Institut für Ernährungswissenschaften

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Aktivierung eines neuartigen Apoptose-Signalweges durch den Proteinkinaseinhibitor Staurosporin
(2009) Daubrawa, Merle; Graeve, Lutz
The protein kinase inhibitor staurosporine induces apoptosis via the activation of the intrinsic pathway. First staurosporine was described as a specific PKC inhibitor. Today it is known as a broad range kinase inhibitor and is used as a potent apoptosis inductor. However, the mechanism of the apoptotic effect remains elusive. Furthermore, staurosporine obviously exhibit the potential to eliminate chemotherapy resistant tumors by the induction of a novel intrinsic apoptotic signaling pathway. Different derivatives of staurosporine, e.g. UCN-01, PKC-412 or Enzastaurin are already tested in clinical trials phase I-II for cancer therapy. In the present work it could be shown that overexpression of Bcl-2 does not impede the caspase-dependent induction of apoptosis in J16- and JE6.1-Jurkat T-lymphocytes or in DT40 B-lymphocytes following staurosporine treatment . After generation of apaf-1 -/- DT40 cells it was demonstrated that staurosporine induces apoptosis despite the absence of Apaf-1 and therefore independently of the apoptosome. Together with the generated caspase-9 -/- DT40 cells, caspase-9 was identified as the central effector protein of both staurosporine-induced apoptotic pathways. The involvement of published and putative caspase-9 kinases could not be confirmed by the usage of specific inhibitors. Using phospho-mimicking and phospho-deficient caspase-9 variants, S183 could be identified as an essential phosphorylation site during staurosporine-induced apoptosis. In addition, after treatment with anticancer drugs apoptosome formation was blocked by an N-terminal tag of caspase-9. However, this tag could not prevent staurosporine-induced apoptosis. In further studies the potential role of cathepsines for this novel apoptosis signaling pathway could be analysed by their specific inhibition. In order to investigate the involvement of multiple kinases in this novel apoptotic signaling pathway, combination experiments with specific inhibitors of the respective kinases should be accomplished. Further investigations should clarify whether the influence of S183 on staurosporine-induced apoptosis is based on conformational alteration or on phosphorylation of caspase-9. The generation of additional caspase-9 variants including deltaCARD-caspase-9 or non-cleavable caspase-9 could lead to a deeper understanding of the role of caspase-9 for staurosporine-induced apoptosis. For this purpose caspase-9 -/- DT40 cells and cells reconstituted with different caspase-9 variants could be employed. The phosphorylation pattern of caspase-9 could be determined by mass spectrometric analysis. Xenograft or chorio allantois membrane models were used to investigate if the staurosporine derivative UCN-01 is also able to induce this novel apoptosis signaling pathway in vivo. The identification of both the mechanisms and the effector proteins of this staurosporine-induced apoptotic signaling pathway should provide the opportunity to develop novel agents for the elimination of chemotherapy-resistant tumors.
Antioxidants Attenuate Heat Shock Induced Premature Senescence of Bovine Mesenchymal Stem Cells
(2022) Nir, Dana; Ribarski-Chorev, Ivana; Shimoni, Chen; Strauss, Carmit; Frank, Jan; Schlesinger, Sharon
Mesenchymal stem cells (MSC) have many roles that are important for the body’s proper functioning. When the MSC pool is damaged, it is often correlated with impaired development or health of the organism. MSC are known for their anti-inflammatory, immunomodulatory and trophic characteristics that play an important role in the physiological homeostasis of many tissues. Heat shock impairs MSC capacity by inducing the generation of reactive oxygen species and mitochondrial dysfunction, which, in turn, send the cells into a state of premature senescence. Here, we pre-exposed MSC to melatonin, resveratrol, or curcumin, which are natural antioxidative compounds, and tested the protective effects of these substances from oxidative stress and aging. Our data showed that pre-exposure of MSC to antioxidants decreased reactive oxygen species while mitochondrial damage remained high. Additionally, although the proliferation of the cells was slow, antioxidants protected the cells from premature senescence, and subsequent cytokine release was prevented. We conclude that while elevated temperatures directly cause mitochondrial damage, senescence is induced by elevated ROS levels. We suggest that heat shock alters cell and tissue homeostasis by several independent mechanisms; however, reducing tissue senescence will reduce damage and provide a pathway to overcome physiological challenges in animals.
Bioinformatische Analyse und funktionelle Charakterisierung von strukturellen Genvarianten in ADME-Genen in humaner Leber
(2016) Tremmel, Roman; Zanger, Ulrich M.
Pharmacogenetics is the study about inter-individual genetic variation that influences the response to drugs and other xenobiotics. A major part of this variation is due to hepatic drug metabolism with enzymes, transporters and receptors involved in the ab-sorption, distribution, metabolism and the excretion of drugs, xenobiotics and endoge-nous substances and collectively defined as ADME-genes. Genetic factors along with environmental and endogenous factors, including gender, age, inflammation processes and others are known to influence the expression and activity of ADME-genes. These influences can affect drug response, side effects or toxicity. According to newly published data, the human genome of any subject differs from a reference genome at 4.1 to 5.0 million positions. More than 99.9% of these differences are single nucleotide polymorphisms (SNP) or short insertions or deletions. Further-more, a person carries up to 2,500 structural variants, including copy number variations (CNV) affecting ~20 million bases (1000 Genomes Project Consortium et al., 2015). Thus structural variants affect more bases than SNPs. Per definition the CNVs are du-plicated or deleted DNA segments greater than 1kb and it was shown that they cover at least 12-30% of the human genome. Genome-wide studies investigating the function-ality of CNVs in the fruit fly, the mouse and in humans showed that there are genes whose expression is clearly affected by CNVs (dosage-sensitve), but also genes show-ing lower expression with increased copy number (dosage reversed) or genes without any expression alterations despite different copy number (dosage-insensitive). A prominent example of CNVs influencing drug metabolism is the phase I gene CYP2D6. Carriers of reduced or amplified gene copies show significantly altered ex-pression and enzyme activity levels and also a different drug metabolism of substrates like codeine (opioid) or tamoxifen (selective estrogen receptor antagonist) in compari-son to carriers with normal copy status of two. Genotyping of CYP2D6 gene copy num-ber may thus help to adjust drug dosage in a genotype dependent manner. In this work I investigated if further ADME-genes are affected by CNVs and if these variants have a functional impact on the expression phenotype and drug metabolism. The distribution of CNVs in the most important ADME-genes (n=340) was investigated in three independent cohorts using CNV data in a public accessible database of ge-nomic variants (DGV; dgv.tcag.ca), processed SNP microarray data of paired samples of healthy (n=269) and tumor (n=351) liver tissue of the TCGA project (http://cancergenome.nih.gov/) and ADME-panel based exon next generation sequenc-ing (NGS) applied on 150 well documented human liver samples of an in-house cohort (IKP148). For the NGS data analysis a method was developed and optimized to esti-mate the relative copy number of the ADME genes or every single exon via the read depth. The results were validated using qPCR with specific TaqMan assays. RNA-sequencing data of 50 healthy TCGA liver samples, and normalised expression data from microarray experiments applied to lymphoblastoid cell lines (LCL) from the HapMap samples and the 150 human liver samples (IKP148) were used to analyse the association between CNVs and the mRNA expression. Furthermore, in the IKP148 liver samples protein and enzymatic activity levels were available or measured using West-ernBlot and mass spectrometry for selected ADME-genes. All pharmacologically important CNVs of phase I and phase II genes, including CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1 and UGT2B17 could be confirmed in all datasets. CNVs which were known, but so far not functionally assessed were found in the phase I and II genes CES1, CYP2E1, CYP21A2, UGT2B15 and UGT2B28. In this work rare CNVs (<1%) were mainly found for transporters like ABCA2, SLC2A4 and SLC47A1. The analysis of the read depth in the IKP148 samples data revealed hybrid genes for CYP2A6 and CYP2D6 with their pseudogenes and allowed a fine mapping of the different alleles. The functional analysis further confirmed the positive association between CNVs and the mRNA expression of CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1 and UGT2B17 in all three cohorts. The combination of all data from the NGS project in the IKP148 liver subjects, including SNP and CNV genotypes showed that 11% and 53% of the variability of CYP2A6 and CYP2D6 enzyme activity were explained by the genetic factors. In contrast the mRNA expression of the genes CES1 and CYP2E1 was not dependent of the CNV pattern in healthy liver tissue (IKP148 and TCGA) and lymphoblastoid cell lines. A detailed analysis of the protein and enzyme activity levels (chlorzoxazone-6-hydroxylation) of CYP2E1 confirmed the dosage-insensitivity in the IKP148 liver sub-jects. The dosage compensation can be principally explained by different mechanisms and could be tissue or tumor specific. Furthermore, CNV-linked genetic variants, altered miRNA regulation, incomplete inclusion of regulatory elements or coding sequences, hybridgenes, monoallelic expression, feedback loops or epigenetics could be factors which mask the CNV effect. In this work a haplotype analysis of the CYP2E1 region identified SNPs which were linked to the duplication and a reduced expression phenotype in persons with European ancestry. Using in silico prediction tools we found a relation of one of the linked SNPs in the 3’UTR with additional predicted miRNA bind-ing sites potentially regulating additional CYP2E1 gene copies. The CNV influence on the mRNA expression of the genes CYP21A2, UGT2B25 and UGT2B28 was inconsistent. Although CYP21A2 deletions were associated with a de-creased expression, gene duplications showed normal expression levels compared to samples with two copies. A significant influence of UGT2B28 CNVs was found in LCLs but not in human liver samples (IKP148 and TCGA). In total 7 of 17, 2 of 12 and 3 of 14 ADME genes showed a significant association between expression and CNV type in the IKP148, TCGA and LCLs of the HapMap samples, respectively. In the TCGA cancer tissue nearly all ADME-genes carry CNVs and in 30% of the genes a significant correlation was observed. With cooperation partners further polymorphisms and phenotypes of SULT1A1 and CYP2E1 were analyzed. CYP2E1: In this part of the thesis factors influencing the risk of developing differentiat-ed thyroid carcinoma (DTC) were investigated. Known risk factors for the progression of DTC are genetic and environmental factors, including ionizing radiations, previous thyroid diseases, and hormone factors. It has been speculated that dietary acrylamide intake correlates with the DTC formation. The acrylamide molecule is metabolized by CYP2E1 to the reactive carcinogenic glycidamide. The enzymatic reaction is probably dependent on the CYP2E1 genotype. Together with a cooperation partner (Prof. Dr. Landi, University of Pisa, Pisa, Italy) we investigated, whether CYP2E1 variants influ-ence the DTC risk. Prof. Landi and colleagues used a case-control-cohort and a haplo-type approach and observed a significant association between a tag-SNP rs2480258 (A allele), which covers variants in intron eight and the 3’UTR, and an increased DTC risk. In the human liver samples (IKP148) the rs2480258 genotypes were assessed using an imputation analysis and it was shown that particularly the A allele of the SNP reduce significantly the mRNA and protein expression and the enzyme activity. An in silico prediction for the molecular mechanism suggested that miR570 specifically down regulates the transcripts in carriers of the A allele. These results indicated that the inter-individual CYP2E1 activity as well as acrylamide (similar to glycidamide) influences the risk for DTC. SULT1A1: Methyleugenol, a secondary metabolite present in herbs such as basil or laurel is metabolized in humans by sulfation to a reactive product which can covalently bind to DNA. The resulting DNA adducts are mutagenic and can promote carcinogene-sis. Our cooperation partner Prof. Dr. Hans-Rudolf Glatt (German Institute for Human Nutrition Potsdam-Rehbruecke (DIfE), Nuthetal, Germany) had shown, that the meth-yleugenol metabolism takes place in the liver of mice and is mainly catalyzed by the phase II enzyme SULT1A1. To investigate these facts in humans, the methyleugenol DNA-adduct levels were measured by the cooperation partner in liver tissues (n=121; IKP148) using mass spectrometry. In this work the SULT1A1 protein levels were de-termined using western blot analysis and the relation between the DNA adducts as well as the SULT1A1 expression and the SULT1A1 CNVs was assessed. The SULT1A1 mRNA and protein expression were significantly correlated to the DNA adducts, e.g. higher SULT1A1 expression resulted in higher adduct levels. This emphasized the role of SULT1A1 in the in vivo metabolism in human liver samples. As mentioned above, there were individuals (IKP148) carrying one, two, three, four and five copies of SULT1A1. Deletions were found less frequent (4%) than duplications (36%). The CNVs were significantly associated with the SULT1A1 mRNA and protein expression. This result was consistent to previous studies investigating the association between SULT1A1 CNVs and enzyme activity. The methyleugenol DNA adduct levels were also significantly associated to the SULT1A1 copy number. Carriers of at least three gene copies exhibited a 2.8-fold higher DNA adduct level compared to donors carrying only one SULT1A1 gene copy. As a consequence this could mean that individuals with mul-tiple SULT1A1 copies reach faster, more often and more easily critical and ultimate adduct levels which increase the risk for developing cancer. Future studies should clari-fy whether methyleugenol intake as well as the individual SULT1A1 CNV make-up in-fluences the risk of cancer.
Charakterisierung von Interaktionspartnern des Kernrezeptors CAR ("Constitutive Androstane Receptor") mittels MALDI-TOF Massenspektrometrie
(2010) Melgar, Clint; Graeve, Lutz
The human nuclear receptor "constitutive androstane receptor" (CAR; NR1I3) plays a pivotal role in the induction of drug metabolism and transport by phenobarbital-type inducers. The hepatic expression of phase I and phase II drug metabolizing enzymes and of transporters is activated by CAR in response to structurally diverse chemicals. In addition to xenobiotic detoxification, activation of CAR is also involved in other hepatic functions like fatty acid oxidation, gluconeogenesis, clearance of steroid hormones and bilirubin. In primary hepatocytes, CAR resides predominantly in the cytoplasm associated with other proteins in a multimeric complex of which some components still remain to be identified. Upon exposure to inducers CAR dissociates from the already identified proteins of the complex, the cytosolic CAR retention protein (CCRP) and HSP 90 resulting in its translocation into the nucleus, where it heterodimerizes with the retinoid X receptor (RXR). The CAR-RXR heterodimer binds to its respective response elements in the regulatory region of target genes and recruits coactivators like SRC-1 (steroide receptor co-aktivator), GRIP1 (glutamate receptor interacting protein) and PGC 1 (peroxysom-proliferator-aktivated receptor γ co-activator 1) to induce gene transcription. To better understand the function of CAR, this study was focused on the identification of proteins which associate with CAR. The aim of this work was to identify putative interaction partners of the nuclear receptor CAR which are expressed in liver to get additional information on structure and regulation of the native protein multimer complex und to obtain a better understanding of the functionality of CAR. Hence, we have established an in vitro pulldown assay with liver homogenate to analyze protein-protein interactions of CAR. As a first step we generated GST and GST-CAR fusion proteins which were expressed in E. coli followed by affinity purification. Then we incubated the fusion proteins with total liver homogenate and separated bound proteins with SDS-PAGE. After visualization with silver staining, the protein bands were excised and prepared for mass spectrometry. For identification of new interaction partners of CAR in liver, the Matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF) was used. After optimization of the pulldown assay we could identify several proteins like cytoskeletal proteins (e.g. lamin A), enzymes (e.g. pyruvate carboxylase, GAPDH) and chaperones (e.g. HSP 70) as binding to CAR. As an especially interesting interaction partner of CAR we decided to further investigate the interaction of CAR with BRG1-associated factor (BAF) 155. This protein is a component of the mammalian SWI / SNF chromatin remodeling complex that plays an important role in fundamental cellular processes such as transcription, replication, and the repair of chromatin. Interaction of GST-CAR-LBD fusion protein was confirmed by additional methods like pulldown assay of GST-CAR with (35S)-methionine labeled BAF 155 in vitro and co-immunoprecipitation of the two proteins. Additionally, we could confirm BAF 155 interaction with CAR by Western blotting of the original pulldown samples. Analyzing the interaction of CAR and BAF 155 with RNA interference was not successful, since the method could not be optimized and validated appropriately, due to time constraints. In conclusion, we were able to establish a highly reliable and reproducible assay to investigate protein interactions resulting in the significant identification of new interaction partners of CAR. Regarding the identified CAR interaction partner BAF 155, this protein or the complete SWI / SNF complex could play a functional role in CAR-mediated transcriptional activation, however further research is needed to establish the role of BAF 155 in CAR function.
The coconut water antimicrobial peptide CnAMP1 is taken up into intestinal cells but does not alter P-glycoprotein expression and activity
(2020) Anaya, Katya; Podszun, Maren; Franco, Octavio Luiz; de Almeida Gadelha, Carlos Alberto; Frank, Jan
Coconut antimicrobial peptide-1 (CnAMP1) is a naturally occurring bioactive peptide from green coconut water (Cocos nucifera L.). Although biological activities have been reported, the physiological relevance of these reports remains elusive as it is unknown if CnAMP1 is taken up into intestinal cells. To address this open question, we investigated the cytotoxicity of CnAMP1 in intestinal cells and its cellular uptake into human intestinal cells. Considering the importance of the P-glycoprotein (P-gp) to the intestinal metabolism of xenobiotics, we also investigated the influence of CnAMP1 on P-gp activity and expression. Both cell lines showed intracellular fluorescence after incubation with fluorescein labelled CnAMP1, indicating cellular uptake of the intact or fragmented peptide. CnAMP1 (12.5–400 μmol/L) showed no signs of cytotoxicity in LS180 and differentiated Caco-2 cells and did not affect P-gp expression and activity. Further research is required to investigate the identity of CnAMP1 hydrolysis fragments and their potential biological activities.
Dietary intake, nutritional status of lactating women and their 6-23-months-old children in Genta Afeshum District, Rural Ethiopia : adaptation and validation of calculator for inadequate micronutrient intake (CIMI)
(2020) Desalegn, Beruk; Frank, Jan
Religious fasting is one of the categories of dietary or food taboos, which may affect the dietary intake and nutritional status of individuals. In Ethiopia, about half of the population are followers of Ethiopian Orthodox Tewahedo religion, and approximately 250 days per annum are fasting days. In these fasting days, lactating and pregnant women and children are exempted from fasting. However, lactating and pregnant women fast and are not also happy to prepare non-fasting foods for their children during the fasting days due to fear of contamination of family food. Early identification of micronutrient deficiencies in Ethiopia are flouted, as most often the quantitative dietary data are not available. As a result, the hidden hunger might have not been addressed properly, where it remains high and persistent. Therefore, easy to use, less costly and applicable assessment tool which can estimate the quantitative dietary intake of an individual or a community is urgently needed to achieve the national and international goals set for eradicating malnutrition. The Calculator for Inadequate Micronutrient Intake (CIM) is a simple, easy-to-use, informative, web-based application of quantitative dietary assessment method, which was first developed in Indonesia for Indonesian population. It estimates energy and nutrient intake correctly, and identifies nutrient inadequacy according to FAO/WHO recommended nutrient intake (RNI) regarding age, sex and physiological stage. Thus, the present study was conducted with the aim of assessing and comparing the nutritional status and dietary intake of lactating women and their 6-23-months-old children in fasting and non-fasting periods, and to adapt and validate the CIMI program for Ethiopian population. This study was conducted in rural Genta Afeshum district, in Tigray, Ethiopia, where almost all people in the woreda are followers of Ethiopian Orthodox Christianity. A longitudinal study was conducted using575 and 522 lactating women and their 6-23-months-old children in the lent fasting and non-fasting. In the present study it was found out that the prevalence of underweight (BMI < 18.5 kg/m2) in fasting lactating women was high (50.6%) which is associated with maternal age, maternal illness within four weeks preceding the fasting survey, fasting status during their pregnancy and lactation period of their children included in this study. Additional predictor variables for maternal underweight were grandfathers‘ as household decision maker, use of non-improved water source, household aid experience and the absence of chicken in the household. The average number of meals, diet diversity, and animal source foods consumption scores were significantly higher in non-fasting compared to fasting periods, regardless of the fasting status (p < 0.001, p < 0.05 and p < 0.001, respectively). Whereas, 31.6–33.7%, 11.7–15.7% and 4.4–4.8% of the 6-23-months-old children in the study population were stunted, underweight and wasted, respectively. In the fasting period, the weight-for-length (WLZ) and length-for-age (LAZ) values for the 6-23-months-old children of non-fasting mothers were significantly higher (p < 0.05) than the children of the fasting mothers‘ group. Similarly, the median weight-for-age (WAZ) and diet diversity score (DDS) of children of fasting mothers were also significantly lower in fasting compared to non-fasting period. The proportion of the 6-23-months-old children who met the minimum acceptable diet (MAD) was small (2.3-6.7%) in the study population; however, this proportion was significantly (p < 0.001) higher in the non-fasting than fasting period in the children of fasting mothers. Age of the child, maternal fasting status during pregnancy and lactation periods, maternal education and occupation were associated with child underweight. Likewise, age of the child, colostrum intake status, maternal fasting status during pregnancy and lactation period and toilet presence in the household were associated with child stunting. But, maternal fasting status during lactation period and maternal education predicted wasting in the children precisely. The average energy, protein and almost all micronutrients intakes of children and women were lower in fasting compared to non-fasting period. At the same time, the prevalence of inadequate intakes of energy, protein and most micronutrients were higher in both the children and lactating women during fasting than non-fasting period. The result of this study revealed that the correlation coefficients for the average dietary nutrient intake calculated by CIMI and the reference software NutriSurvey (NS) were between 0.741-0.956 for the children and between 0.779-0.920 for the lactating women groups. As a conclusion, the dietary pattern and nutritional status of lactating women and their breastfed children are affected during the fasting period. Therefore, the existing multi-sectoral nutrition intervention strategies in Ethiopia should include religious institutions in a sustainable manner. CIMI adapted for the rural Ethiopian setting estimates the average nutrient intake accurately; and identifies inadequate micronutrient intake of individuals enabling enumerators to provide feedback and suggest improvements. Thus, CIMI can be used in Ethiopia, as a simple dietary assessment tool by nutrition and related researchers, policy makers, implementers and evaluators.
Der Einfluss des Osteoblasten- und Tumorzellfibronektins auf die Immunzellen
(2014) Kraft, Sabrina; Nakchbandi, Inaam
The function of the osteoblastic niche for the hematopoesis is an aim of recent research. This work shows, that fibronectin originating from osteoblasts leads to an increase of hematopoetic stem cells and granulocyte monocyte progenitors (GMP). The most abundant fibronectin isoform in OB is EDA. This thesis gives first evidence that there is a relationship between the differentiation of HSPC, GMPs and EDA. These results were associated with a changed expression in two intergrins, α4ß7 and α5ß1. The impaired differentiation of GMPs also leads to a decrease of myeloid and granulocyte progenitor cells. Additionally, myeloid progenitors displayed an increased production of cytokines inhibiting tumor growth. There also was a decrease of myeloid derived suppressor cells (MDSC), which are normally contributers of tumor growth through a t-cell suppression dependent mechanism and the production of tumor proliferating cytokines. The decrease of these cells should be benefical for the inhibition of tumor growth and indeed there was a decrease of tumor growth in two tumor models (murine melanoma and human breast cancer bone metastases). These results are due to the decreased number of MDSCs and the increased production of tumor proliferating cytokines. This effect was t-cell independent. A correlation between OB-FN and the inhibited tumor growth were verified by adoptive transfer experiments with in vitro differentiation of myeloid progenitor cells in presence and absence of EDA and the induction of melanoma. The presence of EDA during the differentiation was able to induce an increase of tumor growth again. This thesis presents for the first time the influence of osteoblast fibronectin in immune cell differentiation and indirectly on tumor growth. The second part of this work suggests, that the deletion of fibronectin in tumor cells, especially of EDA, leads to an inhibted growth of human breast cancer cells in bone and in an increased number of macrophages in the tumor. The analysis of the cytokine production of tumor-associated macrophages showed a tumor inhibiting cytokine profil, which suggest, that the macrophages in the tumor are so called M1-macrophages. M1-macrophages are known to inhibit tumor growth. The depletion of macrophages with clodronate lipsomes in mice, leads indeed to an increase of tumor growth in absence of the tumor cell fibronectin. For the first time it was therefore show an influence of a changed extracellular matrix production on macrophages in tumors.
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.
Der Einfluss verschiedener Ölemulsionen und der IL-6-Typ-Zytokine CNTF und LIF auf die Differenzierung von 3T3-L1-Präadipozyten zu Fettzellen
(2009) Stäbler, Antje; Graeve, Lutz
Type 2 diabetes is a disease of civilization spreading all over the world like an epidemic. Among other things it is characterized by increased glucose and triglyceride levels of the blood. Thus the differentiation of preadipocytes into adipocytes that are specialized in storing large fat depots is essential for the removal of excess nutrients from the circulation. This process can be simulated through the differentiation of 3T3-L1 cells by treatment with insulin, IBMX, and dexamethasone. In this study a compensative effect of olive oil as well as soy bean oil on the differentiation of 3T3-L1 cells was found. The differentiation triggered by insulin, IBMX, and dexamethasone was somewhat inhibited by incubating the cells with one of the two oils, whereas both olive and soy bean oil had a certain potential to act as differentiating agents them-selves when insulin, IBMX, and dexamethasone were not added. This points to benefits of certain oils contained in food compared to thiazolidinediones that are used as antidiabetics and lead to weight gain in the long term. Furthermore the inhibitory effect of the proinflammatory cytokine TNFα on the differentiation process could be compensated to some degree by either oil. The transcription factor PPARγ was used to measure the state of differentiation. Caveolin1 is involved in both buildup and degradation of intracellular fat stores and was induced by oil incubation in differentiated as well as in undifferentiated 3T3-L1 cells. Moreover the signal transduction of the IL-6-related cytokine CNTF in 3T3-L1 cells and its influence on the differentiation process of these cells was investigated. In recent years CNTF was spotlighted because of its leptin-like properties that persist even in leptin resistant states. In this study an increasing sensitivity to CNTF during the differentiation of 3T3-L1 cells was shown. Furthermore the positive effects of the cytokine on fatty acid metabolism and adipocyte development were verified. CNTF favored the emergence of many small adipocytes compared to fewer, but larger adi-pocytes under different conditions. Thereby the functionality of the cells is influenced in a positive way and the protection from the development of insulin resistance is increased. The effect of LIF - another IL-6-related cytokine - was not similar to that of CNTF but was more comparable to the effect of the oils.
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.
Eliminierung apoptotischer Zellen durch professionelle Phagozyten: Generierung, Freisetzung und Erkennung des monozytären Attraktionssignals Lysophosphatidylcholin und Bedeutung von Annexin I als Brückenprotein in der phagozytotischen Synapse
(2007) Waibel, Michaela; Graeve, Lutz
The efficient elimination of apoptotic cells by neighbouring cells or professional phagocytes is essential for tissue homeostasis in multicellular organisms. Therefore, the apoptotic cell displays different so-called ?eat-me?-signals on its cell surface that are crucial for its recognition and engulfment. Especially in higher organisms, where the dying cell and the phagocyte are usually not located in immediate proximity, the release of soluble attraction signals is of special importance. Only recently, the phospholipid lysophosphatidylcholine (LPC) could be identified as a central ?find-me?-signal that is generated by the calcium-independent phospholipase A2 (iPLA2)-mediated hydrolysis of phosphatidylcholine. During apoptosis iPLA2 is processed in a caspase-3-dependent fashion. In the present thesis it could be demonstrated that iPLA2 is cleaved directly by caspase-3 and that this processing leads to its activation. The active iPLA2 is essential for the production of the phospholipid-?find-me?-signal LPC in apoptotic cells. However, the observation that overexpression of the active form of iPLA2 alone was not sufficient for the release of the attraction signal from vital cells implied that other apoptotic events might contribute to the generation and export of the ?find-me?-signal LPC. It turned out that the reactive oxygen species-driven oxidation of membrane lipids like phosphatidylcholine is an additional factor that leads to the enhanced production of LPC, probably because oxidized lipids are more susceptible for PLA2-mediated hydrolysis than non-oxidized lipids. Further studies about the detailed export mechanism of LPC revealed that the ATP-binding cassette transporter (ABC)-family member ABCA1 is essential for the release of the attraction signal during apoptosis. Thus, the oxidation of membrane lipids and the ABCA1-driven export of LPC could be identified as important elements of LPC-generation and its subsequent release during apoptosis. After the generation and the release of the attraction signal LPC could be demonstrated in more detail the consequent question arose which receptors might mediate the effects of LPC on the phagocytes. In the present thesis it could be demonstrated that the G-protein-coupled receptor G2A is responsible for the LPC-stimulated migration of monocytic cells. However, the molecular mechanisms that ultimately lead to the LPC-driven, G2A-mediated migration, are not known so far. Accordingly, a participation of other receptors or the existence of further chemotactic signals cannot be ruled out at this point. Moreover, there are some hints for chemotactically active proteins in literature. If these or other factors contribute to the LPC-mediated chemotaxis of monocytic cells is completely unknown and needs to be clarified in future studies. The recognition and internalization of dying cells is mediated by the interaction between different ?eat-me?-signals that are displayed on apoptotic cells, and specific surface receptors on phagocytes. In this scenario, the interaction can be of a direct nature ore rather indirect via bridging molecules. In this context, here it could be demonstrated that the calcium- and phospholipid-binding protein annexin I gets externalized by dying cells independently of the apoptotic stimulus applied, but dependent on the cell type. On the surface of the apoptotic cell, annexin I binds in a calcium-dependent fashion via its annexin-boxes to externalized phosphatidylserine, which represents a central ?eat-me?-signal. Thereby, annexin I is able to stimulate the elimination of these cells by professional phagocytes and thus fulfills the function of a bridging molecule in the phagocytic synapse. In contrast, the receptors that are responsible for the binding of annexin I to phagocytes are not known so far. As a conclusion it can be stated that the phenomena studied in this thesis represent important steps in the process of apoptotic cell elimination. The physiological relevance of apoptotic cell clearance is the fact that apoptosis, in contrast to necrotic cell death, is highly regulated at all stages and usually turns out without any harmful consequences to the organism. If this complex, multistep process is disturbed, non-cleared apoptotic cells can become a source for inflammatory reactions. In different animal models it could be demonstrated that defects in the attraction of phagocytes as well as deficiencies in the recognition and internalization via ?eat-me?-signals and the subsequent degradation of the apoptotic prey can be a reason for the onset of severe autoimmune disorders. In this context, the development of human systemic lupus erythematosus and of chronic arthritis is discussed to be initiated by the inefficient elimination of apoptotic cells.
Establishment of defined culture conditions for the differentiation, long-term maintenance and co-culture of adipose-derived stem cells for the setup of human vascularized adipose tissue
(2018) Volz, Ann-Cathrin; Kluger, Petra Juliane
Most current attempts in engineering adipose tissue are based on the supplementation with human or animal-derived sera. However, especially the use of animal-derived serum is linked to many disadvantages, like potential contaminations, ethical issues and in general the missing identification of many ingredients. Therefore, serum supplementation impedes the actual application of engineered adipose tissue constructs as implants, to substitute lost tissue after tumor resection, severe burnings or trauma. Equally, due to a potential cover up of the cellular response by unidentified components, it impairs the in vitro use of such models as test systems to elucidate mechanisms of disease development, screen for new drugs or generally assess pharmaceutical safety levels. To be capable for functional anastomosis with the host tissue after implantation and for the use in time- and maturation-dependent in vitro purposes, engineered constructs have to exhibit a minimum sustainability. So far, only few authors addressed the serum-free, defined differentiation of adipocytes. And there are hardly any trials available on the defined maintenance of adipocytes. In this study, the development of a defined culture medium for the adipogenic differentiation of primary human adipose-derived stem cells (ASCs) was aimed. Based on the addition of specific factors for the replacement of serum, ASCs were differentiated to viable and characteristic adipocytes for 14 days, which was proven through the accumulation of lipids, the expression of perilipin A and by the release of leptin and glycerol. Furthermore, a defined maintenance medium was developed, which supported the maturation and stability of cells for a long-term period of additional 42 days until day 56. In order to pursue the goal of a physiological tissue substitute of relevant size, the integration of a vascular component is of fundamental importance to allow sufficient nutrient supply of all peripheral tissue areas. For this purpose, a natural vascular system based on a cellular component would be ideal. Due to the lack of an adequate co-culture medium, a major challenge in adipose tissue vascularization is represented by the setup of an adipocyte/endothelial cell (EC) co-culture. In this study, the development of a tissue-tailored co-culture medium based on adipocyte- and EC-factors was developed. Thereby the critical role of epidermal growth factor (EGF) and hydrocortisone (HC) in adipocyte/microvascular (mv)EC co-culture was determined. Through the adjustment of their supplementation, a functional co-culture of adipocytes and mvECs was achieved. In there, mvECs maintained the cell-specific expression of von Willebrand factor (vWF) and cluster of differentiation 31 (CD31). Additionally, cells kept their ability to incorporate acetylated low density lipoprotein (acLDL). By combining the experiences from both mentioned attempts, a defined adipocyte/EC co-culture medium was developed. Next to the maintenance of functional and characteristic adipocytes, the medium facilitated the formation of vascular-like structures in the direct co-culture. To be able to establish tissue constructs of relevant size, current in vitro attempts have to be transferred to a three-dimensional (3D) environment. In this trial, a 3D adipose tissue model was set up based on the differentiation of ASCs in a collagen type I hydrogel in co-culture with mvECs for 21 days in total. The comparison of these models with native adipose tissue demonstrated high accordance in the gene expression levels related to differentiation and fatty acid metabolism. Some deviations were found mostly in maturation-dependent genes linked to tissue functionality and angiogenic mediation. Differentiation and the maintenance of a homeostatic tissue state highly rely on the physical and chemical characteristics of the applied scaffold. As another part, the influence of a novel cellulose-based material (CBM) on defined adipogenic differentiation of ASCs and the defined maintenance of mvECs was investigated in this thesis. An accelerating effect of CBM on the defined differentiation of ASCs was proven by enhanced release of leptin and the increased expression of perilipin A. CBM was further shown to facilitate the formation of vascular-like structures by mvECs under defined conditions in the absence of another supporting cell type. Additionally, the successful co-culture of adipocytes and mvECs was demonstrated on CBM under defined conditions. Summarized, defined culture media for the differentiation, maintenance and co-culture of primary ASC and mvECs were developed. The supporting effect of CBM on the defined establishment of cultures was proven. Further the successful setup of a 3D adipocyte/mvEC co-culture model with a high predictive power was shown. Combined these achievements can be used for the in vitro setup of a 3D vascularized adipose tissue under defined conditions.
Functional validation of genetic changes discovered in high-throughput mutational and copy number screens with respect to carcinogenesis and treatment sensitivity for Head and Neck Cancer
(2015) Keck, Michaela-Kristina; Graeve, Lutz
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease regarding both anatomic location and molecular characteristics. It is comprised of two distinct disease entities - human papillomavirus (HPV)-positive and HPV-negative HNSCC - that differ regarding primary site of disease as well as aetiology/pathogenesis. Persistent infection with high-risk HPV is causally related to oropharyngeal HNSCC, whereas tobacco and alcohol consumption are linked to the development of HPV-negative head and neck cancers. HPV(+) and HPV(-) HNSCC differ regarding their clinical behavior as well as prognosis with HPV(+) tumors being associated with a more favorable prognosis than HPV(-) tumors. However, besides Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), which is used with modest success as no predictive biomarkers have been identified, no targeted treatments are available that consider the biologic heterogeneity and no classification is used in clinical practice that reflects the underlying biology of the disease. As a consequence, all patients are treated uniformly with a combination of surgery, radiation, and chemotherapy depending on stage and location of the tumor. In this study, a homogeneous and clinically annotated cohort of 134 treatment-naive HNSCC specimens, including 59 HPV(+) samples (44%), were processed, subjected to Agilent 4X44Kv2 gene expression arrays and Nanostring copy number assays, and analyzed together with all available HNSCC cohorts (n=938). As a result, a clinically meaningful classification was identified for HNSCC - which was hampered in previous studies due to a limited inclusion of HPV(+) samples, unknown HPV status, small sample size, or missing clinical annotation. Three HNSCC supergroups were identified - basal (BA), classical (CL), inflamed/mesenchymal (IMS) - and further subdivided by HPV status into five HNSCC subtypes - BA, CL-HPV, CL-nonHPV, IMS-HPV, IMS-nonHPV - that differ regarding survival and show different expression profiles and copy number (CN) aberrations. Importantly, two HPV-associated subtypes with different molecular profile and prognosis were identified - CL-HPV and IMS-HPV. The IMS subtypes show a strong immune phenotype with expression of immune response genes and may benefit from immune therapies such as programmed cell death protein 1 (PD-1) inhibitors. The BA subtype shows markedly increased expression of EGFR and hypoxia related genes, which might render these tumors susceptible to EGFR-targeting and hypoxia-targeting therapies. Besides differences regarding their biological and clinical behavior, HPV(+) and HPV(-) HNSCC show a distinct mutational landscape. The incidence of HPV(+) tumors is rising, however, studies are missing that investigate a sufficient number of HPV(+) tumors to derive meaningful information and assign targetable genomic changes to either disease entity. 120 (51 HPV(+), 69 HPV(-)) out of the cohort of 134 samples were further used to build DNA sequencing libraries and processed to next generation sequencing (NGS) on the Illumina platform (Illumina HiSeq 2000/2500 sequencers) to detect novel or targetable genomic changes. The overall mutational burden is similar in both disease entities, however, the mutational landscape differs. In addition to known aberrations, novel targetable aberrations with predicted driver potential are identified in various genes, among which are EGFR, CCND1, and FGFR1 in HPV(-) tumors, and FGFR2/FGFR3, KRAS, BRCA1/2 in HPV(+) tumors, as well as PIK3CA and PI3K pathway genes in both and rare aberrations in DDR2 and EPHA2. The fibroblast growth factor receptor 3 (FGFR3) S249C mutation is recurrently found in six HPV(+) tumors, which makes FGFR3 the second most mutated gene in HPV(+) tumors - after the well-known oncogene PIK3CA. Therefore, functional validation of FGFR3 S249C was performed in vitro in the two HNSCC cell lines SCC47 and 93VU147T. Two different FGFR3 isoforms were identified in all six FGFR3 mutated HNSCC tumor samples - FGFR3IIIb and the soluble FGFR3Delta8-10. Both isoforms were used to stably transfect the S249C mutation into SCC47 and 93VU147T and to study its effects on proliferation and migration as well as FGFR3 signaling. No effect on proliferation or migration rate is seen beyond an increase in proliferation rate in SCC47 cells transfected with wild type (WT) FGFR3IIIb. No constitutive activation of downstream pathways is seen in unstimulated SCC47 cells. Investigation of different downstream signaling pathways identifies the MAPK and the PI3K/Akt pathway as the two important signaling pathways downstream of FGFR3 in HNSCC cell lines. FGFR3 S249C cannot be confirmed as an oncogenic driver in SCC47 and 93VU147T. However, its possible role as an oncogenic co-driver besides other receptor tyrosine kinases and its function in drug resistance mechanisms - especially resistance to EGFR inhibitors - remain to be investigated.
Glucocorticoid-induziertes Wachstum von Tumorzellen : systematische Quantifizierung, Signalmechanismen und Inhibition
(2010) Gündisch, Sibylle; Jeremias, Irmela
Glucocorticoids (GCs) like Dexamethasone (Dex) are widely used in cancer patients, as cytotoxic drugs in hematopoetic tumors or adjuvants in solid tumors to reduce severe side effects. Nevertheless, GCs are accused to reduce anti-cancer treatment efficiency. Due to preliminary works in our research group the suspicion arose that GCs are able to induce proliferation of tumor cells. The present work provides the first systematic quantification of the proproliferative effects of GCs on tumor cells. Enhanced tumor cell growth was validated by repetitive microscopy, impedance analysis, investigation of DNA synthesis rate, enzymatic activity as well as absolute cell number. It could be proven that 6 out of 10 cell lines from solid tumors showed enhanced proliferation after stimulation with Dex, whereas this phenotype was not limited to one tumour entity or a common origin. In vivo, Dex significantly promoted tumor cell growth in a preclinical mouse model with a lung carcinoma cell line. Furthermore the effect of GCs was detected on 139 primary, patient-derived acute childhood leukemia cells. In 15% GCs were able to increase the in vitro survival of the tumor cells and one sample showed even GC-induced proliferation. Accordingly the anti-apoptotic and pro-proliferative effects of GCs could be proven not only on established solid tumor cell lines but also on primary hematopoetic tumor cells. Knockdown studies in cells of solid tumors showed that GC-induced proliferation was mediated by the glucocorticoid receptor and was further transmitted by the proteinkinases Akt and p38-MAPK. GC-induced proliferation could be prevented by induction of apoptosis which was caused either by clinically applicable substances, as for example Vincristine, or by inducible expression of the pro-apoptotic molecule Caspase-3. To sum up, the present work identified GC-induced proliferation of tumor cells as a new, tumor cell directed side effect of GCs. Of direct translational relevance, our data argue towards a restricted use of GCs during anti-cancer therapy as well as the need for preclinical and clinical studies which demonstrate a more effective and safer application of GCs during anti-cancer therapy.
GLUT-1 content and interaction with stomatin in red blood cells from species without vitamin C biosynthesis and their relevance for diabetes mellitus type 1
(2016) Frey, Tabea Caroline; Biesalski, Hans-Konrad
Ascorbic acid is commonly known as vitamin C. By definition, vitamins, with the exception of vitamin D, are substances which can not be synthesized, but are essential for the organism. In this light, vitamin C is special. It is hypothesized that millions of years ago some species, like primates, Guinea pigs and fruit bats, lost the ability to synthesize ascorbate from glucose due to an inactivation of an enzyme called L-gulono-g-lactone oxidase. Since then, these species have been dependent on dietary intake of this micronutrient. Ascorbate is not only the most efficient watersoluble antioxidant, but also an important cofactor in neurotransmitter or collagen biosynthesis. An inadequate intake of this vitamin leads to scurvy. In 2008, a French researcher documented that all species that lost the ability to synthesize ascorbic acid express a different facilitative glucose transporter isoform(GLUT) in their erythrocytes. The expressed GLUT-1 transports not only glucose but also dehydroascorbate which is the oxidized form of vitamin C. Was that different GLUT expression the keystone event for the evolutionary success of these species? To examine the questions regarding the evolutionary benefit of this transporter expression, the kinetics of ascorbate and dehydroascorbate transport into erythrocytes from four different species were evaluated. Further, recycling and disposal of the transported vitamin as well as a possible accumulation were observed. The results demonstrate that there are three different transport types of dehydroascorbate. One which does not transport the vitamin at all, one whose transport of dehydroascorbate competes with glucose and one which absorbs dehydroascorbate completely independent of the extracellular glucose concentration. After absorption, vitamin C is not recycled and is not disposed back into the extracellular fluid. Additionally, it is not stored in the cell until the erythrocyte undergoes apoptosis. The evolutionary benefit is found in an electron transfer across the erythrocyte membrane from intracellular ascorbate to the extracellular, oxidized form of vitamin C. In an energetic light, this recycling of extracellular vitamin C is more efficient than the de novo synthesis of the micronutrient. Therefore, the erythrocyte acts not as a reservoir for vitamin C storage, but as a reservoir for electron storage to prevent degradation and loss of dehydroascorbate in times of high oxidative stress. This electron reservoir becomes more important in diseases with high levels of oxidative stress. A metabolic disorder, which is frequently described to be accompanied by high levels of oxidative stress and lowered vitamin C levels in plasma and cells, is Diabetes mellitus. The decreased plasma concentrations do not result from a smaller dietary intake. Probably, the uptake of dehydroascorbate into erythrocytes, and, therefore, the extracellular ascorbate recycling is disordered. Investigations of the distribution of GLUT-1 in different erythrocyte membrane subdomains showed that the regulation of this transporter is altered in subjects with diabetes mellitus type 1 compared to healthy controls. In vitro, no differences in dehydroascorbate transport rate could be observed, but significantly decreased intra-erythrocyte vitamin C concentrations were detected in vivo. In conclusion, the altered regulation of GLUT-1 in the erythrocyte membrane in the case of diabetes can affect vitamin C recycling in plasma. A decreased ascorbate pool in the cells leads to a decreased recycling capacity, and, therefore, to a lower antioxidant defense outside the cell. Due to that knowledge, the recommended dietary intake of vitamin C in the case of diabetes mellitus must be reconsidered to prevent further complications.
Health enhancing traditional foods in Brazil : an interdisciplinary approach to food and nutritional security
(2012) Abadio Finco, Fernanda; Graeve, Lutz
The Brazilian nutritional profile is currently characterized by the so-called "nutrition transition process" i.e. the population presents nutritional status characteristics of both developing and developed countries. Therefore, malnutrition is present not only in the form of undernutrition but increasingly also presents as overweight and obesity. Some studies suggest that this is not only a particular problem of urban societies but also of rural communities. Recently, Brazil has impressively advanced on issues which address nutrition, agriculture and health within a sustainable framework. One of the recent initiatives encompasses the Brazilian Food and Nutrition Security Policy, which could be considered as the vanguard of this theme by covering different dimensions of nutritional issues, as defined hereunder: ?Food and Nutrition Security is the achievement of the right of all people to access food regularly and permanently, with quality and enough quantity, without compromising the access to other basic needs, based on food practices to promote health, with respect to cultural differences and being social, economic and environmentally sustainable?. Since the Brazilian Food and Nutritional Policy is characterized by a broad view on food and nutrition, different components related to food and nutrition have to be considered. Therefore, the health side of the food, in a pluralistic vision has to be taken into account. Thus, food and their consumers are unavoidably connected. Beyond classical nutrients, much attention has recently been focused on bioactive compounds and their preventive role on non-communicable diseases such as diabetes, cardiovascular diseases and cancer. Therefore researchers are increasingly interested to unfold the preventive biochemical processes of these compounds. Hence, the current research aimed to investigate health enhancing properties of traditional Brazilian fruits within the Food and Nutrition Security definition of the country. Given the interdisciplinary feature of the topic Food and Nutrition Security, the work was performed in two stages. The first one encompasses a nutritional survey with two rural communities in APA ? Cantão, Tocantins State, Brazil and the second part comprises experimental laboratory research. The outcome from the nutrition survey showed a high level of food insecurity among the families (84.2%). The nutritional profile of the study population expressed a high prevalence of overweight for the adults (53.7%). Regression analysis showed that the high Body Mass Index (BMI) is influenced by the consumption of an imbalanced diet and the physical activity level. Furthermore, women had a higher prevalence of overweight and obesity in comparison to men. Another observation is that rural communities have a monotonous diet with very low consumption of fruits and vegetables. Besides the negative effect on their body composition, this last result points to the risk of developing micronutrient deficiencies, i.e. hidden hunger. Based on the outcome and the demand presented by the participants in the nutrition survey, two fruits available in the region were chosen to investigate their possible biofunctional properties. Different assays were performed with Bacaba (Oenocarpus bacaba Mart.) and Jenipapo (Genipa americana L.) phenolic extracts. Extracts from both fruits showed antioxidant and antiproliferative capacities. Since bacaba displayed higher activities than Jenipapo, this fruit was chosen for a more detailed investigation of the biochemical mechanisms involved. The results showed that bacaba phenolic extracts induced apoptosis in MCF-7 breast cancer cells through the mitochondrial pathway. Caspase-6, -8 and -9 were activated when compared to the untreated control in a dose dependent manner (p<.05). However, caspase-9 showed the highest activation. Since MCF-7 cells do not express caspase-3 and based on additional investigations on PARP (Poly (ADP-ribose) polymerase) - cleavage, the experiments suggest that caspase-9 plays an important role in the observed apoptotic effect. The laboratory work thus emphasizes the potential healthy properties of traditional fruits from the Brazilian biodiversity with high antioxidant activities. Altogether, the results indicate the need of a better nutritional education with the involved communities in order to promote healthy eating practices and to increase the consumption of fruit and vegetables. Based on this, it is suggested for government and policy makers to take action in rural communities. Indeed, it is undeniable that the biodiversity available in Brazil is a huge treasure and source of novel ?superfruits?. Therefore, the current work reinforces the development of research in this area in order of identify health enhancing neglected traditional fruits and to promote their consumption, add value and generate income to small farmers and traditional communities with not only the improvement of their economic power, but also of their diets and health respecting their tradition and culture. Not to mention the contribution to biodiversity preservation since plants that were merely discarded could now have a multifactor value in line with the Brazilian Food and Nutritional Security policy.
Identification and functional studies of two novel serine phosphorylation sites of insulin receptor substrate (IRS)-2: Ser 675 and Ser 907
(2010) Fritsche, Louise; Schleicher, Erwin D.
Insulin receptor substrate (IRS) proteins are major transducers of the insulin and IGF-1 signal into the PI-3 kinase/PKB and the MAP kinase pathway. In addition to tyrosine phosphoryla-tion, a large number of serine/threonine phosphorylation sites enable the IRS proteins to inte-grate different extra- and intracellular stimuli resulting in positive and negative modulation of the insulin and IGF-1 signal. Chronic hyperphosphorylation of serine/threonine sites of IRS-1 is involved in the development of insulin resistance. IRS-2 is of great importance for β-cell survival and for the regulation of hepatic metabolism. The study of serine/threonine phos-phorylations is required to understand the physiological and pathophysiological regulation of this important mediator of insulin signaling. In this thesis two novel IRS-2 serine phosphoryla-tion sites have been identified and characterized (mouse amino acid numbering): Ser 675, which is located in the kinase regulatory loop binding (KRLB) domain unique to IRS-2 and Ser 907, which is adjacent to the Grb2 binding site Tyr 911. Using phospho-site specific antibod-ies both sites were demonstrated to be phosphorylated upon insulin, phorbol ester and ani-somycin treatment in Fao rat hepatoma cells. The phosphorylation was also detected in pri-mary human hepatocytes and in liver tissue of insulin treated or refed mice. The insulin-induced phosphorylation of Ser 907 was mediated by the MAP kinase ERK1/2. Simulation of a permanent phosphorylation of this site in BHK cells expressing IRS-2 Glu 907 led to a slight decrease of IRS-2 tyrosine phosphorylation with no apparent effect on insulin downstream signaling. The insulin-induced association of IRS-2 with Grb2 in HEK293 cells was abrogated by mutation of the adjacent Tyr 911 to Phe, but not influenced by mutation of Ser 907 to Ala. Of note, the activation of MAP kinase signaling was not impaired in HEK293 cells expressing IRS-2 Phe 911 and not regulated by the expression level of IRS-2 wildtype, but completely dependent on IR expression, indicating the importance of an alternative, IRS-2-Grb2-independent pathway for the activation of MAP kinase signaling in these cells. The insulin-induced phosphorylation of Ser 675 was dependent on mTOR, but not on the downstream kinase p70 S6K1. Prevention of this phosphorylation in BHK cells or HEK293 cells expressing IRS-2 Ala 675 had no effect on proximal or distal insulin signal transduction. But compared with IRS-2 wildtype, the mutated IRS-2 protein Ala 675 showed increased half life in cycloheximide-treated HEK293 cells. Thus, phosphorylation of Ser 675 could have a similar function as its homologous site Ser 632 in IRS-1 and could be involved in the regula-tion of mTOR-dependent IRS-2 proteasom-mediated protein degradation.
Identification and quantification of tocomonoenol isomers in plants and microalgae and investigation of their metabolism in liver cells
(2022) Montoya Arroyo, Alexander; Frank, Jan
Tocopherols (T), tocomonoenols (T1), and tocotrienols (T3) are tocochromanols, a group of bioactive compounds composed of a chromanol ring and a 16-carbon sidechain with biological functions, such as the protection of lipid membranes from oxidation and the modulation of cellular signaling. T have saturated sidechains, while T1 and T3 have a single or three double bonds in theirs, respectively. The prefixes alpha-, beta-, gamma-, and delta- are assigned based on the number and positions of methyl groups on the chromanol ring. alpha-, beta-, gamma-, and delta-congeners of T1 have been reported, with alpha-T1 being the predominantly identified congener. Two different alpha-T1 isomers are known, 11-alpha-T1, which has been mainly found in land plants, and 12-alpha-T1, which has been mostly detected in marine organisms. However, little is known regarding the occurrence of T1 in photosynthetic organisms and their metabolism in the liver, a strong determinant of bioavailability and bioactivity. The aim of this thesis was to evaluate underutilized plant-based food sources, cyanobacteria, and microalgae as potential sources of T1 and to characterize the uptake and conversion into metabolites of T1 in cultured liver cells in comparison to T and T3. Acrocomia aculeata fruits were analyzed for alpha-T1 due to its phylogenetic relationship with Elaeis spp, the most common source of this congener. No alpha-T1 was detected in oils from endosperm and mesocarp of wild fruits of Acrocomia aculeata from Costa Rica. Aerial parts of the local underutilized leafy vegetable Urtica leptophylla were evaluated as source of T1 due to its agronomical potential and previous reports of T1 in leaves of plants. LC-MS analyses indicated that leaves and flowers of Costa Rican Urtica leptophylla contain minor amounts of alpha-T1 and gamma-T1. Cyanobacteria and microalgae from different species and origins were analyzed as source of alpha-T1 due to their role as primary producers in aquatic ecosystems and the reported presence of 12-alpha-T1 in marine phytoplankton. alpha-T1 in cyanobacteria and microalgae ranged from traces up to 17% of the total tocochromanol content. alpha-T1 concentrations alone were higher than the sum of all four T3. 11-alpha-T1 was the major alpha-T1 isomer in cyanobacteria and microalgae, as determined by GC-MS. Hence, 11-alpha-T1 is a product of biosynthetic pathways even in aquatic organisms. The effect of nitrogen depletion during the cultivation of microalgae on their alpha-T1 content was investigated. Nitrogen depletion did neither significantly affect the relative or absolute content of alpha-T1, despite an increase in tocochromanol content, nor the proportion of 11-alpha-T1/12-alpha-T1 in microalgae. The uptake and conversion into metabolites of 11-alpha-T1 in HepG2 liver cells was compared to those of alpha-T3 and alpha-T. Cellular uptake of alpha-T1 in liver cells was higher than that of alpha-T. 11-alpha-T1, similar to alpha-T, was converted mostly to alpha-carboxymethylhydroxychroman in a time dependent manner, but to lower extend than alpha-T3. The effect of both ring methylation and sidechain saturation on the uptake and metabolism of the alpha- and gamma-congeners of T1, T and T3 was studied in HepG2 cells. gamma-Congeners were metabolized at higher extent than alpha-congeners and metabolite production increased with increasing number of double bonds in the sidechain independently of chromanol ring methylation. In conclusion, alpha-T1 is present with up to 17% of total tocochromanols in cultured microalgae, which thus are an important new source of this congener. gamma-T1 is only a minor tocochromanol in U. leptophylla flowers. 11-alpha-T1, and not 12-alpha-T1, is the major alpha-T1 isomer in cyanobacteria and microalgae and nitrogen depletion of microalgae does not significantly affect alpha-T1 concentration. The metabolic conversion of alpha-T1 into alpha-carboxymethylhydroxychroman in HepG2 cells is similar to that of alpha-T and significantly lower than that of alpha-T3, suggesting that it may be handled by the organism similar to alpha-T. In conclusion, novel potential food sources of alpha-T1 have been identified and, because of similarities with alpha-T, its pharmacokinetics and biological activities warrant further investigation.
Interaktion des bakteriellen Quorum Sensing Moleküls N-(3-oxododecanoyl)-L-Homoserin-Lacton (AHL-12) mit humanen neutrophilen Granulozyten
(2013) Kahle, Nadine; Hänsch, Gertrud Maria
To evade immune-defense mechanisms of the host, Pseudomonas aeruginosa form so called biofilms. To coordinate this complex process, the bacteria communicate via quorum sensing signals, such as N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12). AHL-12 is also recognized by human polymorphonuclear neutrophils (PMN). It is chemotactic for PMN and thus might cause their directed migration to the site of infection in order to eliminate infiltrated microorganisms. Through different approaches, the aim of this work was to find out in which way AHL-12 interacts with PMN. As a first step, an AHL-12 induced signaling-pathway should be elucidated in in vitro experiments using primary, human PMN. In a further approach using radioactive- (3H-AHL-12) and fluorescence-labeled (AHL-12-FITC) AHL-molecules, the interaction of AHL-12 with PMN should be examined, especially with regard to the expression of a specific receptor for AHL-12 on PMN. In this work it was confirmed that AHL-12 induced chemotaxis of human PMN. After stimulation of PMN with AHL-12, MAPK-p38 was phosphorylated and hence activated. Furthermore, it was shown that MAPK-p38 phosphorylated MAPKAP-Kinase 2, which in turn phosphorylated and hence activated leukocyte specific protein 1 (LSP1). LSP1 finally affected chemotaxis via binding to F-Actin and by modulating the intensity of integrin-mediated adherence. The AHL-12 induced chemotaxis was inhibited by SB203580, an inhibitor of MAPK-p38. The question, whether there is a specific receptor for AHL-12 in PMN, could not be answered conclusively, because it was not possible to isolate a protein with receptor function using conventional methods such as immunoprecipitation and crosslinking. In uptake studies with 3H-AHL-12 and AHL-12-FITC, it was shown, that the major portion of AHL-12-FITC bound to the cell membrane. However, another portion got into the cells, which raises the possibility that an intracellular receptor or an intracellular binding protein for AHL-12, for example IQGAP1, exists. Taken together these data provide evidence that AHL-12 is chemotactic for PMN. Assuming that AHL-12 is released in the early phase of biofilm formation, before bacteria are embedded in the EPS, PMN have the chance to kill bacteria and thus could contribute to prevent biofilm formation.
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.

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