT of mice (Chapter VI). With IVET, active promoters are selected by expression of an "essential growth factor" (in our system the erythromycin resistance mediated by ErmGT) that allows the organism to colonize and/ or grow in the ecosystem (Rainey, 1999, Walter et al., 2003). Expression of ivi promoters in particular ecosystems must therefore be permanent and strong in order to allow comparable growth rates of ivi clones and clones bearing constitutive promoters, especially in the GIT, where inactive bacteria are washed out. The findings of Chapter V and VI indicate that L. reuteri LTH5531 does not possess strongly expressed "GIT inducible" genes, while possessing 38 ones specifically induced in sourdough. Ivi genes are more likely to contribute to the ecological performance of an organism in a specific environment than genes expressed equally in a broad range of habitats (Rainey, 1999, Gal et al, 2003, Walter et al., 2005). Therefore, traits encoded by ivi genes are likely to be adaptive and the extent of their expression would be shaped by natural selection to improve ecological fitness. The presence of thirty-eight "sourdough specific" ivi fusions in L. reuteri LTH5531 probably reflects the long term adaptation of LTH5531 to the sourdough environment, just as ivi genes detected in strain 100-23 reflect adaptation of this GIT isolate to the rodent GIT (Walter et al., 2003). Indeed, LTH5531 was isolated from an experimental sourdough that had been inoculated with an industrial starter. This industrial starter has been propagated over several years, giving the organisms present sufficient time to adapt. In accordance with this, by using RAPD-PCR, Meroth et al. (2003) showed that strain LTH5531 was present in a commercial type II sourdough starter collected 10 years prior isolation of LTH5531, thus indicating that this strain has adapted to the sourdough environment for at least 10 years. The results of Chapter V clearly demonstrated that knowledge of gene expression and metabolic activities of bacteria during food fermentations can be obtained by applying IVET. The results collected provide an important molecular basis on which improved starter strains can be developed for industrial exploitation. Moreover, the results of Chapter VI show the importance of working with highly adapted, autochthonous strains in studies of microbial ecology in order to reveal the adaptive interactions responsible for the ecological success of these bacteria in their natural environment or during food fermentations.
Development of a genetically defined diploid yeast strain for the application in spirit production
(2005) Schehl, Beatus; Heinisch, Jürgen
Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. In this work two haploid laboratory strains with suitable auxotrophic markers were used for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Different fruit mashes were fermented, subjected to distillation and analysed for fermentation parameters including growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols, uretahne and glycerol. All spirits produced were tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques adjusting yeast physiology to special production schemes.
Changes in the concentration of particular hormones and carbohydrates in apple shoots after "bending" respectively chemical treatments and relationship to the flower induction process
(2005) Boonplod, Nopporn; Bangerth, Fritz
SUMMARY Apples are cultivated commercially throughout the temperate zone. A regular production however does not seem possible because of irregular yields from year to year. Main causes for this are the so called "alternate bearing" behavior which is the result of profuse flowering in one year but few or no flowers in the following year. It is reported that too vigorously growing shoots are part of the reasons for alternate bearing in apple trees. Applications of chemicals or conventional cultural practices, such as bending shoots have been widely used to restrict shoot growth and promote flower induction. However, the physiological mode of action of these methods in FI is still unknown. Phytohormones are thought to be involved in the process of flower induction (FI). In the above experiments, we investigated changes in endogenous hormones, starch and sugar contents after bending upright shoots into a horizontal position and spraying apple trees with the growth regulators Alar plus Ethrel to improve FI. The experiments were carried out during the years 2001 to 2003 at the Experiment Station, of the University of Hohenheim, Germany, whereby the apple cvs. ?Golden Delicious?, ?Boskoop?, ?Elstar? and ?Idared? were used. The apical part of growing shoots and non-growing bourse shoots, beside bark, wood and shoot diffusates were collected. Plant samples were frozen immediately in liquid nitrogen and freeze dried. Phosphate buffer 0.1M, pH 6.2 was used for collecting auxin in the shoot diffusates. All samples were stored at ?20C until extraction and purified, identified and quantified by Radio Immuno Assay (RIA). The results revealed, in general, that shoot bending and spraying with Alar plus Ethrel changed the endogenous hormone concentrations in the apical part of shoots, as well as in wood, bark and shoot exudates of apple trees. The ?Golden Delicious? cultivar and vigorously growing shoots showed clearer tendencies of hormonal changes than the other cvs. and non-growing bourse shoots. Cytokinin concentrations in the apical part of shoots, and in wood and bark increased after both treatments. Contrary to that, GAs and IAA concentrations in the apical part of shoots and in shoot exudates showed the opposite results. Both treatments had no effect on the concentration of ABA. Ethylene production in shoot tips was considerably stimulated by the combined treatment of Ethrel plus Alar probably due to Ethrel being a "synthetic precursor" of ethylene. Considerable variation existed in the mentioned hormonal changes in respect to the year of examination and the cv. under investigation. Time of treatments and in particular climatic conditions were probably the most influential variables. In spite of all this and on the basis of the above results the conclusion can be drawn that higher concentrations of cytokinins and lower concentrations of gibberellins and auxin are favorable for FI. Spraying with Alar plus Ethrel and bending of shoots seemed to decrease the reducing-sugars, as well as sucrose and starch concentrations in growing shoots and their leaves. In non-growing shoots, spraying seemed to reduce starch but to increase reducing-sugars and sucrose concentrations. A correlation between changes in carbohydrate contents (reducing sugar, sucrose and starch) caused by the spraying treatments and FI does not seem to exist. All the observed changes in the carbohydrate concentrations caused by spraying treatments were not particular impressive and did not really support the often published claim that the effect of spraying growth regulators, bending shoots or other cultural practices may mediate their stimulatory effect on FI via a change in carbohydrates. In contrast to that the above observed experimental results rather suggest that hormones are more effectively involved in the flower induction process of fruit trees.
Effekt der Überproduktion von Enzymen des Glucosestoffwechsels auf das Wachstum und die Alkoholbildung in der Hefe Saccharomyces cerevisiae
(2006) Emili, Markus; Heinisch, Jürgen
The wine-, beer- and baker's yeast Saccharomyces cerevisiae is the major source in world wide alcohol production. Regarding the research in bioethanol production, the work presented here was aimed to examine the effect of the in vivo overproduction of all enzymes contributing to the conversion of glucose to ethanol in the yeast Saccharomyces cerevisiae with the prospect of increasing ethanol formation. S. cerevisiae is probably the best studied eucaryotic organism with respect to both classical and molecular genetics. It turned out to be of great advantage that two different multi-copy-vectors could be employed in these studies. Each of them was used in the first part of the work to insert half of the set of genes intended for overexpression. The first genes were inserted by restriction and ligation and later on a combination of the PCR-technique, with which the genomic fragments of interest were amplified, and the efficient homologous recombination in vivo was used. With these methods, the gene encoding a hexose transporter (HXT1), all the genes encoding glycolytic enzymes (HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH1 bzw. TDH2, PGK1, GPM1, ENO2, PYK1), as well as the genes encoding enzymes needed for the conversion of pyruvate to ethanol (PDC1, ADH1), were cloned. Following the isolation from yeast, the plasmids were amplified in E. coli and characterized by restriction analysis. The measurement of specific enzyme activity in crude extract of yeast transformants with such plasmids showed a slight overproduction (factor 1,5 to 3,0) for all enzymes, except for glyceraldehyde-3-phosphate dehydrogenase. For HXT1, an increased mRNA level (factor 14 in contrast to the control) was taken as evidence for overproduction. In the enzymatic determinations a clear tendency showing a lower overproduction with an increasing number of genes on the plasmids was observed. These findings suggest a negative feedback on glycolytic flux regulation. The the growth rates obtained in the second part of the work also showed a clear reduction with increasing numbers of plasmid-encoded genes. Regarding the physiological parameters, no changes in the coefficients for glucose consumption and ethanol formation could be found in comparison to a wild-type control, and the yield remained basically unchanged as well. Interestingly, abolishing the ATP-inhibition of phosphofructokinase by expression of a mutant allele of PFK1, resulted in a faster growth of transformants with an otherwise isogenic background. This result indicates the physiological relevance of the allosteric regulation at this essential glycolytic step. A lack of enzyme activity in one of the glycolytic steps in deletion mutants normally leads to growth inhibition on hexoses. On this basis, the construction of a yeast strain was initiated with the objective to obtain stable multi-copy transformants simply by growing cells on different sugars as carbon sources. In detail, this was done by crossing a strain carrying a pgi1-deletion with a strain carrying a pyk1-deletion followed by sporulation and tetrad dissection. Preliminary data with intermediate strain constructs indicate a clear increase in plasmid stability after growing cells on complex media. From the results of this thesis, valuable insights into the regulation of the glycolytic flux in vivo can be deduced, which may serve as a basis for ongoing research on the improvement of ethanol formation by yeast.
Einfluss von Karotten- und Tomatensaft-Konsum auf Coloncarcinogenese-relevante Faecesmarker beim Menschen
(2006) Schnäbele, Kerstin; Briviba, Karlis
Colorectal cancer is one of the most common tumor diseases in the world. Most of the colorectal tumors are sporadic and develop somatically in epithelial cells. Nutritional factors can markedly affect tumor development. A high intake of fruits and vegetables is often associated with a reduced risk of colorectal cancer. Protective effects of fruits and vegetables are attributed to ingredients, such as fibers, vitamins, and secondary plant products (e.g. carotenoids), which have potential anticarcinogenic properties. The aim of this study was to investigate, by means of a human intervention trial with carrot and tomato juice consumption, whether a diet rich in carotenoids, especially high in beta-carotene and lycopene, can modify processes relevant to colon carcinogenesis in the gastrointestinal lumen. Therefore, several faecal markers had to be established and used in this study. In the randomized crossover trial, 22 healthy male subjects on a low-carotenoid diet consumed 330ml of carrot or tomato juice daily for a period of two weeks. The two juice intervention periods were preceded by two-week depletion phases. At the end of each study period the stool of twelve volunteers was collected over a 48-hour period. This stool was used to produce some preparations such as non-filtered and sterile-filtered faecal water, as well as faecal lipid extracts, in order to use them in cell culture systems. Spectral photometric and flow cytometric methods were used to determine the effects of the above-mentioned preparations on colon adenocarcinoma cells (HT-29), as well as to determine the activities of the bacterial enzymes beta- glucosidase and beta-glucuronidase in faecal water. HPLC methods were used to measure the concentrations of several bile acids in faecal water, as well as to determine the concentrations of carote-noids and malondialdehyde (MDA) in faecal samples. The concentrations of the major short chain fatty acids (SCFA) were measured via gas chromatography. Consumption of carrot juice led to a marked increase of beta-carotene and alpha-carotene in faeces and in non-filtered faecal water, as did lycopene after consumption of tomato juice. In the succeeding depletion phases, the contents of those carotenoids in faeces and faecal water returned to their initial values. Changes in faecal MDA concentrations by carrot and tomato juice interventions could not be observed. Faecal water showed high, dose-dependent cytotoxic effects on HT-29 cells. Those effects were, however, not markedly changed by carrot and tomato juice consumption. Neither bile acid concentrations nor the bile acid profile in faecal water changed after carrot and tomato juice consumption. Bacterial activities of beta-glucosidase and beta-glucuronidase also did not change. While tomato juice consumption did not significantly affect the pH value of faecal water, this value was, however, decreased by carrot juice consumption. Although faecal water concentrations of acetate and butyrate contributed to the decrease in faecal water pH values, SCFA were probably not responsible for the observed pH changes after carrot juice consumption. SCFA concentrations in faecal water and SCFA proportions did not change significantly. Neither bile and SCFA concentrations, nor the activities of tested bacterial enzymes, had any influence on the cytotoxic effects of sterile-filtered faecal water. These cytotoxic effects, however, decreased with increasing proportions of the primary bile acids cholic and chenodesoxycholic acid, independent of the study phases. As determined by multiple regression analysis, the most probable leading factors for the growth inhibitory effects of faecal water are the faecal MDA content and bacterial beta-glucosidase activity. Further studies should investigate whether the parameters mentioned directly influence cytotoxic and antiproliferative effects of faecal water or if those parameters are indirect markers for the activity of individual microflora. Carrot and tomato juice consumption strongly increased the cytotoxic effects of faecal lipid extracts in HT-29 cells, likely caused by the induction of apoptosis. Which mechanisms account for these effects and the consequences of these effects in the in vivo situation should be investigated in further studies. This work shows that two-week interventions with carotenoid-rich juices lead only to minor changes in luminal processes relevant to colon carcinogenesis in young healthy volunteers on an energy- and macronutrient-balanced diet. Lacking effects on 1) the toxic and antiproliferative properties of faecal water, 2) lipid peroxidation in faeces, 3) the bile and SCFA concentrations in faecal water, and 4) bacterial enzyme activities indicate that related physiological effects can not be influenced by a diet rich in carotenoids under the just described conditions. Other anticarcinogenic mechanisms seem to be of greater importance.
Mechanismen der Mastzellaktivierung durch gram-negative Bakterien und Bakterienprodukte aus der Darmflora.
(2006) Krämer, Sigrid; Bischoff, Stephan C.
The role of mast cells (MC) as effector cells in IgE dependent processes like the type 1 allergy has been known for a long time. During the decade, it has been shown that MC are also involved in other pathophysiological processes such as mucosal polyposis, rheumatoid arthritis, inflammatory bowl disease, tissue fibrosis, and atherosclerosis. Furthermore, MC play an important role in the regulation of host defense against microbes, tissue remodeling processes, and neuro-immunology-interaction. The first aim of the present study was to clarify the question whether human intestinal MC express toll-like receptors (TLR), which recognize conserved bacterial and viral components, and can MC be activated through TLR-ligands. The second major focus of the present study was to investigate if the stimulation of human intestinal MC with different E. coli and Shigella strains, respectively, results in an activation of MC and to identify the underlying mechanism(s). Accordingly, human intestinal MC were isolated from surgery tissue with a mechanical and enzymatical protocol. The purity of the MC cultures used in all experiments was between 98 and 100% which was achieved by positive selection (MACS). We could show, that human intestinal MC express mRNA for TLR 1, 2, 3, 4, 5, 6, 8, and 9. However, neither the stimulation with LPS (lipopolysaccharide, TLR 4 ligand), LTA (lipoteichoic acid, TLR 2 ligand), Zymosan (TLR 2 ligand), poly I:C (polyinosinic-polycytidylic acid, TLR 3 ligand), R848 (TLR 7/8 ligand), CpG (C poly G oligo-desoxy-nucleotide, TLR 9 ligand) and non CpG, respectively resulted in a release of histamine, leucotriens, TNF-alpha, or IL-8. Furthermore, mRNA expression levels of TNF-alpha and IL-8 were not induced by any of the treatments. Similar results where found when human intestinal MC were stimulated with E. coli (O101:H-) isolated from human faeces or the probiotic strain E. coli Nissle 1917. Even after stimulation with pathogenic bacteria strains such as the invasive S. flexneri M90T and the fimbriated E. coli, respectively, no induction of any of the parameters mentioned above was found. However, E. coli strains activate the intracellular signal molecule and transcription factors ERK1/2, c-Fos, and AP1, but this activation failed to induce a complete immune answer. In contrast, the hemolytic E. coli stains ATCC 25922 and ATCC 35218 provoked strong activation of intestinal MC. Using the isogenic hemolysin negative E. coli mutants and the hemolysin positive transformants of the probiotic E. coli Nissle 1917 it was shown, that human intestinal MC are sensitive target cells for E. coli alpha hemolysin. Stimulation of MC with sublytic concentration of hemolysin resulted in an induction of TNF-alpha, IL-3, IL-5, IL-6, IL-8 mRNA expression, the release of histamine as well as leucotrien. This activation was found to be regulated by calcium dependent signal cascades. Inhibition of intracellular signal molecules showed that the activation depends on L-typ calcium channels, calcineurin, NFAT and NFkappaB. Prolonged infection with hemolytic E. coli strains resulted in lysis of intestinal MC indicating a biphasic activation of hemolysin.
Die kleine GTPase RagA und der potentielle Pankreastumormarker TCTP, zwei Bindungspartner des Kerntransport-Proteins RanBP3
(2006) Schleicher Lilia; Biesalski, Hans-Konrad
RanBP3 is a component of the Crm1-mediated protein export from the nucleus. It favors the binding of export substrate to the RanGTP-Crm1 complex in the nucleoplasm. Upon translocation through the nuclear pore into the cytoplasm, the export complex is dissociated by RanBP1 and RanGAP. It was the objective of this thesis to advance the understanding of RanBP3 function by identifying novel binding partners. Using Yeast-Two-Hybrid Screening, two new interactors of nuclear protein RanBP3 were found, the small GTPase RagA and the translationally controlled tumour protein (TCTP). As a minimal requirement a short C-terminal part of RagA (residues 275-313) binds to the N-terminal region of RanBP3. Full length recombinant RagA was expressed. To substantiate the two-hybrid interactions, the binding of RagA to RanBP3 was verified by in vitro binding assays using recombinant proteins. RagA competes with exportin Crm1 for binding to RanBP3. Likewise, the nucleotide exchange factor for Ran, RCC1, binds to the RanBP3-Ran complex and inhibits RagA binding. Expression of GFP-fusion proteins established the cellular localisation of RagA in living and fixed cells. It is found predominantly in and at the nucleus. A model of RagA-import and -export is suggested. Residues 100-172 in the C-terminal part of TCTP form the binding site for its interaction with RanBP3. Using Northern Blot analysis, two mRNAs of TCTP were found in all human tissues examined. The TCTP sequence was completed using an ovarian cDNA library and the TCTP was cloned and expressed. By means of quantitative PCR it was shown that TCTP belongs to the small group of very abundant mRNA molecules in the cell. The TCTP-RanBP3 interaction was verified by in vitro binding assays using recombinant proteins. A polyclonal antibody to TCTP was generated. By using a GFP-TCTP fusion protein and anti-TCTP antibody the cellular localisation of TCTP was analysed. In Cos7-cells TCTP is found in and around the nucleus. In human cell lines most of the TCTP is localised at the nuclear membrane. Using specific anti-TCTP antibody it was shown that the TCTP level is increased in pancreatic tumours. A differential diagnosis of pancreatitis and pancreas tumour is feasible. TCTP has the potential to become a pancreatic tumour marker or a prognostic factor for ductal type adenocarcinomas of the exocrine pancreas.
Einfluss kurzkettiger Fettsäuren und mikrobieller Fermentationsprodukte neuartiger Oligosaccharide auf Cytotoxizität, Proliferation und Apoptose von humanen Coloncarcinom-Zelllinien
(2006) Roser, Silvia; Rechkemmer, Gerhard
Colon cancer is the second most common cancer in Germany. The role of dietary fibre in the prevention of colon cancer is still controversial: Promising results from in vitro and animal studies are contradictory to inconsistent results from epidemiological stu-dies. Functional carbohydrates as constituents of prebiotic food can modify the colonic microflora for the benefit of short chain fatty acid (SCFA)-producing microbial strains. The SCFA-concentrations should also be increased in the distal part of the colon where most colon carcinomas are developing. SCFA are considered to be preventive against colon cancer. For this study, three different new functional oligosaccharides (OS, made of Isomaltulose and resistant starch) were produced from the Südzucker company and fermented in vitro with human feces of healthy test subjects. The resulting fermentation supernatants (FS) were tested in a cell culture system, using colon carcinoma cell lines of various degrees of differentiation (HT29, HT29 Clone 19A, T84). Cytotoxicity, proliferation, the induction of apoptosis, influences on the cell cycle and electrophysiological parameters were measured. Spectral photometric and flow cytometric methods were performed, as well as measurements in vertical diffusion chambers (Ussing chambers). The parallel testing of SCFA-mixtures with the same SCFA-concentrations as in the FS was included, as well as the testing of a FS ?Control? which was produced without OS-fermentation. Several independent fermentations revealed reproducible results regarding the SCFA-concentrations of the FS. After OS-fermentation, the ratio of the three major SCFA in the FS, acetate, propionate, and butyrate, was similar to that observed in vivo. The FS and SCFA-mixtures tested had a cytotoxic effect on all cell lines at the con-centration of 50 %. A dose dependent decrease in cell proliferation could be found, as well as the induction of apoptosis at a concentration of 50 %. Parallel testing of the analogous SCFA-mixtures showed that cytotoxic and proliferation inhibiting effects of the FS could be primarily attributed to their SCFA-content. This could not be confirmed for apoptosis induction: the SCFA-mixtures were mostly able to induce a higher apoptosis rate than the FS. Similarly, the effects of FS and SCFA-mixtures on the cell cycle were different: The SCFA-mixtures showed more potent inhibition of DNA-synthesis than the analogous FS, which generally led to an arrest in the G2-phase of the cell cycle. Neither FS nor SCFA-mixtures had an impact on transepithelial resistance or short circuit current of differentiated cell monolayers in Ussing chambers. The difference in the fermentation patterns of the various FS and the SCFA-concentrations of the SCFA-mixtures was not great enough to achieve significantly different results in the test systems used. Also, the various differentiation grades of the cell lines showed inconsistent results after treatment with FS and their SCFA-mixtures, so that no correlation could be found between degree of differentiation and test compound action. This study shows that the in vitro fermentation of OS with human feces results in reproducible SCFA-patterns in the FS, similar to the in vivo situation. For the screening of FS and their SCFA-mixtures, respectively, a spectrum of methods was established for the incubation with colon carcinoma cell lines of various differentiation states and of all stages of growth (exponential, subconfluent, confluent, fully differentiated monolayer). Indeed, the effects measured after incubation with FS could only in part been ascribed to their SCFA content. Other FS components than SCFA that play a role, especially regarding to their apoptosis inhibiting and cell cycle influencing effects, remain to be identified. Also, this study allows no conclusions to be drawn, which of the fermented OS is more promising in it?s beneficial influence on colon cancer preventing factors, e.g. the induction of apoptosis, than the other. Future studies should investigate FS with greater differences in their SCFA-concentrations. The same OS which were used for the in vitro fermentation, should also be tested in animal studies and human intervention studies to elucidate their fermentation patterns in vivo.
Hydrostatic high pressure treatment of casein to generate defined particle and gel structures
(2006) Merel-Rausch, Eva; Hinrichs, Jörg
The focus of the work was to study the influence of pressure treatment conditions on pressure-induced casein structures in detail. The influence of process parameters like pressure build-up, pressure level, holding time and release rate but also temperature, ionic strength and casein concentration were determined. This work showed that the structure formation of casein under high pressure treatment depends on numerous factors. Sols but also gels can be formed and could be used for different applications particularly with the choice of the release rate and the milieu conditions, even if pressure conditions and casein concentration are kept constant.
Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.
Funktionen charakteristischer Sequenzmotive endogener und toxischer mitochondrialer Proteine
(2006) Papatheodorou, Panagiotis; Rassow, Joachim
In the course of their biogenesis, mitochondria take up nuclear encoded proteins from the cytosol continuously. Protein import at the mitochondrial outer membrane is mediated by TOM proteins and by TIM proteins at the inner membrane, respectively. Now and then, toxical proteins released by pathogenic bacteria to infected tissue can also reach mitochondria. The present dissertation provides new findings on the role of characteristical sequence motifs that can be identified in endogenous and toxical mitochondrial proteins. In an extensive project the importance of sequence motifs from mitochondrial metabolite carrier proteins in their biogenesis and function was investigated in more detail. It could be shown, that the positively charged presequence of the citrate carrier from Rattus norvegicus is not involved in mitochondrial targeting but rather serves as an internal chaperone. A conserved sequence motif, PX(D/E)XX(R/K), the Carrier Signature, which can be found in all mitochondrial carrier proteins, does also not represent a mitochondrial targeting signal, as could be proven by using the dicarboxylate carrier from Saccharomyces cerevisiae as a model protein. Even the translocation across the outer membrane, the insertion into the inner membrane and the following dimerization of the dicarboxylate carrier are processes occuring independently of the Carrier Signature. Instead, it was discovered, that the Carrier Signature is primarily necessary for the function of metabolite carrier proteins in the inner membrane. In another project it could be shown for the Map toxin from enteropathogenic Escherichia coli strains (EPEC), that it is directed to the mitochondrial matrix, mediated by its typical N-terminal presequence and by the TOM and TIM complexes, respectively. The Map toxin leads then to the fragmentation of the mitochondrial network independent of the mitochondrial fission machinery and to the loss of the mitochondrial membrane potential. Moreover, it could be proven, that an internal conserved sequence motif, WXXXE, is essential for cytotoxicity of the Map toxin in the cytosol and for fission of mitochondria. A lysine residue within the WXXXE sequence serves probably as a locus of sumoylation. The investigations show, that mechanisms of intracellular protein transport are not only important for the biogenesis of mitochondria, but can also be relevant for pathological processes.
Determination of Laterality in the Rabbit Embryo: Studies on Ciliation and Asymmetric Signal Transfer
(2007) Feistel, Kerstin; Blum, Martin
The midline of the vertebrate embryo plays a pivotal role in the regulation of left-right (LR) asymmetry. In mammals recent interest has focused on a structure situated at the caudal part of the notochord, the posterior notochord (PNC), which is homologous to Kupffer?s vesicle (KV) in fish and the gastrocoel roof plate (GRP) in frog. Despite highly diverging embryonic architecture, the PNC/KV/GRP is the site where motile monocilia set up a directional fluid flow, an event indispensable for the generation of LR asymmetry. Signals created at the PNC/KV/GRP need to be transferred to the periphery of the embryo, where they initiate the left-specifying program in the left lateral plate mesoderm (LPM). In this study morphogenesis and ciliogenesis of the notochordal plate as well as the signaling processes between midline and LPM were studied in the rabbit embryo. Rabbit development progresses through a flat blastodisc phase and represents the typical mode of mammalian embryogenesis. Transcription of ciliary marker genes, the first sign of beginning ciliogenesis, initiated in Hensen?s node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen?s node anteriorly to form the notochordal plate. Cilia lengthened to about 5µm and polarized from an initially central position to the posterior pole of cells. Electron microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. These data showed that the ciliogenic gene program essential for laterality determination is conserved at the midline of the rabbit embryo. The present study also provided evidence that initiation as well as repression of the Nodal cascade crucially depended on communication between midline and lateral plate (LP). Separation of LP tissue from the midline before, during and after the 2 somite stage demonstrated that signals from the PNC induced and maintained the competence of LPM to express Nodal. Signals from the midline were necessary after the 2 somite stage to maintain a right-sided identity, i.e. absence of Nodal expression. Gap-junction-dependent intercellular communication (GJC) was shown to play a central role in this process. Previously, GJC had been involved in LR axis determination in cleavage stage frog embryos and early blastodisc stages in chick. This study for the first time demonstrates the role of GJC in mammalian embryos. GJs regulate the signaling between midline and periphery: permeable gap junctions were required specifically at the 2 somite stage to repress Nodal induction in the right LPM, whereas closed GJs were a prerequisite for Nodal signaling on the left side. Establishment of the right-sided fate depended on FGF8, the signaling of which was regulated by the opening status of GJs. A 3-step model is proposed for symmetry breakage and induction of the LR signaling cascade in vertebrates: (1) Nodal protein synthesized at the lateral edges of the PNC diffuses bilaterally and confers competence for the induction of the Nodal cascade to the LPM, (2) at the same time the left-specific cascade is actively repressed by action of the GJC/FGF8 module, and (3) following the onset of leftward flow at the PNC repression gets released specifically on the left side at the 2 somite stage, presumably by transient inhibition of GJC. This model not only is consistent with the presented data, but also with published work in other model organisms.
Epidemiologische Aspekte der Falschen Mehltauinfektion durch Plasmopara viticola an Vitis
(2007) Keil, Sven; Spring, Otmar
The obligate biotrophic oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni causes downy mildew on grapevine. Plasmopara viticola is one of the economically most important pathogens in viticulture, with severe losses in yield of up to 70%. Existing prognosis models for plant protection in viticulture only allow yes/no statements on possible infection events in the vineyard. On the basis of these models, the severity of infections remain uncertain, although this represents a crucial point for an efficient application of fungicides. At low infestation severity, the application of protective fungicides at the end of the incubation period usually is sufficient. The low level of infestation can be tolerated and only further propagation of the pathogen must be prevented. In contrast, at high infestation severity curative fungicides have to be applied as soon as possible, because otherwise too much host tissue would be destroyed. Based on epidemiological studies and field experiments a prognostic concept has been designed, which enables the user to evaluate the relevance of infection events of grapevine downy mildew. This work has been carried out in the context of the research project ?Optimierung der Peronospora-Bekämpfung im Rebschutz auf der Basis eines erweiterten Prognosemodells (Forschungsvorhaben des Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz Nr. 514-33.54/01HS048)?. The developed concept was then integrated into the existing prognosis model and should support both consultants and winegrowers in using plant protection agents only in case of an expected increase of infestation frequency and severity. In this way, an effective and more economical use of fungicides is possible, which contributes to economic savings and reduces pesticide contamination of the environment. In the present study, aspects of sporangiogenesis, infestation of host tissue and hibernation, spreading of sporangia, interaction between vine leaves and sporangia as well as the climatic conditions during infection were analysed and evaluated with respect to the impact on epidemiology. The results improve existing literature data and deliver new insight to the epidemiology and biology of Plasmopara viticola.
Funktionelle Untersuchung der Sensorkinase KdpD von Escherichia coli mit Hilfe verschiedener KdpD-Deletionsmutanten
(2007) Rothenbücher, Marina; Kuhn, Andreas
The high affinity K+ transport system KdpFABC is one of several uptake systems that accumulate K+ in Escherichia coli. Expression of the kdpFABC operon is under control of the regulatory proteins KdpD and KdpE, which constitute a typical sensor kinase/response regulator system. KdpD is an integral protein of the cytoplasmic membrane. The N-terminal domain, the 4 helices and 200 amino acids of the cytoplasmic C-terminal domain are accredited to be involved in the signal input function. Surprisingly, a mutant (KdpD-C) lacking the N-terminal domain, helix 1 and 2 is a functional K+ sensor, which is able to detect the changes in K+ concentration in the medium. To investigate which parts of the KdpD protein are essential for signal transduction, various truncated KdpD variants were constructed and analyzed. The results show that the fragment C499-894, which contains only the cytoplasmic C-terminal domain of KdpD, is able to recognise the increase in K+ concentration in medium and reduce the level of activity. This mini sensor is also able to discriminate between Li+, Rb+ and K+ ions like the wild-type KdpD. A plasmid coding for this mini sensor allows a kdpD deletion strain to grow under K+-limited conditions in medium. Presumably, the signal perceived by KdpD is in the periplasm, but how is the signal transmitted to the cytoplasmic domain of KdpD that controls activity? Perhaps KdpD is not the direct sensor, an additional component in the membrane might sense the signal and communicate with KdpD. Further research with sodium carbonate extraction to determine the membrane localisation of C499-894 showed the protein mainly found in the supernatant, suggesting C499-894 is a soluble protein, although C499-894 could be attached to the membrane to come in contact with an unknown membrane protein. Looking for the unknown component two protein candidates were found by co-purification, the cytochrome oxidase A (CyoA) and the glucosamine-6-phosphate synthase (GlmS). To find an interaction between these proteins and KdpD more research has to be done.
Transepitheliale Stimulation humaner Leukozyten durch Bakterien und ihre Oberflächenbestandteile
(2007) Bäuerlein, Annette; Parlesak, Alexandr
Background: The intestinal mucosa plays an important role in the discrimination of immune response between pathogenic and non-pathogenic bacteria as well as in mediating the systemic immunity. To adress the question whether probiotic, commensal, pathogenic germs and bacteria of food origin as well as their membrane components modify the immune response of the intestinal mucosa, we co-cultivated enterocyte-like CaCo-2 cells with human blood leucocytes in transwell cultures. Of further interest was the sequence of enteroxyte-leukocyte activation. Methods: PBMC (Peripheral blood mononuclear cells) were stimulated transepithelially in CaCo-2/PBMC co-cultures and directly challenged with probiotic, commensal, enteropathogenic and food-originating bacteria as well as with membrane components of grampositive and gramnegative bacteria. The expression of inflammatory cytokines (TNF-α, IL-8, IL-6, IL-10 and IFN-γ) was studied by enzyme linked immunosorbant assay (ELISA). The ratio of IL-8/18S mRNA was detected using quantitative reverse transcription polymerase chain reaction (qRT PCR). The permeation of endotoxin was quantified via Limulus amobocyte (LAL) assay and the integrity of the CaC-2 cell monolayer was detected via fluorescein-dextran and transepithelial electric resistance (TEER). Results: Grampositive bacteria did not activate immunocompetent cells in leukocyte-enterocyte co-cultures whereas a stimulation with the gramnegative probiotic E. coli Nissle resulted in higher expressions of TNF-α, IL-8, IL-6, IL-10 and IFN-γ than stimulation with the enteropathogenic E. coli (EPEC). The feature ?probiotic? results not necessarily in an enhanced production of inflammatory cytokines. Differences in epithelial permeability were not necessarily associated with an enhanced activation of immunocompetent cells. There was no activation of immunocompetent cells after direct or transepithelial stimulation with lipoteichoic acid (LTA) of E. faecalis. In contrast, endotoxin depending on its structure was a very potent (E. coli K12, E. coli Nissle, S. Typhimurium) or a moderately potent (B. vulgatus, B. vulgatus MPK) inducer of an inflammatory cytokine response. A neutralisation of endotoxin permeating into the basolateral compartment with Polymyxin B and Colisin resulted in a nearly total inhibition of inflammatory response. Furthermore, directly stimulated PBMC with comparable amounts of the permeating endotoxin in CaCo-2/PBMC co-cultures showed the same activation status as transepithelially stimulated cells. The probiotic, nonstimulating bacteria (Lb. rhamnosus GG, B. vulgatus, B. bifidum) were not able to reduce the E. coli K12 induced TNF-α, IL-8, IL-6, IL-10 and IFN-γ production. A co-stimulation of LPS from E. coli K12 (1 µg/ml) with non-stimulating endotoxins (B. vulgatus and B. vulgatus MPK) 100 µg/ml tended to reduce cytokine expression. Conclusion: These results show that the attribute ?probiotic?does not result in an obligatory activation of immunocompetent cells. The ability to stimulate immunocompetent cells is preferentially dependent on the presence of endotoxin, regarding the structure which is responsible for the stimulating capacity and is not mediated by enterocytes in first line. Activation of the basolaterally located lymphocytes occurs via permeating endotoxin. The permeability of the intestinal epithelial layer is only relevant when permeating endotoxin is able to stimulate immunocompetent cells to due its structural features.
Improving food safety of sprouts and cold-smoked salmon by physical and biological preservation methods
(2007) Weiss, Alexander; Hammes, Walter
The safety of raw, ready-to-eat foods is of paramount importance and is in the focus of the food industry, consumers as well as food scientists. To improve the food safety status of the products, efficient decontamination as an important processing step and/or the use of protective microorganisms as biocontrol agents are promising approaches. In our work we successfully used these approaches for raw sprouts and cold-smoked salmon as examples for RTE foods. Therefore the set goals have been successfully performed and essential scientific knowledge has been contributed. The results have been published and are described in the following in form of the respective abstracts. Thermal seed treatment to improve the food safety status of sprouts: Alexander Weiss and Walter P. Hammes. 2003. Thermal seed treatment to improve the food safety status of sprouts. (Journal of Applied Botany. 77: 152-155) Efficacy of heat treatment in the reduction of salmonellae and Escherichia coli O157:H? on alfalfa, mung bean and radish seeds used for sprout production: Weiss Alexander and Hammes, Walter P. 2005. Efficacy of heat treatment in the reduction of salmonellae and Escherichia coli O157:H? on alfalfa, mung bean and radish seeds used for sprout production. (Eur. Food Res. Tech. 211, 187-191) Characterization of the microbiota of sprouts and their potential for application as protective cultures: Alexander Weiss, Christian Hertel, Silke Grothe, Diep Ha and Walter P. Hammes 2006. Characterization of the microbiota of sprouts and their potential for application as protective cultures. (Applied and Environmental Microbiology. Submitted for publication) Lactic acid bacteria as protective cultures against Listeria spp.on cold-smoked salmon: Weiss Alexander and Hammes, Walter P. 2006. Lactic acid bacteria as protective cultures against Listeria spp.on cold-smoked salmon. (Eur. Food Res. Tech. 222, 343-346)
Probiotic bacteria enhance the antibacterial barrier of enterocytes: insights into their mechanism of action
(2007) Schlee, Miriam; Bode, Christiane
In the healthy intestine there is a stable balance of luminal bacteria and host factors to prevent infections or inflammatory bowel diseases (IBD). A complex network of environmental, genetic, and immunoregulatory factors may precipitate the onset of ulcerative colitis (UC) and Crohn's disease (CD), the primary manifestations of inflammatory bowel disease (IBD). It is currently believed that IBD results from an aberrant immune response of the intestinal mucosa towards the normal commensal bacterial flora. Alternatively, a primary defect in the mucosal barrier might permit bacterial invasion and trigger inflammation. In our research group the hypothesis was proposed that the defective barrier in Crohn´s disease may be due to a lack of defensins which form a chemical barrier against luminal bacteria. A major gut defensin is the human beta defensin-2 (hBD-2) which is an inducible antimicrobial peptide synthesized and secreted by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. In recent studies, Fellermann et al demonstrated that the defective expression of hBD-2 which was measured in the gut mucosa of patients with Crohn´s disease was due to a reduced copy number of the hBD-2 gene. In patients with ulcerative colitis beta-defensin expression is low in the colon during remission, but readily inducible during inflammation. Probiotic bacteria might act beneficially in the human gut by inducing the expression of defensins and thereby reinforcing the mucosal barrier. Recently, our group has been the first to describe hBD-2 induction by the probiotic strain E. coli Nissle (Mutaflor®) which is an effective treatment for ulcerative colitis during remission. The aim of the present work was to determine the underlying molecular mechanisms. We determined a time- and dose-dependent expression pattern of hBD-2 in Caco-2 cells upon stimulation with IL-1 beta;, E. coli Nissle culture supernatant and diverse other probiotic strains. We further investigated the transcriptional regulation of hBD-2 expression mediated by probiotics. The hBD-2 promoter contains several elements known to be involved in transcriptional upregulation such as the NF-kappa B element, which is believed to be one of the main regulators of the hBD-2 gene expression. However, for certain signals, the expression of the hBD-2 gene has been reported to depend on the activation of a second transcription factor, such as AP-1. Most importantly, E. coli Nissle was shown to shed or secrete factors, contained in the bacterial supernatant, which were sufficient to trigger activation of NF-kappa B and AP-1 and to induce hBD-2. Our results indicated further that the supernatant-induced activation of the MAP kinase pathways ERK½, JNK, and p38 may be directly responsible for the probiotic supernatant-induced activation of the transcription factors AP-1 and NF-kappa B and subsequent synthesis of hBD-2. A further aim of the present study was to identify and isolate the bacterial components which are responsible for E. coli Nissle mediated hBD-2 induction. As E. coli Nissle culture supernatant was found to be a more potent stimulant than the bacterial pellet, we investigated the characteristics of the unknown soluble or shed molecules in the bacterial culture media. The first analysis revealed the factor as a heat resistant and proteinase sensitive molecule. Both, E. coli Nissle specific lipopolysaccharide (LPS) and bacterial DNA, which might contain immunostimulatory DNA motifs, failed to trigger hBD-2 expression. Based on the knowledge of the surface composition several E. coli Nissle deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin, the flagella filament protein, specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Next, we isolated flagellins from different bacteria strains (Salmonella enterica serovar Enteritidis, E. coli ATCC 25922, E. coli Nissle and the uropathogenic E. coli strain CFT073 Delta hly, whose genome structure resembles closely that of E. coli Nissle). In the Western blot anti-H1 flagellin displayed immunoreactivity against the different types of flagellins, due to the highly conserved central region of the flagellin filament structure. Incubation of Caco-2 cells with isolated E. coli Nissle flagellin (molecular size 60.81 kDa) induced hBD-2 promoter activation in a dose-dependent manner. The induction of hBD-2 expression by flagellin was confirmed with a positive control (Salmonella flagellin). Interestingly, the serotype-identical CFT073 Delta hly flagellin expressed only moderate hBD-2 inducing ability compared to E. coli Nissle flagellin. Thus, differences in extracellular matrix e.g. the glycosilation degree might underlie the differentially modulated hBD-2 response of Caco-2 cells by the two flagellins. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle. In conclusion, flagellin of E. coli Nissle provides reinforcement of mucosal antimicrobial function, apparently without inducing inflammation. This might explain the beneficial effects of E. coli Nissle on remission maintenance in ulcerative colitis. In patients with Crohn´s disease there is evidence against a therapeutic effect of probiotics and this may be explained by a defective defensin system. Future investigations about strain-specific beneficial functions might contribute to the therapeutic application of science-based probiotic products.
Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas
(2007) Aichler, Michaela; Graeve, Lutz
Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers in humans. It is the fourth leading cause of cancer related deaths in Germany and in the United States. Most PDA occurs sporadically, but there are also approximately 5-10% of patients with a family history of pancreatic cancer. The high mortality of PDA is attributed to a lack of early detection methods and poor efficacy in therapies for advanced disease. As an alternative, preventive strategies in individuals with familial pancreatic carcinoma should be considered. Several epidemiological studies showed an inverse correlation between selenium-intake and mortality of certain types of cancer and particularly in gastrointestinal cancers. To this end, in the first part of this study, the influence of selenium as a preventive nutritional additive was investigated in a genetically defined pancreatic cancer mouse model, the EL-TGFatg/+;p53+/- mouse strain. As a major finding, the differentiation grade of the pancreatic carcinomas was heavily influenced by the selenium status. In the selenium-deficient group there were more non-differentiated pancreatic carcinomas than in the selenium-adequate group, which highlighted the implication of selenium or selenoproteins in tumour differentiation. Unexpectedly, however, there was no protective effect of selenium on total or pancreatic tumour latency. Within the selenoproteins, the thioredoxin reductases are strong candidates which may influence cell death and differentiation in pancreatic carcinogenesis. Their function is generally associated with tumour proliferation and also linked to the activation of the tumour suppressor p53. Consequently, the role of the thioredoxin reductases in the pancreas was studied in the second part of this thesis. The enzymatic activity of cytosolic (TXNRD1) and mitochondrial (TXNRD2) thioredoxin reductase in the pancreas and other organs was determined in relation to the selenium-status. TXNRD1 activity in the pancreas was moderate and decreased under selenium deficiency. TXNRD2, instead, showed very high pancreatic activity in relation to other organs and its activity was even increased under selenium-deficiency emphasising its special role in this organ. To further investigate the function of Txnrd1 and Txnrd2 in the pancreas, tissue-specific knockout mice were created and characterized. The Txnrd1 knockout mice did not show an overt phenotype. Interestingly although, pancreatic acinus cells in one year old mice showed a disturbed rough endoplasmic reticulum and alterations in serum amylase and lipase. These mice also had an impaired glucose tolerance. The pancreas of Txnrd2 knockout mice showed severe chronic pancreatitis and pancreatic atrophy at the end of an observation period of one year. The progressive pathogenic process started with mild pancreatitis, developing spontaneously at an age of four weeks. The chronic stage was characterized by the formation of different types of acinar-to-ductal metaplastic lesions, which could be classified in part as early precursor lesions of pancreatic carcinomas. The endocrine pancreas was not affected. The pancreas-specific Txnrd2 knockout mouse strain is the first genetically modified mouse model spontaneously developing acute and chronic pancreatitis. This strain constitutes a unique and powerful tool to model pancreatic pathogenesis, especially the yet unresolved process of transformation from inflammatory to malignant disease.

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