Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.     

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca²⁺-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca²⁺ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca²⁺-triggered exocytosis by increasing the Ca²⁺ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca²⁺-triggered release apparatus.     

Mnl2, a novel component of the ER associated protein degradation pathway

In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY^* in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.     

Markers of enhanced cholesterol absorption are a strong predictor for cardiovascular diseases in patients without diabetes mellitus

Objective

Hypercholesterolemia is a major risk factor for cardiovascular disease (CVD), and diabetes mellitus and statin treatment affect cholesterol metabolism. The aim of the present study was to evaluate markers of cholesterol metabolism and determine their relationship with CVD in patients without diabetes mellitus who were not receiving statin treatment.

Methods

In addition to conventional CVD risk factors, plasma levels of campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (an indicator of cholesterol synthesis) were determined in 835 consecutive patients referred for coronary angiography. Coronary artery disease was evaluated by coronary angiograms, carotid atherosclerosis and peripheral vascular disease were assessed by Doppler ultrasound, and cerebrovascular accidents and transient ischemic attacks were identified by medical history.

Results

After excluding patients with known diabetes mellitus and those receiving statin treatment, 177 patients were included in the analysis. Compared to patients without CVDs (n = 111), patients with concomitant CVDs (n = 66) had a reduced lathosterol-to-cholesterol ratio (1.25 ± 0.61 vs. 1.38 ± 0.63, P < 0.05) and an increased campesterol-to-cholesterol ratio (1.81 ± 1.04 vs. 1.50 ± 0.69, P < 0.05), indicating that enhanced absorption and reduced synthesis of cholesterol is associated with CVD development. Logistic regression analysis including all established cardiovascular risk factors (age, sex, total cholesterol, arterial hypertension, body mass index and smoking) revealed that campesterol and the campesterol-to-cholesterol ratio were significant predictors of concomitant CVD in this patient population.

Conclusion

In patients without diabetes mellitus, markers of enhanced cholesterol absorption were a strong predictor for concomitant CVD.     

Der Einfluß der Umweltfaktoren auf das Wachstum und den Häutungsrhythmus der StrandkrabbeCarcinides maenas

Zusammenfassung 1. Junge Strandkrabben von 4–16 mm Carapaxbreite wurden bis zur Geschlechtsreife unter konstanten Umweltbedingungen aufgezogen.2. Die Dauer ihrer Häutungsintervalle nimmt bei konstanter Temperatur mit der Körpergröße stetig zu.3. Die Dauer der Häutungsintervalle hängt von der Temperatur und der Ernährung ab. Von der Tageslänge scheint sie weitgehend unabhängig zu sein.4. Der relative Grösßenzuwachs bei jeder Häutung ist im gesamten untersuchten Größenbereich und bei den verschiedenen Temperaturen bei allen Häutungen gleich: Bei den Häutungen verdoppelt sich jeweils das Körpervolumen.5. Augenstielamputationen und Verlust von Extremitäten wirken auf den Häutungsrhythmus in gleicher Weise: Die Schwankungsbreite in der Dauer der Häutungsintervalle ist vermindert. Die Häutungsintervalle sind in 20° C deutlich, in 10° C nur geringfügig verkürzt.6. Durch die Anwesenheit größerer Artgenossen werden die Häutungen verzögert. Die optische Wahrnehmung spielt dabei keine Rolle.7. Aus diesen Ergebnissen wird folgendes geschlossen: Der ausschlaggebende Faktor für die Auslösung von Häutungen ist ein bestimmter Größenzuwachs. Temperatur und Ernährung beeinflussen den Häutungsrhythmus dadurch, daß sie das Tempo des Wachstums bestimmen. Die winterliche Häutungsruhe in Freilandpopulationen wird nicht durch den Kurztag bedingt, sondern durch die Kälte. Diese hemmt lediglich das Wachstum, sie verhindert nicht die Häutungen über das häutungshemmende Hormon. Dieses vermindert vielmehr die Temperaturabhängigkeit des Häutungsrhythmus, indem es die Häutungen im Warmen stärker verzögert als im Kalten. Es gestattet die Anpassung des Häutungstermins an die individuelle Lage der Tiere. Es hemmt in Anwesenheit größerer Artgenossen die Häutung. Beim Verlust mehrerer Gliedmaßen wird seine Sekretion eingestellt, so daß die nächste Häutung vorzeitig erfolgt. Das häutungshemmende Hormon bedingt dementsprechend die große individuelle Variation in der Dauer der Häutungsintervalle.

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The effect of environmental factors on growth and moulting rhythm in the shore crab,Carcinides maenas

Young crabs (carapace width 4 to 16 mm) were raised under controlled conditions in the laboratory. The time intervals between subsequent moults increase at all test temperatures with increasing body size. The length of intermoult periods varies with temperature and feeding. It is not affected by day length. Moulting takes place as soon as a certain increase in size is attained. In comparable size groups, the amount of this increase is identical in all test temperatures. Moreover, the relation of increase to initial size is constant over the whole size range investigated. The body volume doubles at each moult. Eyestalk amputations and loss of extremities have similar effects: They shorten the intermoult periods at 20° C considerably, but at 10° C they do so only slightly; furthermore, the amplitude of fluctuations is narrowed. The presence of large specimens tends to retard moulting in smaller ones; this response is independent of visual stimuli. The following assumptions are made: Low temperatures retard the moulting rhythm directly by slowing down growth. They are not acting via the moult inhibiting hormone. Loss of several extremities causes a stop of hormone delivery resulting in shortened intermoult periods. Recognition by touch of a larger specimen causes increased hormone delivery and thus retardation of the subsequent moulting process.