@phdthesis{Kraus2020,
  author    = {Kraus, Michael},
  title     = {The Conversion of Bifidobacterium adolescentis Sucrose Phosphorylase into a Polyphenol Transglucosidase via Structure-based Enzyme Engineering},
  doi       = {10.25972/OPUS-19247},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192477},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The initial goal was the conversion of Bifidobacterium adolescentis Sucrose Phosphorylase (BaSP) into a polyphenol glucosidase by structure based enzyme engineering. BaSP was chosen because of its ability to utilize sucrose, an economically viable and sustainable donor substrate, and transfer the glucosyl moiety to various acceptor substrates. The introduction of aromatic residues into the active site was considered a viable way to render it more suitable for aromatic acceptor compounds by reducing its polarity and potentially introducing π-π-interactions with the polyphenols. An investigation of the active site revealed Gln345 as a suitable mutagenesis target. As a proof of concept BaSP Q345F was employed in the glycosylation of (+)-catechin, (-)-epicatechin and resveratrol. The variant was selective for the aromatic acceptor substrates and the glucose disaccharide side reaction was only observed after almost quantitative conversion of the aromatic substrates. A crystal structure of BaSP Q345F in complex with glucose was obtained and it displayed an unexpected shift of an entire domain by 3.3 {\AA}. A crystal structure of BaSP D192N-Q345F, an inactive variant in complex with resveratrol-3-α-D-glucosid, the glucosylation product of resveratrol, synthesized by BaSP Q345F was solved. It proved that the domain shift is in fact responsible for the ability of the variant to glycosylate aromatic compounds. Simultaneously a ligand free crystal structure of BaSP Q345F disproved an induced fit effect as the cause of the domain shift. The missing link, a crystal structure of BaSP Q345F in the F-conformation is obtained. This does not feature the domain shift, but is in outstanding agreement with the wildtype structure. The domain shift is therefore not static but rather a step in a dynamic process. It is further conceivable that the domain shifted conformation of BaSP Q345F resembles the open conformation of the wild type and that an adjustment of a conformational equilibrium as a result of the Q345F point mutation is observed. An investigation into the background reaction, the formation of glucose-glucose disaccharides of BaSP Q345F and three further variants that addressed the same region (L341C, D316C-L341C and D316C-N340C) revealed the formation of nigerose by BaSP Q345F.},
  subject      = {Phosphorylase},
  language  = {en}
}
@article{FrischholzBerberichBoecketal.2020,
  author    = {Frischholz, Sebastian and Berberich, Oliver and B{\"o}ck, Thomas and Meffert, Rainer H. and Blunk, Torsten},
  title     = {Resveratrol counteracts IL-1β-mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs},
  series = {Journal of Tissue Engineering and Regenerative Medicine},
  volume    = {14},
  journal   = {Journal of Tissue Engineering and Regenerative Medicine},
  number    = {7},
  doi       = {10.1002/term.3031},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-215471},
  pages     = {897 -- 908},
  year      = {2020},
  abstract  = {When aiming at cell-based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL-1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti-inflammatory properties. However, long-term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long-term model cultures for cell-based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL-1β (1-10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL-1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL-1β-treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL-1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL-1β-mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long-term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.},
  language  = {en}
}
@phdthesis{Frischholz2021,
  author    = {Frischholz, Sebastian},
  title     = {Resveratrol Counteracts IL-1β-mediated Impairment of Extracellular Matrix Deposition in 3D Articular Chondrocyte Constructs},
  doi       = {10.25972/OPUS-23745},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-237453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Articular cartilage is an exceptional connective tissue which by a network of fibrillar collagen and glycosaminoglycan (GAG) molecules allows both low- friction articulation and distribution of loads to the subchondral bone (Armiento et al., 2018, Ulrich-Vinther et al., 2003). Because of its very limited ability to self-repair, chondral defects following traumatic injury increase the risk for secondary osteoarthritis (OA) (Muthuri et al., 2011). Still, current OA treatments such as common nonsteroidal anti-inflammatory drugs (NSAIDs) and joint replacement primarily address end-stage symptoms (Tonge et al., 2014). As low-grade inflammation plays a pivotal role in the pathogenesis of OA (Robinson et al., 2016), there is a strong demand for novel therapeutic concepts, such as integrating application of anti-inflammatory agents into cartilage cell- based therapies in order to effectively treat OA affected joints in early disease stages. The polyphenolic phytoalexin resveratrol (RSV), found in the skin of red grapes, berries, and peanuts, has been shown to have effective anti-inflammatory properties (Shen et al., 2012). However, its long-term effects on 3D chondrocyte constructs cultured in an inflammatory environment with regard to tissue quality have remained unexplored so far. Therefore, in this study, pellets made from expanded porcine articular chondrocytes were cultured for 14 days with either the pro-inflammatory cytokine interleukin-1β (IL-1β) (1 - 10 ng/ml) or RSV (50 μM) alone, or a co-treatment with both agents. Constructs treated with chondrocyte medium only served as control. Treatment with IL-1β at 10 ng/ml resulted in a significantly smaller pellet size and reduced DNA content. However, RSV counteracted the IL-1β-induced decrease and significantly enhanced diameter and DNA content. Also, in terms of GAG deposition, treatment with IL-1β at 10 ng/ml resulted in a tremendous depletion of absolute GAG content and GAG/DNA. Again, RSV co-treatment counteracted the inflammatory stimulus and led to a partial recovery of GAG content. Histological analysis utilizing safranin-O staining confirmed these findings. Marked expression of the cartilage-degrading enzyme matrix metalloproteinase 13 (MMP13) was detected in IL-1β-treated pellets, but none upon RSV co- treatment. Moreover, co-treatment of IL-1β-challenged constructs with RSV significantly increased absolute collagen content. However, under non- inflammatory conditions, RSV induced gene expression and protein accumulation of collagen type X, a marker for undesirable hypertrophy. Taken together, in the present thesis, RSV was demonstrated to elicit marked beneficial effects on the extracellular matrix composition of 3D cartilaginous constructs in long-term inflammatory culture in vitro, but also induced hypertrophy under non-inflammatory conditions. Based on these findings, further experiments examining multiple concentrations of RSV under various inflammatory conditions appear desirable concerning potential therapeutic applicability in OA.},
  subject      = {Resveratrol},
  language  = {en}
}
@phdthesis{Glaser2012,
  author    = {Glaser, Nina},
  title     = {Influence of natural food compounds on DNA stability},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72872},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Cancer is one of the leading causes of death all over the world. Malnutrition and toxic contaminations of food with substances such as mycotoxins have been thought to account for a high percentage of cancers. However, human diet can deliver both mutagens and components that decrease the cancer risk. Genomic damage could be reduced by food components through different mechanisms such as scavenging of reactive oxygen species. In the first part of this study we tried to investigate the effects of patulin and resveratrol on DNA stability in V79 cells. Patulin is a mycotoxin, which is frequently found in spoiled apples and other fruits. The WHO has established a safety level of 50 µg/L, which is indeed not observed by all manufacturers. The acute toxicity of patulin in high concentrations is well known, however its potential carcinogenicity is still a matter of debate. Therefore we wanted to investigate further steps in the mechanism of patulin-induced genotoxicity. Patulin caused the formation of micronuclei and nucleoplasmic bridges in a dose-dependent manner. Further analysis revealed that patulin induced both kinetochore-negative and positive micronuclei. Time course of incubation indicate a new mechanism for patulin-induced nucleoplasmic bridge formation. We hypothized a mechanism via cross-linking of DNA, which was confirmed by a modified version of comet assay. Incubations of cells with patulin led to an increased number of multinucleated cells and multipolar mitoses. Cell cytometry revealed a G2 arrest by patulin, which might explain the amplification of centrosomes and patulin-induced aneuploidy. Patulin cause a dose-dependent DNA damage in comet assay which was influenced by the cellular GSH content. However, an induction of oxidative stress was just seen with higher concentrations of patulin. Levels of cellular glutathione were increased after 24 h incubation indicating an adaptive response to patulin-induced stress. There is growing interest in polyphenols such as resveratrol which have shown many positive effects on human health. The beneficial properties are partially attributed to their ability to scavenge reactive oxygen species. Co-incubation of V79 cells with patulin and 10 µM of the antioxidant resveratrol led to a slight reduction of micronucleus frequency compared to cells which were just treated with patulin. However, in higher concentrations resveratrol themselves caused the formation of micronuclei in V79 cells. Kinetochore analysis indicated only clastogenic properties for resveratrol but no disturbance of mitosis. The antioxidant properties of resveratrol were shown in ferric reducing antioxidant power (FRAP) assay. However, in cellular system resveratrol in higher concentrations revealed also prooxidative properties, as shown in 2,7-dichlordihydrofluorescein (DCF) assay. The increased level of glutathione after resveratrol treatment might reflect an adaptive response to resveratrol-induced oxidative stress. For the second part of this thesis we investigated the effects of an anthocyanin-rich grape extract on hypertensive Ren-2 rats. Ren-2 rats are an accepted genetically modified rat model for the investigation of hypertension and increased oxidative stress. We divided 23 female Ren-2 rats into three groups. One group was fed with an anthocyanin-rich Dacapo grape extract, one group was treated with the angiotensin converting enzyme (ACE) inhibitor ramipril and the third group was kept without medication during the experiment. After one week untreated group showed a clear increase in systolic and diastolic blood pressure compared to the ramipril treated rats. This was in part attenuated in the animals fed with anthocyanin-rich Dacapo grape extract. Effects on blood pressure were also reflected in an increased thirst of untreated and extract fed animals. Comet assay with cells of kidney and liver revealed a slight protective impact of Dacapo extract on DNA damage compared to the other groups. Similar results were obtained after evaluation of ɣ-H2AX-staining of kidney and heart sections. However, in the small intestine oppositional effects were seen, indicating an increased number of double strand breaks probably due to the high local concentration of polyphenols after oral ingestion. Antioxidative properties of the extract were shown in FRAP assay. However, this effect was not reflected in an increased antioxidative capacity in serum or a protective impact in the dihydroethidium (DHE) assay. The extract showed protective effects on DNA damage in comet assay and ɣ-H2AX-staining, but was not able to reduce hypertension back to the control level of ramipril treated animals. High local concentrations could also result in an increased damage of the affected tissue. Therefore, the administration of such concentrated compounds should be handled with care.},
  subject      = {Patulin},
  language  = {en}
}
@article{AwadOthmanStopper2017,
  author    = {Awad, Eman and Othman, Eman M. and Stopper, Helga},
  title     = {Effects of resveratrol, lovastatin and the mTOR-inhibitor RAD-001 on insulin-induced genomic damage in vitro},
  series = {Molecules},
  volume    = {22},
  journal   = {Molecules},
  number    = {12},
  doi       = {10.3390/molecules22122207},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159260},
  pages     = {2207},
  year      = {2017},
  abstract  = {Diabetes mellitus (DM) is one of the major current health problems due to lifestyle changes. Before diagnosis and in the early years of disease, insulin blood levels are elevated. However, insulin generates low levels of reactive oxygen species (ROS) which are integral to the regulation of a variety of intracellular signaling pathways, but excess levels of insulin may also lead to DNA oxidation and DNA damage. Three pharmaceutical compounds, resveratrol, lovastatin and the mTOR-inhibitor RAD-001, were investigated due to their known beneficial effects. They showed protective properties against genotoxic damage and significantly reduced ROS after in vitro treatment of cultured cells with insulin. Therefore, the selected pharmaceuticals may be attractive candidates to be considered for support of DM therapy.},
  language  = {en}
}