Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction^{1, 2}. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells^{1, 2}. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells^{1, 2}. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide^{1, 2}. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.Download video file.^{(152M, mp4)}     

Methanol production from lignin-related substances by Phanerochaete chrysosporium

Methanol production resulting from the demethoxylation of lignin-related substances by Phanerochaete chrysosporium K-3 was studied in the presence or absence of glutamic acid in order to determine if methanol formation involved the ligninolytic system of the fungus. The general pattern was that methanol formation, calculated as percentage of theoretical yield, decreased in the order guaiacyl > syringyl > veratryl (3,4-dimethoxy) compounds. Methoxyhydroquinone and vanillic acid were most easily demethoxylated, while methanol production decreased with increasing molecular weight for the same type of structure (i.e. guaiacyl). Glutamic acid inhibited the demethoxylation of many of the compounds tested. The demethoxylation of the 4-methoxy group of veratric acid was particularly inhibited by glutamic acid suggesting a participation of the ligninolytic system, while the 3-methoxy group was influenced to a lesser extent.
The demethoxylating enzyme acting on lignin-related phenols is probably a peroxidase, while the identity of the enzyme demethoxylating dimethoxy compounds is not known with certainty, although a peroxidase type of enzyme reaction is anticipated also here.     

Two timescales control the creation of large protein aggregates in cells

Protein aggregation is of particular interest because of its connection with many diseases and disorders. Many factors can alter the dynamics and result of this process, one of them being the diffusivity of the monomers and aggregates in the system. Here, we study experimentally and theoretically an aggregation process in cells, and we identify two distinct physical timescales that set the number and size of aggregates. The first timescale involves fast aggregation of small clusters freely diffusing in the cytoplasm, whereas in the second one, the aggregates are larger than the pore size of the cytoplasm and thus barely diffuse, and the aggregation process is slowed down. However, the process is not entirely halted, potentially reflecting a myriad of active but random forces that stir the aggregates. Such a slow timescale is essential to account for the experimental results of the aggregation process. These results could also have implications in other processes of spatial organization in cell biology, such as phase-separated droplets.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Antiviral action of the tumor suppressor ARF

Oncogenic viruses frequently target the pathways controlled by tumor suppressor genes, suggesting an extra function for these proteins as antiviral factors. The control exerted by the tumor suppressor Arf on cellular proliferation is crucial to restrict tumor development; however, a potential contribution of Arf to prevent viral infectivity has remained unexplored. In the present study, we investigated the consequences of loss or increased expression of Arf on viral infection. Our results reveal that ARF expression is induced by interferon and after viral infection. Furthermore, we show that ARF protects against viral infection in a gene dosage-dependent manner, and that this antiviral action is mediated in part by PKR through a mechanism that involves ARF-induced release of PKR from nucleophosmin complexes. Finally, Arf-null mice were hypersensitive to viral infection compared to wild-type mice. Together, our results reveal a novel and unexpected role for the tumor suppressor ARF in viral infection surveillance.