Uptake and Toxicity of Copper Oxide Nanoparticles in C6 Glioma Cells

Copper oxide nanoparticles (CuO-NPs) are frequently used for many technical applications, but are also known for their cell toxic potential. In order to investigate a potential use of CuO-NPs as a therapeutic drug for glioma treatment, we have investigated the consequences of an application of CuO-NPs on the cellular copper content and cell viability of C6 glioma cells. CuO-NPs were synthesized by a wet-chemical method and were coated with dimercaptosuccinic acid and bovine serum albumin to improve colloidal stability in physiological media. Application of these protein-coated nanoparticles (pCuO-NPs) to C6 cells caused a strong time-, concentration- and temperature-dependent copper accumulation and severe cell death. The observed loss in cellular MTT-reduction capacity, the loss in cellular LDH activity and the increase in the number of propidium iodide-positive cells correlated well with the specific cellular copper content. C6 glioma cells were less vulnerable to pCuO-NPs compared to primary astrocytes and toxicity of pCuO-NPs to C6 cells was only observed for incubation conditions that increased specific cellular copper contents above 20 nmol copper per mg protein. Both cellular copper accumulation as well as the pCuO-NP-induced toxicity in C6 cells were prevented by application of copper chelators, but not by endocytosis inhibitors, suggesting that liberation of copper ions from the pCuO-NPs is the first step leading to the observed toxicity of pCuO-NP-treated glioma cells.     

A scoring system for coat and tail condition in ringtailed lemurs,Lemur catta

Coat condition can be influenced by a wide variety of disorders and thus provides a useful tool for noninvasive health and welfare assessments in wild and captive animals. Using Lemur catta as an exemplar, we offer a 6‐step scoring system for coat and tail condition, ranging from perfectly fluffy to half or more of body and tail being hairless. The categories are described in detail and illustrated with sample pictures from a wild population in Berenty Reserve, Madagascar. Furthermore, we elaborate on intermediate conditions and discoloration of fur. Coat condition scoring allows the comparison between years, seasons, and the effect of toxin, disease or stress. Although this system was developed for wild L. catta, we believe it can also be of value for other species. We recommend scoring coat condition in healthy wild mammal populations to give a baseline on yearly and seasonal variations vs. deteriorating health conditions or pathology. Am. J. Primatol. 71:183–190, 2009. © 2009 Wiley‐Liss, Inc.     

Spectro-Temporal Weighting of Loudness

Real-world sounds like speech or traffic noise typically exhibit spectro-temporal variability because the energy in different spectral regions evolves differently as a sound unfolds in time. However, it is currently not well understood how the energy in different spectral and temporal portions contributes to loudness. This study investigated how listeners weight different temporal and spectral components of a sound when judging its overall loudness. Spectral weights were measured for the combination of three loudness-matched narrowband noises with different center frequencies. To measure temporal weights, 1,020-ms stimuli were presented, which randomly changed in level every 100 ms. Temporal weights were measured for each narrowband noise separately, and for a broadband noise containing the combination of the three noise bands. Finally, spectro-temporal weights were measured with stimuli where the level of the three narrowband noises randomly and independently changed every 100 ms. The data consistently showed that (i) the first 300 ms of the sounds had a greater influence on overall loudness perception than later temporal portions (primacy effect), and (ii) the lowest noise band contributed significantly more to overall loudness than the higher bands. The temporal weights did not differ between the three frequency bands. Notably, the spectral weights and temporal weights estimated from the conditions with only spectral or only temporal variability were very similar to the corresponding weights estimated in the spectro-temporal condition. The results indicate that the temporal and the spectral weighting of the loudness of a time-varying sound are independent processes. The spectral weights remain constant across time, and the temporal weights do not change across frequency. The results are discussed in the context of current loudness models.     

Formation of dAMP-glycerol and dAMP-Tris derivatives by Thermococcus kodakaraensis DNA primase

In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.     

Characterization of DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis

In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.     

New vistas for treatment of obesity and diabetes? Endocannabinoid signalling and metabolism in the modulation of energy balance

Growing evidence suggests that pathological overactivation of the endocannabinoid system (ECS) is associated with dyslipidemia, obesity and diabetes. Indeed, this signalling system acting through cannabinoid receptors has been shown to function both centrally and peripherally to regulate feeding behaviour as well as energy expenditure and metabolism. Consequently, modulation of these receptors can promote significant alterations in body weight and associated metabolic profile. Importantly, blocking cannabinoid receptor type 1 function has been found to prevent obesity and metabolic dysfunction in various murine models and in humans. Here we provide a detailed account of the known physiological role of the ECS in energy balance, and explore how recent studies have delivered novel insights into the potential targeting of this system as a therapeutic means for treating obesity and related metabolic disorders.     

Dynamics of the actin-binding protein drebrin in motile cells and definition of a juxtanuclear drebrin-enriched zone

The actin-binding protein (ABP) drebrin, isoform E2, is involved in remodelling of the actin cytoskeleton and in formation of cell processes, but its role in cell migration has not yet been investigated. Therefore, we have studied the organization of drebrin in motile cultured cells such as murine B16F1 melanoma and human SV80 fibroblast cells, using live cell confocal microscopy. In cells overexpressing DNA constructs encoding drebrin linked to EGFP, numerous long, branched cell processes were formed which slowly retracted and extended, whereas forward movement was halted. In contrast, stably transfected B16F1 cells containing drebrin-EGFP at physiological levels displayed lamellipodia and were able to migrate on laminin. Surprisingly, in such cells, drebrin was absent from anterior lamellipodia but was enriched in a specific juxtanuclear zone, the "drebrin-enriched zone" (DZ), and in the tail. In leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was specifically enriched along posterior portions of the microspikes, together with tropomyosin. Drebrin knock-down by small interfering RNAs did not impair movements of SV80 cells. Our results confirm the role of drebrin E2 in the formation of branching processes and further indicate that during cell migration, the protein contributes to retraction of the cell body and the tail but not to lamellipodia formation. In particular, the novel, sizable juxtanuclear DZ structure will have to be characterized in future experiments with respect to its molecular assembly and cell biological functions.