Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

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.     

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 muM and remained constant upon further increases of the copper concentration to 120 muM. The cell growth rate (mu), on the other hand, decreased over the entire range (10-120 muM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 muM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.     

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.