Based on the knowledge that many diseases are caused by defects in the metabolism of the cells and, in particular, in defects of the mitochondria, mitochondrial medicine starts precisely at this ...point. This new form of therapy is used in numerous fields of human medicine and has become a central focus within the field of medicine in recent years. With this form of therapy, the disturbed cellular energy metabolism and an out-of-balance antioxidant system of the patient are to be influenced to a greater extent. The most important tool here is mitotropic substances, with the help of which attempts are made to compensate for existing dysfunction. In this article, both mitotropic substances and accompanying studies showing their efficacy are summarized. It appears that the action of many mitotropic substances is based on two important properties. First, on the property of acting antioxidantly, both directly as antioxidants and via activation of downstream enzymes and signaling pathways of the antioxidant system, and second, via enhanced transport of electrons and protons in the mitochondrial respiratory chain.
Understanding the influence of secondary metabolites from fungi on the mitochondria of the host plant during infection is of great importance for the knowledge of fungus-plant interactions in ...general; it could help generate resistant plants in the future and in the development of specifically acting plant protection products. For this purpose, it must first be possible to record the mitochondrial parameters in the host plant. As of the date of this protocol, no measurements of mitochondrial respiration parameters have been performed in wheat paleae. The protocol shown here describes the measurements using the XF24 analyzer, which measures the rate of oxygen consumption in the sample by changes in the fluorescence of solid-state fluorophores. This procedure covers the preparation of samples for the XF24 analyzer and the measurement of mitochondrial parameters by adding specific mitochondrial inhibitors. It also shows the necessary approach and steps to be followed to obtain reliable, reproducible results. This is a robust protocol that allows the analysis of mitochondrial respiration directly in the wheat paleae. It demonstrates an important add-on method to existing screenings and also offers the possibility to test the effects of early infection of plants by harmful fungi (e.g., Fusarium graminearum) on mitochondrial respiration parameters. Key features This protocol offers the possibility of testing the effects of early infection of plants by pathogens on mitochondrial respiration parameters. This protocol requires a Seahorse XF24 Flux Analyzer with Islet Capture Microplates and the Seahorse Capture Screen Insert Tool. Graphical overview.
The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein ...does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses.
The process of aging is characterized by the increase of age-associated disorders as well as severe diseases. Due to their role in the oxidative phosphorylation and thus the production of ATP which ...is crucial for many cellular processes, one reason for this could be found in the mitochondria. The accumulation of reactive oxygen species damaged mitochondrial DNA and proteins can induce mitochondrial dysfunction within the electron transport chain. According to the “mitochondrial theory of aging,” understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on aging in general, but the measurement of mitochondrial respiration in skin cells from cell cultures cannot completely reflect the real situation in skin. Here, we describe a new method to measure the mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using a XF24 extracellular flux analyzer to evaluate the effect of coenzyme Q10. We observed a decrease in mitochondrial respiration and ATP production with donor age corresponding to the “mitochondrial theory of aging.” For the first time ex vivo in human epidermis, we could show also a regeneration of mitochondrial respiratory parameters if the reduced form of coenzyme Q10, ubiquinol, was administered. In conclusion, an age-related decrease in mitochondrial respiration and ATP production was confirmed. Likewise, an increase in the respiratory parameters by the addition of coenzyme Q10 could also be shown. The fact that there is a significant effect of administered coenzyme Q10 on the respiratory parameters leads to the assumption that this is mainly caused by an increase in the electron transport chain. This method offers the possibility of testing age-dependent effects of various substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.
Human skin is exposed to a number of harmful agents of which the ultraviolet (UV) component of solar radiation is most important. UV‐induced damages include direct DNA lesions as well as oxidative ...damage in DNA, proteins and lipids caused by reactive oxygen species (ROS). Being the main site of ROS generation in the cell, mitochondria are particularly affected by photostress. The resulting mitochondrial dysfunction may have negative effects on many essential cellular processes. To counteract these effects, coenzyme Q10 (CoQ10) is used as a potent therapeutic in a number of diseases. We analyzed the mitochondrial respiration profile, the mitochondrial membrane potential and cellular ATP level in skin fibroblasts after irradiation. We observed an accelerated regeneration of cellular ATP level, a decrease in mitochondrial dysfunction as well as a preservation of the mitochondrial membrane potential after irradiation in human skin fibroblasts by treatment with CoQ10. We conclude that the faster regeneration of the ATP level was achieved by a preservation of mitochondrial function by the addition of CoQ10 and that the protective effect of CoQ10 is primarily mediated via its antioxidative function. We suggest also that it might be further dependent on a stimulation of DNA repair enzymes by CoQ10.
Being the main site of ROS generation in the cell, mitochondria are particularly affected by photostress. The resulting mitochondrial dysfunction may have negative effects on many essential cellular processes. To counteract these effects, CoQ10 is used as a therapeutic in a number of diseases. We observed that CoQ10 is able to preserve the mitochondrial membrane potential after SSL‐UVA irradiation (d, e), decrease the level of mitochondrial dysfunction (a, b) and lead therefore to a more rapid regeneration of the cellular ATP levels in human fibroblasts (c). We conclude that the protective effect of CoQ10 is primarily mediated via its antioxidative function.
7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the ...mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead to the discovery of new targets and thus be of great importance for the development of preventive therapies. Because of this, we developed a real-time base excision repair assay with a specially designed double-stranded reporter oligonucleotides to measure the activity of hOGG1 in lysates of isolated mitochondria. This system presented here differs from the classical assays, in which an endpoint determination is performed via a denaturing acrylamide gel, by the possibility to measure the hOGG1 activity in real-time. In addition, to determine the activity of each enzymatic step (N-glycosylase and AP-lyase activity) of this bifunctional enzyme, a melting curve analysis can also be performed. After isolation of mitochondria from human fibroblasts using various centrifugation steps, they are lysed and then incubated with specially designed reporter oligonucleotides. The subsequent measurement of hOGG1 activity is performed in a conventional real-time PCR system.
Skin cells are constantly exposed to environmental influences such as air pollution, chemicals, pathogens and UV radiation. UV radiation can damage different biological structures, but most ...importantly cellular DNA. Mitochondria contain their own genome and accumulate UV-induced DNA mutations to a large extent. This can result,
, in accelerated skin aging. Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on the development of age-related diseases. Previous studies have been carried out on cell cultures derived from primary cells, which does not fully represent the real situation in the skin, while the mitochondrial parameters were considered barely or not at all. Here we describe a method to measure mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using an Agilent Seahorse XF24 Flux Analyzer. Before the assay, epidermis and dermis are separated enzymatically, we then used the XF24 Islet capture microplates to position the epidermis samples to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). In these plates, small nets can be fixed to the plate bottom. The epidermis was placed with the vital-basal-side on the net. Active ingredients in the three ports were injected consecutively to determine the effect of each compound. This allows determining the efficiency of the individual complexes within the respiratory chain. This protocol enables the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.
Zusammenfassung
Mitochondrien, die Kraftwerke der Zelle – diese geläufige Bezeichnung entstammt ihrer bekanntesten Funktion, nämlich der Produktion von Adenosintriphosphat (ATP), dem universellen ...Energieträger für alle Zellen. Dies spiegelt jedoch nicht mal annähernd das wider, was Mitochondrien imstande sind zu leisten. Neben der täglichen Produktion an Adenosintriphosphat im Kilogrammmassstab spielen sie eine zentrale Rolle beim programmierten Zelltod, der Anpassung des zellulären Redoxpotenzials sowie der Synthese von Eisen-Schwefel-Clustern (Fe-S-Cluster), welche für das Überleben einer Zelle von grosser Bedeutung sind. Dies alles geschieht bei elektrischen Spannungen, die einem buchstäblich die Haare zu Berge stehen lassen müssten, ohne dass wir davon etwas mitbekommen. Mitochondrien – so winzig, so unscheinbar und dennoch voller Wunder. Daher verwundert es nicht, dass Funktionsstörungen dieser Organellen weitreichende Folgen für die Gesundheit haben können. Die mitochondriale Medizin, welche sich in den letzten Jahren zu einem zentralen Schwerpunkt innerhalb der gesamten Medizin entwickelt hat, versucht, diese Folgen zu verhindern, abzumildern und zu beseitigen. Um dies zu bewerkstelligen, bedarf es einer modernen und innovativen Diagnostik, welche sowohl zu Beginn als auch während einer Therapie Anwendung finden sollte.
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