Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing

AbstractTwo phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzymic activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358–386, 862–882) are detectable. Furthermore, a helix loop helix motif (amino acids 716–729) as well as two nuclear localization signals (amino acids 838–854, 345–349) indicate the presence of the type 3 isoform in the nucleus

Factors influencing participation in a randomized controlled resistance exercise intervention study in breast cancer patients during radiotherapy

Background: Over the past years knowledge about benefits of physical activity after cancer is evolving from randomized exercise intervention trials. However, it has been argued that results may be biased by selective participation. Therefore, we investigated factors influencing participation in a randomized exercise intervention trial for breast cancer patients. Methods: Non-metastatic breast cancer patients were systematically screened for a randomized exercise intervention trial on cancer-related fatigue. Participants and nonparticipants were compared concerning sociodemographic characteristics (age, marital status, living status, travel time to the training facility), clinical data (body-mass-index, tumor stage, tumor size and lymph node status, comorbidities, chemotherapy), fatigue, and physical activity. Reasons for participation or declination were recorded. Results 117 patients (52 participants, 65 nonparticipants) were evaluable for analysis. Multiple regression analyses revealed significantly higher odds to decline participation among patients with longer travel time (p = 0.0012), living alone (p = 0.039), with more comorbidities (0.031), previous chemotherapy (p = 0.0066), of age ≥ 70 years (p = 0.025), or being free of fatigue (p = 0.0007). No associations were found with BMI or physical activity. By far the most frequently reported reason for declination of participation was too long commuting time to the training facility. Conclusions: Willingness of breast cancer patients to participate in a randomized exercise intervention study differed by sociodemographic factors and health status. Neither current physical activity level nor BMI appeared to be selective for participation. Reduction of personal inconveniences and time effort, e.g. by decentralized training facilities or flexible training schedules, seem most promising for enhancing participation in exercise intervention trials. Trial registration: Registered at ClinicalTrials.gov: NCT01468766 (October 2011)

Verification of German methane emission inventories and their recent changes based on atmospheric observations

Continuous methane concentration records and stable isotope observations measured in the suburbs of Heidelberg, Germany, are presented. While delta13C-CH4 shows a significant trend of -0.14 permil per year, towards more depleted values, no trend is observed in the concentration data. Comparison of the Heidelberg records with clean air observations in the North Atlantic at Izana station (Tenerife) allows the determination of the continental methane excess at Heidelberg, decreasing by 20% from 190 ppb in 1992 to 150 ppb in 1997. The isotope ratio which is associated with this continental methane pile-up in the Heidelberg catchment area shows a significant trend to more depleted values from delta13C (source) = -47.4 ± 1.2 permil in 1992 to 52.9 ± 0.4 permil in 1995/96, pointing to a significant change in the methane source mix. Total methane emissions in the Heidelberg catchment area are estimated using the 222Radon (222Rn) tracer method: from the correlations of half hourly 222Rn and CH4 mixing ratios from 1995 to 1997, and the mean 222Rn exhalation rate from typical soils in the Rhine valley, a mean methane flux of 0.24 ± 0.5 g CH4 km-2 s-1 is derived. For the Heidelberg catchment area with an estimated radius of approximately 150 km, Core Inventories Air 1990 (CORINAIR90) emission estimates yield a flux of 0.47 g CH4 km-2 s-1, which is about 40% higher than the 222Rn derived number if extrapolated to 1990. The discrepancy can be explained by over-estimated emissions from waste management in the CORINAIR90 statistical assessment. The observed decrease in total emissions can be accounted for by decreasing contributions from fossil sources (mainly coal mining) and from cattle breeding. This finding is also supported by the observed decrease in mean source isotopic signatures

Second M-3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium

Background and Purpose The bronchodilator tiotropium binds not only to its main binding site on the M-3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting beta-adrenoceptor agonists, as combination therapy with anticholinergic agents and beta-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. Experimental Approach ACh, tiotropium, and atropine binding to M-3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. Key Results Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M-3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the beta-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct beta-adrenoceptor-M-3 receptor crosstalk in this effect. Conclusions and Implications We propose that tiotropium has two binding sites at the M-3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with beta-adrenoceptor agonists

Diesel exhaust particles distort lung epithelial progenitors and their fibroblast niche

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by inflammation and impaired tissue regeneration, and is reported as the fourth leading cause of death worldwide by the Centers for Disease Control and Prevention (CDC). Environmental pollution and specifically motor vehicle emissions are known to play a role in the pathogenesis of COPD, but little is still known about the molecular mechanisms that are altered following diesel exhaust particles (DEP) exposure. Here we used lung organoids derived from co-culture of alveolar epithelial progenitors and fibroblasts to investigate the effect of DEP on the epithelial-mesenchymal signaling niche in the distal lung, which is essential for tissue repair. We found that DEP treatment impaired the number as well as the average diameter of both airway and alveolar type of lung organoids. Bulk RNA-sequencing of re-sorted epithelial cells and fibroblasts following organoid co-culture shows that the Nrf2 pathway, which regulates antioxidants' activity, was upregulated in both cell populations in response to DEP; and WNT/β-catenin signaling, which is essential to promote epithelial repair, was downregulated in DEP-exposed epithelial cells. We show that pharmacological treatment with anti-oxidant agents such as N-acetyl cysteine (NAC) or Mitoquinone mesylate (MitoQ) reversed the effect of DEP on organoids growth. Additionally, a WNT/β-catenin activator (CHIR99021) successfully restored WNT signaling and promoted organoid growth upon DEP exposure. We propose that targeting oxidative stress and specific signaling pathways affected by DEP in the distal lung may represent a strategy to restore tissue repair in COPD

SK channel activation potentiates auranofin-induced cell death in glio- and neuroblastoma cells

Brain tumours are among the deadliest tumours being highly resistant to currently available therapies. The proliferative behaviour of gliomas is strongly influenced by ion channel activity. Small-conductance calcium-activated potassium (SK/KCa) channels are a family of ion channels that are associated with cell proliferation and cell survival. A combined treatment of classical anti-cancer agents and pharmacological SK channel modulators has not been addressed yet. We used the gold-derivative auranofin to induce cancer cell death by targeting thioredoxin reductases in combination with CyPPA to activate SK channels in neuro- and glioblastoma cells. Combined treatment with auranofin and CyPPA induced massive mitochondrial damage and potentiated auranofin-induced toxicity in neuroblastoma cells in vitro. In particular, mitochondrial integrity, respiration and associated energy generation were impaired. These findings were recapitulated in patient-derived glioblastoma neurospheres yet not observed in non-cancerous HT22 cells. Taken together, integrating auranofin and SK channel openers to affect mitochondrial health was identified as a promising strategy to increase the effectiveness of anti-cancer agents and potentially overcome resistance

Assembling proteomics data as a prerequisite for the analysis of large scale experiments

<p>Abstract</p> <p>Background</p> <p>Despite the complete determination of the genome sequence of a huge number of bacteria, their proteomes remain relatively poorly defined. Beside new methods to increase the number of identified proteins new database applications are necessary to store and present results of large- scale proteomics experiments.</p> <p>Results</p> <p>In the present study, a database concept has been developed to address these issues and to offer complete information via a web interface. In our concept, the Oracle based data repository system SQL-LIMS plays the central role in the proteomics workflow and was applied to the proteomes of <it>Mycobacterium tuberculosis</it>, <it>Helicobacter pylori</it>, <it>Salmonella typhimurium </it>and protein complexes such as 20S proteasome. Technical operations of our proteomics labs were used as the standard for SQL-LIMS template creation. By means of a Java based data parser, post-processed data of different approaches, such as LC/ESI-MS, MALDI-MS and 2-D gel electrophoresis (2-DE), were stored in SQL-LIMS. A minimum set of the proteomics data were transferred in our public 2D-PAGE database using a Java based interface (Data Transfer Tool) with the requirements of the PEDRo standardization. Furthermore, the stored proteomics data were extractable out of SQL-LIMS via XML.</p> <p>Conclusion</p> <p>The Oracle based data repository system SQL-LIMS played the central role in the proteomics workflow concept. Technical operations of our proteomics labs were used as standards for SQL-LIMS templates. Using a Java based parser, post-processed data of different approaches such as LC/ESI-MS, MALDI-MS and 1-DE and 2-DE were stored in SQL-LIMS. Thus, unique data formats of different instruments were unified and stored in SQL-LIMS tables. Moreover, a unique submission identifier allowed fast access to all experimental data. This was the main advantage compared to multi software solutions, especially if personnel fluctuations are high. Moreover, large scale and high-throughput experiments must be managed in a comprehensive repository system such as SQL-LIMS, to query results in a systematic manner. On the other hand, these database systems are expensive and require at least one full time administrator and specialized lab manager. Moreover, the high technical dynamics in proteomics may cause problems to adjust new data formats. To summarize, SQL-LIMS met the requirements of proteomics data handling especially in skilled processes such as gel-electrophoresis or mass spectrometry and fulfilled the PSI standardization criteria. The data transfer into a public domain via DTT facilitated validation of proteomics data. Additionally, evaluation of mass spectra by post-processing using MS-Screener improved the reliability of mass analysis and prevented storage of data junk.</p

A meta‐analysis of change in applicants' perceptions of fairness

Using an event‐triggered multi‐stage framework, this random‐effects meta‐analysis examined the changes in applicants' perceptions of fairness between consecutive stages and throughout the entire personnel selection process. We integrated findings of studies with at least two measurement points, resulting in 45 effect sizes (overall N = 3,038). Trajectories of perceptions of fairness decreased nonlinearly across the process, with a steeper decrease for people who held high levels of initial fairness expectations. Unjust treatment produced a decrease in perceptions of fairness from pretest to posttest and an increase from posttest to postdecision. Furthermore, the length of the time interval moderated the changes in fairness perceptions between the posttest and postdecision stage. Practical implications and an agenda for future research are discussed

SK channel-mediated metabolic escape to glycolysis inhibits ferroptosis and supports stress resistance in C. elegans

Metabolic flexibility is an essential characteristic of eukaryotic cells in order to adapt to physiological and environmental changes. Especially in mammalian cells, the metabolic switch from mitochondrial respiration to aerobic glycolysis provides flexibility to sustain cellular energy in pathophysiological conditions. For example, attenuation of mitochondrial respiration and/or metabolic shifts to glycolysis result in a metabolic rewiring that provide beneficial effects in neurodegenerative processes. Ferroptosis, a non-apoptotic form of cell death triggered by an impaired redox balance is gaining attention in the field of neurodegeneration. We showed recently that activation of small-conductance calcium-activated K+ (SK) channels modulated mitochondrial respiration and protected neuronal cells from oxidative death. Here, we investigated whether SK channel activation with CyPPA induces a glycolytic shift thereby increasing resilience of neuronal cells against ferroptosis, induced by erastin in vitro and in the nematode C. elegans exposed to mitochondrial poisons in vivo. High-resolution respirometry and extracellular flux analysis revealed that CyPPA, a positive modulator of SK channels, slightly reduced mitochondrial complex I activity, while increasing glycolysis and lactate production. Concomitantly, CyPPA rescued the neuronal cells from ferroptosis, while scavenging mitochondrial ROS and inhibiting glycolysis reduced its protection. Furthermore, SK channel activation increased survival of C. elegans challenged with mitochondrial toxins. Our findings shed light on metabolic mechanisms promoted through SK channel activation through mitohormesis, which enhances neuronal resilience against ferroptosis in vitro and promotes longevity in vivo