Mechanics of Individual Keratin Bundles in Living Cells

Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed.     

Foxp3+ Regulatory T Cells Delay Expulsion of Intestinal Nematodes by Suppression of IL-9-Driven Mast Cell Activation in BALB/c but Not in C57BL/6 Mice

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3⁺ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3⁺ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.     

Cationic composition and acid–base state of the extracellular fluid,and specific buffer value of hemoglobin from the branchiopod crustacean <Emphasis Type="Italic">Triops cancriformis</Emphasis>

Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein’s native environment. This study determined the cationic composition and acid–base state of the animal’s extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO₂ partial pressure of 1.36 kPa at pH 7.52 and 20°C. The discrepancy between this high CO₂ partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L⁻¹ pH⁻¹. Hb covered 40–60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)⁻¹ pH⁻¹ suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.     

Badger Badger. T. J. Roper. HarperCollins Publishers. London. 2010, 388 pp. ISBN 978-0-00-733977-8

Book Review

Badger Badger. T. J. Roper. HarperCollins Publishers. London. 2010, 388 pp. ISBN 978-0-00-733977-8     

Exosome Secretion Ameliorates Lysosomal Storage of Cholesterol in Niemann-Pick Type C Disease

Niemann-Pick type C1 disease is an autosomal-recessive lysosomal storage disorder. Loss of function of the npc1 gene leads to abnormal accumulation of free cholesterol and sphingolipids within the late endosomal and lysosomal compartments resulting in progressive neurodegeneration and dysmyelination. Here, we show that oligodendroglial cells secrete cholesterol by exosomes when challenged with cholesterol or U18666A, which induces late endosomal cholesterol accumulation. Up-regulation of exosomal cholesterol release was also observed after siRNA-mediated knockdown of NPC1 and in fibroblasts derived from NPC1 patients and could be reversed by expression of wild-type NPC1. We provide evidence that exosomal cholesterol secretion depends on the presence of flotillin. Our findings indicate that exosomal release of cholesterol may serve as a cellular mechanism to partially bypass the traffic block that results in the toxic lysosomal cholesterol accumulation in Niemann-Pick type C1 disease. Furthermore, we suggest that secretion of cholesterol by exosomes contributes to maintain cellular cholesterol homeostasis.     

Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors

Nineteen GABA_A receptor (GABA_AR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABA_ARs is unknown. Here we report the discovery of a new structural class of GABA_AR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABA_AR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABA_ARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA_ARs.     

Calcium imaging of individual erythrocytes: problems and approaches

Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E(2) and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission. Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable.     

The Quantity and Quality of Illegally Imported Products of Animal Origin in Personal Consignments into the European Union Seized at Two German Airports between 2010 And 2014

The import of products of animal origin (POAO) in travellers’ personal consignments presents a considerable risk of introducing animal diseases and emerging zoonoses into the European Union. The current regulation (EU) 206/2009 implements strict measures for illegally imported POAO, whereupon non-complying products have to be seized and destroyed regardless. Especially airports serve as global bottlenecks for illegally imported POAO where passenger controls of non-European flights are performed by customs and veterinary services in collaboration. Results of these control measures have to be submitted in the form of annual reports to the European Commission. However, few data on qualities and quantities of seizures have been published so far. In this study, POAO seized at two German airports between 2010 and 2014 were analysed in terms of quantities, qualitative categories and region of origin. In most years considered, more than 20 tonnes POAO were seized at each airport. However, reported amounts of seizures seem to be only the tip of the iceberg as an all-passenger control is not feasible and therefore travellers are only spot-checked. The analysis suggests that the organisational structures of both customs and official veterinary services and their different risk perceptions interfere in completing an effective ban on the illegal import of POAO.