Effects of hyperoxia and hyperoxic oscillations on the proteome of murine lung microvascular endothelium

Patients presenting with insufficient tissue oxygenation and impaired lung function as in acute respiratory distress syndrome (ARDS) frequently require mechanical ventilation with supplemental oxygen. Despite the lung being used to experiencing the highest partial pressure of oxygen during healthy breathing, the organ is susceptible to oxygen-induced injury at supraphysiological concentrations. Hyperoxia-induced lung injury (HALI) has been regarded as a second hit to pre-existing lung injury and ventilator-induced lung injury (VILI) attributed to oxidative stress. The injured lung has a tendency to form atelectasis, a cyclic collapse and reopening of alveoli. The affected lung areas experience oxygen conditions that oscillate between hyperoxia and hypoxia rather than remaining in a constant hyperoxic state. Mechanisms of HALI have been investigated in many animal models previously. These studies provided insights into the effects of hyperoxia on the whole organism. However, cell type-specific responses have not been dissected in detail, but are necessary for a complete mechanistic understanding of ongoing pathological processes. In our study, we investigated the effects of constant and intermittent hyperoxia on the lung endothelium from a mouse by an in vitro proteomic approach. We demonstrate that these oxygen conditions have characteristic effects on the pulmonary endothelial proteome that underlie the physiological (patho)mechanisms

Precursors of Cytochrome Oxidase in Cytochrome-Oxidase-Deficient Cells of Neurospora crassa

Three different cell types of Neurospora crassa deficient in cytochrome oxidase were studied: the nuclear mutant cni-1, the cytoplasmic mutant mi-1 and copper-depleted wild-type cells. * 1. The enzyme-deficient cells have retained a functioning mitochondrial protein synthesis. It accounted for 12–16% of the total protein synthesis of the cell. However, the analysis of mitochondrial translation products by gel electrophoresis revealed that different amounts of individual membrane proteins were synthesized. Especially mutant cni-1 produced large amounts of a small molecular weight translation product, which is barely detectable in wild-type. * 2. Mitochondrial preparations of cytochrome-oxidase-deficient cells were examined for precursors of cytochrome oxidase. The presence of polypeptide components of cytochrome oxidase in the mitochondria was established with specific antibodies. On the other hand, no significant amounts of heme a could be extracted. * 3. Radioactively labelled components of cytochrome oxidase were isolated by immunoprecipitation and analysed by gel electrophoresis. All three cell types contained the enzyme components 4–7, which are translated on cytoplasmic ribosomes. The mitochondrially synthesized components 1–3 were present in mi-1 mutant and in copper-depleted wild-type cells. In contrast, components 2 and 3 were not detectable in the nuclear mutant cni-1. Both relative and absolute amounts of these polypeptides in the enzyme-deficient cells were quite different from those in wild-type cells. * 4. The components of cytochrome oxidase found in the enzyme-deficient cells were tightly associated with the mitochondrial membranes. * 5. Processes, which affect and may control the production of enzyme precursors or their assembly to a functional cytochrome oxidase are discussed

A social role for churches and cultural demarcation:how German MEPs represent religion in the European Parliament

This study deals with the question of how German members of the European Parliament (MEPs) represent the German model of religion–state relations at the European level. Based on a survey and interviews with German MEPs as well as a content-analysis of German MEPs’ speeches, motions and parliamentary questions during the seventh term of the European Parliament (EP), our study demonstrates that this model is represented in three dimensions. First, German MEPs reflect the close cooperation between the churches and the state in Germany, primarily on social issues, through largely church- and religion-friendly attitudes and relatively frequent contacts with religious interest-groups. Second, by referring to religious freedoms and minorities primarily outside the EU and by placing Islam in considerably more critical contexts than Christianity, German MEPs create a cultural demarcation line between Islam and Christianity through their parliamentary activities, which is similar to, though less politicised than, cultural boundaries often produced in public debates in Germany. Third, our study illustrates similar patterns of religious affiliation and subjective religiosity among German parliamentarians in both the EP and the national Parliament, which to some degree also reflect societal trends in Germany. Yet our data also suggest that European political elites are more religious than the average German population. If the presence of religion in terms of religious interest-groups and arguments is included, the EP appears to be more secularist than the German Parliament

Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

BACKGROUND: Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. METHODS: Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. RESULTS: hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. CONCLUSION: Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes

The mitochondrial phosphate carrier: Role in oxidative metabolism, calcium handling and mitochondrial disease

The mitochondrial phosphate carrier (PiC) is a mitochondrial solute carrier protein, which is encoded by SLC25A3 in humans. PiC delivers phosphate, a key substrate of oxidative phosphorylation, across the inner mitochondrial membrane. This transport activity is also relevant for allowing effective mitochondrial calcium handling. Furthermore, PiC has also been described to affect cell survival mechanisms via interactions with cyclophilin D and the viral mitochondrial-localized inhibitor of apoptosis (vMIA). The significance of PiC has been supported by the recent discovery of a fatal human condition associated with PiC mutations. Here, we present first the early studies that lead to the discovery and molecular characterization of the PiC, then discuss the very recently developed mouse models for PiC and pathological mutations in the human SLC25A3 gene. © 2015

Stability of domain structures in multi-domain proteins

Multi-domain proteins have many advantages with respect to stability and folding inside cells. Here we attempt to understand the intricate relationship between the domain-domain interactions and the stability of domains in isolation. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Stability of such folds to exist independently is optimized by evolution. Specific residue mutations in the sites equivalent to inter-domain interface enhance the overall solvation, thereby stabilizing these domain folds independently. A few naturally occurring variants at these sites alter communication between domains and affect stability leading to disease manifestation. Our analysis provides safe guidelines for mutagenesis which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR