Alexander Gosslau

Mitochondrial DNA deletions and the aging heart.

Mitochondrial DNA (mtDNA) mutations appear to be associated with a wide spectrum of human disorders and proposed to be a potential contributor of aging. However, in an age-dependent increase of the common 4977 bp deletion of human mtDNA still many unanswered questions remain. Comparing mtDNA copy levels in different tissues revealed that cardiac muscle had the highest, while the cortex cerebelli showed the lowest copy number of mtDNA in every donor. Intriguingly, mtDNA copy number showed no changes during aging. In heart tissue, the amount of 4977 bp mtDNA deletion increased in an age-dependent manner showing significant differences at the age of 40 years and older (p<0.005). In vitro studies analyzing human normal cells transfected with telomerase (BJ-T) revealed that oxidative stress (OS)--a well accepted promoter of aging--induced 4977 bp deletion and point mutations as demonstrated by real-time PCR and DHPLC analysis. Interestingly, OS induced apoptosis only in transformed human fibroblasts by activation of the intrinsic (mitochondrial-mediated) signalling pathway as indicated by morphological damage of mitochondria, DNA laddering and increase of the Bax/Bcl-2 ratio. In conclusion, in heart tissue, the amount of the 4977 bp deletion increased in an age-dependent manner and it was more detectable after the 4th decade of life, although there was some scatter in the data. Since, apoptosis was induced by the mitochondria-mediated pathway only in transformed cells, the role for apoptosis in normal tissue of the aging heart remains unclear.

The importance of natural product characterization in studies of their anti‐inflammatory activity

The knowledge that natural products provide a rich source for therapeutic discovery has led to the development of many of the worlds most commonly used drugs. In view of the growing need for effective anti-inflammatory agents, the potential for natural products to serve as safe and effective therapeutic agents has gained increasing attention. However, polymolecular extracts must be rigorously evaluated and chemically characterized to insure adequate consistency in performance. The research in this field has been plagued by inconsistencies due in part to inadequate chemical characterization and documentation, making comparison of results across studies very difficult. Analytical chemistry and molecular methods now exist to insure sufficient transparency to avoid this limitation. Further, our understanding of the complexity of inflammation has advanced to enable significant insight into the mechanism of action of these natural extracts. Here, we review the inflammatory pathways targeted by many therapeutic agents, discuss the value of natural products as anti-inflammatory agents, review approaches for their biological and chemical evaluation, and highlight challenges to the field. We present two examples highlighting the rigorous use of cell, molecular, and chemical methods for characterization and quality control as templates for future studies of anti-inflammatory activity of natural products.

Effects of the black tea polyphenol theaflavin-2 on apoptotic and inflammatory pathways in vitro and in vivo

SCOPE Theaflavin-2 (TF-2), a major component of black tea extract, induces apoptosis of human colon cancer cells and suppresses serum-induced cyclooxygenase-2 (COX-2) expression 1. Here, we explored the mechanisms for activation of apoptosis, evaluated the impact on inflammatory genes in a broader panel of cells and tested whether topical anti-inflammatory effects could be observed in vivo. METHODS AND RESULTS TF-2 triggered apoptosis in five other transformed cancer cell lines, inducing cell shrinkage, membrane blebbing, and mitochondrial clustering within 3 h of treatment. Among a set of pro-apoptotic genes, TF-2 quickly induced the up-regulation of P53 and BAX, suggesting mitochondria as the primary target. Using a cell model for inflammatory response, we showed that TF-2 suppressed the 12-O-tetradecanoylphorbol-13-acetate-induced COX-2 gene expression, and also down-regulated TNF-α, iNOS, ICAM-1, and NFκB. A reporter gene assay showed that TF-2 down-regulated COX-2 at the transcriptional level. We also demonstrated that TF-2 exhibited anti-inflammatory activity in two mouse models of inflammation. Topical application with TF-2 significantly reduced ear edema and produced a pattern of gene down-regulation similar to that observed in the cell model. CONCLUSION These results suggest that the anti-inflammatory and pro-apoptotic activity of TF-2 may be exploited therapeutically in cancer and other diseases associated with inflammation.

Oxidativer Stress, altersabhängige Zellschädigungen und antioxidative Mechanismen

Zusammenfassung Zellulärer oxidativer Stress entsteht durch die Produktion von reaktiven oxidativen Spezies (ROS) einerseits und einer unzureichenden antioxidativen Abwehr andererseits. Davon sind besonders stoffwechselaktive Zellen wie Neurone oder Muskelzellen aber im Prinzip auch alle anderen Zelltypen betroffen. Eine zentrale ROS-Verbindung ist das Wasserstoffperoxid, das vor allem in den Mitochondrien produziert wird. In Verbindung mit reduzierten Schwermetallen (z.B. Eisen und Kupfer) entsteht daraus das sehr reaktive Hydroxylradikal, das alle zellulären Makromoleküle schädigt. Infolge von Oxidationsprozessen an den Nucleinsäuren kann es zu Mutationen kommen, während Proteinschäden zu Enzymdefekten und Veränderungen des Cytoskeletts führen. Die Lipidperoxidation verursacht über Schädigungen der Zellmembranen außerdem eine Störung des Ionengleichgewichts. Diese Zellschädigungen können schließlich zum Zelltod führen, wodurch der oxidative Stress verschiedene Zelldefekt-Erkrankungen verursacht. Die Mitochondrien spielen dabei eine zentrale Rolle, da sie sowohl die Hauptquelle des endogenen oxidativen Stresses darstellen als auch als Signalgeber des programmierten Zelltodes (Apoptose) fungieren. Die antioxidative Abwehr wirkt auf verschiedenen Ebenen: Während die Inaktivierung von Schwermetallen durch Chelatbildner, wie Ferritin, erfolgt, werden ROS-Verbindungen enzymatisch vor allem durch die Glutathion-Peroxidase oder nicht-enzymatisch durch Antioxidantien reduziert, wie z.B.Vitamin E, Vitamin C und Glutathion. Stressproteine spielen eine wichtige Rolle bei der Reparatur, dem Abbau denaturierter Proteine sowie bei der Hemmung der Apoptose.Summary Cellular oxidative stress is due to the production of reactive oxygen species (ROS), on the one hand, and weaknesses of the antioxidative defence, on the other. This is particularly true for cells with an active metabolism such as neurons and muscle cells, but it is also relevant for all other cell types. Hydrogen peroxide is an important member of ROS and is generated predominantly by mitochondria. In combination with reduced trace metals such as iron or copper, hydrogen peroxide is transformed into the highly reactive hydroxyl radical which causes damage to virtually all macromolecules. Oxidation of nucleic acids results in mutations while protein denaturation leads to enzyme defects and impairment of the cytoskeleton. Lipid peroxidation in cell membranes is strongly involved in the perturbation of ion homeostasis. Because this cell damage ultimatively causes cell death, oxidative stress initiates several diseases. Mitochondria play a major role in this context because they are the main source of endogenous oxidative stress and additionally function as an inducer of programmed cell death (apoptosis). Several strategies of antioxidative defence exist: While transition metals can be inactivated by chelating proteins (e.g., ferritin), ROS can be reduced enzymatically (e.g., by the glutathione peroxidase) or non-enzymatically by antioxidants (e.g., by vitamin E, vitamin C and glutathione). Stress proteins are implicated in the repair and transport of denatured proteins as well as in the inhibition of apoptosis.

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts.

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.

Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A

Heat‐induced cell death appears to be a cell‐specific event. Chronic heat stress was lethal to human colon cancer cells (Caco‐2, HT29, and HCT116), but not to normal diploid fibroblasts and other cancer cells (BJ‐T, WI38, HeLa, ovarian 2008, WI38VA). Acute heat stress (45–51°C, 30 min) caused cell death of colon cancer cells during recovery at physiological temperature. Thermal killing of Caco‐2 cells was not mediated via oxidative stress since Caco‐2 cells were much more resistant than HeLa and other cancer cells to H2O2‐induced cell death. Acute heat stress caused a striking loss of eukaryotic initiation factor 5A (eIF5A) in colon cancer cells, but not in HeLa and other normal or transformed human fibroblasts. The heat‐induced loss of eIF5A is likely to be due to changes in the protein stability. The half‐life of eIF5A was changed from >20 h to less than 30 min during the acute heat stress. Sequence analysis of the eIF5A gene from Caco‐2 and HeLa cells did not reveal any difference, suggesting that the change in stability in Caco‐2 cells was not due to any eIF5A mutation. Pretreatment of cells with protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) partially blocked the heat‐induced loss of eIF5A and prevented heat‐induced cell death. In light of the essential role of eIF5A in cell survival and proliferation, our results suggest that the stability of eIF5A may have an important role in determining the fate of the particular cell type after severe heat stress. J. Cell. Physiol. 219: 485–493, 2009.

Induction of Hsp68 by oxidative stress involves the lipoxygenase pathway in C6 rat glioma cells

The induction of Hsp68 by heat shock (HS) and oxidative stress (OS) involves different pathways in C6 rat glioma cells. The pathways were analyzed by specific inhibitors of signal transduction cascades. Quercetin (inhibitor of PLA(2) and lipoxygenase) inhibited only the OS-induced but not the HS-induced expression of Hsp68. Preincubation with quinacrine (inhibitor of PLA(2)) before stress also suppressed the expression of Hsp68 only after oxidative stress. Moreover, another inhibitor of lipoxygenase (alpha-tocopherol) exclusively suppressed OS-induced Hsp68 expression. This different regulation was confirmed by exposing the cells to arachidonic acid (AA) during stress which strongly increased the induction of Hsp68 only after OS. PGE(2) (metabolite of cyclooxygenase) and indomethacin (inhibitor of cyclooxygenase) had no influence on Hsp68 expression in response to both stressors. The results suggest that the induction of Hsp68 by oxidative stress is mainly transmitted by the lipoxygenase pathway in C6 rat glioma cells.