Die sog. Asphalt-Flecke der kultivierten Herzmuskelzellen — eine im Phasenkontrastmikroskop sichtbare Formation des Glykogens

Zusammenfassung Bei einer korrespondierenden Betrachtung dreier Asphaltflecke von lebenden Herzmuskelzellen in der Kultur und den identischen Stellen im Elektronenmikroskop erweist sich der Fleckeninhalt als eine Anhäufung locker beieinanderliegender -Teilchen des Glykogens.Präparate für die elektronenmikroskopische Untersuchung konnten hergestellt werden, nachdem der schweren Fixierbarkeit des Fleckeninhaltes mit der Verwendung des Glutaraldehyds in Kakodylatpuffer und der Spülung mit 50%igem Alkohol nach der Reynold'schen Kontrastierung Rechnung getragen war.Mit dieser Diagnose ist zugleich bewiesen, daß Glykogen im Phasenkontrastbild der lebenden Zelle sichtbar werden kann.

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The asphalt coloured spots of living heart muscle cells — glycogen formations visible under phase contrast

Summary The contents of three asphalt coloured spots previously examined in the living heart muscle cells in a culture by a phase contrast microscope and subsequently identified in the electron microscope by the method of Gross and Riedel proved to be accumulations of -particles of glycogen loosely lying together.Suitable sections could be manufactured for electron microscope after complying with the difficulty of the spot contents to be fixated — revealed during the histochemical investigations. One had to use glutaraldehyde in cacodylate buffer as the first fixative and 50% alcohol instead of distilled water for rinsing after the Reynold's staining.By this diagnosis at the same time, it is proven that certain glycogen formations in living cells are visible under phase contrast.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Suppression of oxidative phosphorylation confers resistance against bevacizumab in experimental glioma

Although bevacizumab initially shows high response rates in gliomas and other tumours, therapy resistance usually develops later. Because anti‐angiogenic agents are supposed to induce hypoxia, we asked whether rendering glioma cells independent of oxidative phosphorylation modulates their sensitivity against hypoxia and bevacizumab. LNT‐229 glioma cells without functional mitochondria (rho⁰) and control (rho⁺) cells were generated. LNT‐229 rho⁰‐cells displayed reduced expression of oxidative phosphorylation‐related genes and diminished oxygen consumption. Conversely, glycolysis was up‐regulated in these cells, as shown by increased lactate production and stronger expression of glucose transporter‐1 and lactate dehydrogenase‐A. However, hypoxia‐induced cell death in vitro was nearly completely abolished in the LNT‐229 rho⁰‐cells, these cells were more sensitive towards glucose restriction and the treatment with the glycolysis inhibitor 2‐deoxy‐D‐glucose. In an orthotopic mouse xenograft experiment, bevacizumab induced hypoxia as reflected by elevated Hypoxia‐inducible factor 1‐alpha staining in both, rho⁺‐ and rho⁰‐tumours. However, it prolonged survival only in the mice bearing rho⁺‐tumours (74 days vs. 105 days, p  = 0.024 log‐rank test) and had no effect on survival in mice carrying LNT‐229 rho⁰‐tumours (75 days vs. 70 days, p  = 0.52 log‐rank test). Interestingly, inhibition of glycolysis in vivo with 2‐deoxy‐D‐glucose re‐established sensitivity of rho⁰‐tumours against bevacizumab (98 days vs. 80 days, p  = 0.0001). In summary, ablation of oxidative phosphorylation in glioma cells leads to a more glycolytic and hypoxia‐resistant phenotype and is sufficient to induce bevacizumab‐refractory tumours. These results add to increasing evidence that a switch towards glycolysis is one mechanism how tumour cells may evade anti‐angiogenic treatments and suggest anti‐glycolytic strategies as promising approaches to overcome bevacizumab resistance.

     

Nimodipine confers clinical improvement in two models of experimental autoimmune encephalomyelitis

Multiple sclerosis is characterised by inflammatory neurodegeneration, with axonal injury and neuronal cell death occurring in parallel to demyelination. Regarding the molecular mechanisms responsible for demyelination and axonopathy, energy failure, aberrant expression of ion channels and excitotoxicity have been suggested to lead to Ca²⁺ overload and subsequent activation of calcium‐dependent damage pathways. Thus, the inhibition of Ca²⁺ influx by pharmacological modulation of Ca²⁺ channels may represent a novel neuroprotective strategy in the treatment of secondary axonopathy. We therefore investigated the effects of the L‐type voltage‐gated calcium channel blocker nimodipine in two different models of mouse experimental autoimmune encephalomyelitis (EAE ), an established experimental paradigm for multiple sclerosis. We show that preventive application of nimodipine (10 mg/kg per day) starting on the day of induction had ameliorating effects on EAE in SJL /J mice immunised with encephalitic myelin peptide PLP _139–151, specifically in late‐stage disease. Furthermore, supporting these data, administration of nimodipine to MOG _35–55‐immunised C57BL /6 mice starting at the peak of pre‐established disease, also led to a significant decrease in disease score, indicating a protective effect on secondary CNS damage. Histological analysis confirmed that nimodipine attenuated demyelination, axonal loss and pathological axonal β‐amyloid precursor protein accumulation in the cerebellum and spinal cord in the chronic phase of disease. Of note, we observed no effects of nimodipine on the peripheral immune response in EAE mice with regard to distribution, antigen‐specific proliferation or activation patterns of lymphocytes. Taken together, our data suggest a CNS ‐specific effect of L‐type voltage‐gated calcium channel blockade to inflammation‐induced neurodegeneration.

     

Non‐breeding range size predicts the magnitude of population trends in trans‐Saharan migratory passerine birds

Understanding why populations of some migratory species show a directional change over time, i.e. increase or decrease, while others do not, remains a challenge for ecological research. One possible explanation is that species with smaller non‐breeding ranges may have more pronounced directional population trends, and their populations are thus more sensitive to the variation in environmental conditions in their non‐breeding quarters. According to the serial residency hypothesis, this sensitivity should lead to higher magnitudes (i.e. absolute values) of population trends for species with smaller non‐breeding ranges, with the direction of trend being either positive or negative depending on the nature of the environmental change. We tested this hypothesis using population trends over 2001–2012 for 36 sub‐Saharan migratory passerine birds breeding in Europe. Namely, we related the magnitude of the species' population trends to the size of their sub‐Saharan non‐breeding grounds, whilst controlling for factors including number of migration routes, non‐breeding habitat niche and wetness, breeding habitat type and life‐history strategy. The magnitude of species' population trends grew with decreasing absolute size of sub‐Saharan non‐breeding ranges, and this result remained significant when non‐breeding range size was expressed relative to the size of the breeding range. After repeating the analysis with the trend direction, the relationship with the non‐breeding range size disappeared, indicating that both population decreases and increases are frequent amongst species with small non‐breeding range sizes. Therefore, species with small non‐breeding ranges are at a higher risk of population decline due to adverse factors such as habitat loss or climatic extremes, but their populations are also more likely to increase when suitable conditions appear. As non‐breeding ranges may originate from stochasticity of non‐breeding site selection in naive birds (‘serial‐residency’ hypothesis), it is crucial to maintain a network of stable and resilient habitats over large areas of birds’ non‐breeding quarters.     

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

The N‐end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP‐dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N‐terminal amino acids (N‐degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N‐end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N‐degrons. However, while the N‐degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal‐ClpS binds and discriminates peptides mimicking bona fide N‐end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal‐ClpS localizes to this plastid‐like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti‐malarial drugs aimed at disrupting parasite‐specific protein quality control pathways.     

Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.