Steroid concentrations in atopic dermatitis patients: Reduced plasma DHEAS and increased cortisone levels.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease, which is characterized by a disrupted epidermal barrier function present both in affected skin and in non-affected skin. Mainly glucocorticosteroids were used in topical and systemic atopy treatments because of their potent anti-inflammatory effects, unfortunately with strong side effects. In this study we determined that 2 out of 16 steroids were significantly different in healthy volunteers vs AD-patients. Cortisone, which is higher in AD-patients plasma, is a direct precursor of the bioactive corticosteroid cortisol, which just displays a higher tendency and is known for its potent anti-inflammatory effects. In addition a tendency of reduced levels of the anti-inflammatory ER ligand estrone was found in AD-patients. DHEA is a precursor of testosterone, its levels just display a lower tendency in male AD-patients, while its sulfonation metabolite DHEAS is lower in male and female AD-patients. We found and conclude that altered steroid levels in the plasma of AD-patients indicate altered vitamin D signaling (based on reduced DHEA sulfonation) and increased feedback for anti-inflammatory signaling (increased levels of cortisone) present in AD-patients. This article is protected by copyright. All rights reserved

Distinct roles in autophagy and importance in infectivity of the two ATG4 cysteine peptidases of leishmania major

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite

Reduced Basal Autophagy and Impaired Mitochondrial Dynamics Due to Loss of Parkinson's Disease-Associated Protein DJ-1

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease

Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

DJ-1 Null Dopaminergic Neuronal Cells Exhibit Defects in Mitochondrial Function and Structure: Involvement of Mitochondrial Complex I Assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O2 consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease

Analysis of relevant signalling pathways of radiation induced COX-2 Expression in tumorcells

Zusammenfassung: Krebserkrankungen sind weltweit ein Problem und die Zahl der Neuerkrankungen steigt stetig. Chirurgische Eingriffe, Strahlentherapie und Chemotherapie sind die maßgeblichen Behandlungsformen bei Tumorerkrankungen. Neue Entwicklungen in der Strahlentherapie machen Bestrahlung alleine oder in Kombination mit Chemotherapie zu einer wichtigen Behandlungsmethode, die mehr und mehr an Bedeutung gewinnt. Der Erfolg einer Behandlung ist oftmals Abhängig vom genetischen Profil eines Tumors. Damit wird die Untersuchung der molekularen Abläufe in Zellen nach Bestrahlung zu einem wichtigen Aspekt der Strahlentherapie um effektive Strategien zur Tumorbehandlung zu entwickeln. COX-2 ist in vielen Tumoren überexprimiert und korreliert mit einem schlechten Verlauf der Krankheit. Zudem kann COX-2 durch ionisierende Strahlung induziert werden. Das macht COX-2 zu einem interessanten molekularen ?Target? für die Strahlentherapie und die Tumortherapie im Allgemeinen. Studien mit spezifischen COX-2 Inhibitoren haben in verschiedenen Zelllinien zu unterschiedlichen Ergebnissen geführt. Die Untersuchungen in der hier vorliegenden Arbeit bezogen sich hauptsächlich auf das Überleben und das Wachstum von Prostatakarzinomzelllinien nach Bestrahlung alleine und in Kombination mit dem spezifischen COX-2 Inhibitor Celecoxib sowie die Analyse von Signalwegen über welche die strahleninduzierte COX-2 Expression verläuft. Dies sind die wichtigsten Resultate: 1. Die Behandlung mit Celecoxib hat keinen Einfluss auf die Strahlensensitivität der untersuchten Prostatakarzinomzelllinien. 2. Die Proliferation der untersuchten Zelllinien wurde durch die Behandlung mit Celecoxib gehemmt. 3. Die Inhibition der Proliferation durch Celecoxib scheint unabhängig vom COX-2 Niveau. 4. Apoptose kann in den untersuchten Zelllinien durch Behandlung mit Celecoxib in klinisch relevanten Dosen nicht induziert werden. 5. Die Induktion der COX-2 Expression durch ionisierende Strahlung ist abhängig von der Zelllinie. 6. Bei der Induktion der COX-2 Expression spielen die Signalwege der MAPK-Signalwegfamilie eine maßgebliche Rolle. Die erzielten Ergebnisse führen zu dem Schluss, dass COX-2 ein wichtiges molekulares ?Target? in der Strahlentherapie sein kann. Dies ist jedoch abhängig von der betrachteten Zelllinie. Ebenso sind die Signalwege die zu einer strahleninduzierten COX-2 Expression führen für jede Zelllinien individuell verschieden. So kann auch die Anwendung von Celecoxib in der Strahlentherapie für Behandlung bestimmter Tumore positiv sein.Summary: Cancer is a health problem worldwide and the number of new cases is rising. Surgery, radiotherapy and chemotherapy are the major treatment modalities. New developments in radiotherapy make radiation alone and in combination with chemotherapy to an important therapy becoming more and more mattering. The success of a therapy often depends on the genetic profile of a tumor. This makes analysis of molecular processes in cells after radiation an important aspect in radiotherapy developing an effective strategy for tumor treatment. COX-2 is overexpressed in a lot of tumors and correlates with a poor prognosis. Moreover COX-2 can be induced by ionizing radiation. This makes COX-2 an interesting molecular target in radiation therapy and in cancer therapy in general. Studies with specific COX-2 inhibitors came to different results in different cell lines. The aim of the presented study was to investigate the survival and the proliferation of prostate cancer cells after treatment with ionizing radiation alone and in combination with specific COX-2 inhibitor Celecoxib and the analysis of signaling pathways leading to radiation induced COX-2 expression. The following major results were obtained: 1. Treatment with Celecoxib had no influence on the radiosensitivity of the prostate cancer cell lines investigated. 2. The proliferation of different cell lines was inhibited by the treatment with Celecoxib. 3. The inhibition of the proliferation seems to be independent of the level of COX-2 of the cell lines. 4. Apoptosis can not be induced by Celecoxib in clinical relevant doses in the cell lines investigated. 5. Induction of COX-2 expression by ionizing radiation depends on the cell line investigated. 6. The MAPK-signaling pathways play a major role at COX-2 expression. In conclusion the results of the presented study indicate that COX-2 can be an important molecular target in radiation therapy. Although this depends on the cell line investigated. As well, the signaling pathways leading to a radiation induced expression of COX-2 are individual for each cell line. Thus the application of Celecoxib during radiation therapy can be positive on the treatment of different tumors