Dae-Won Kim

Immunostimulation and anti-DNA antibody production by backbone modified CpG-DNA.

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-DNA) have gained attention as potentially useful therapeutics. However, the phosphorothioate-modified CpG-DNAs (PS-ODN) can induce backbone-related side effects. Here, we compared the immunostimulatory activity of natural phosphodiester CpG-DNA (PO-ODN) from Mycobacterium bovis and PS-ODN in mice. Both PO-ODN and PS-ODN induced production of IL-12. PS-ODN increased spleen weights, spleen cell numbers, and the migration of macrophages into the peritoneal cavity in the mice in a CG sequence-dependent manner. PS-ODN induced anti-PS-ODN antibody production in the mice, and the PS-ODN-specific IgM was cross-reactive with other PS-ODNs in a CG sequence-independent manner. In contrast, PO-ODN did not affect on spleen weights, cell numbers, or IgM production. These results may provide an explanation for the side effects in immunotherapeutic application of PS-ODN. They also suggest that PO-ODN may be more optimal than PS-ODN to enhance innate immune responses without severe side effects.

Constitutive RelA activation mediated by Nkx3.2 controls chondrocyte viability

During endochondral ossification, a process that accounts for the majority of bone formation in vertebrates, hypertrophic chondrocytes display a greater susceptibility to apoptosis when compared to proliferating chondrocytes. However, the molecular mechanisms underlying this phenomenon remain unclear. Nkx3.2, a member of the NK class of homeoproteins, is initially expressed in chondrogenic precursor cells, and later, during cartilage maturation, its expression is restricted to proliferating chondrocytes. Here, we show that the nuclear factor kappa B (NF-κB) pathway is required for chondrocyte viability and that Nkx3.2 supports chondrocyte survival by constitutively activating RelA. Although signal-dependent NF-κB activation has been intensively studied, ligand-independent NF-κB activation is poorly understood. The data presented here support a novel ligand-independent mechanism of NF-κB activation, whereby Nkx3.2 recruits the RelA–IκBα heteromeric complex into the nucleus by direct protein–protein interactions and activates RelA through proteasome-dependent IκBα degradation in the nucleus. Furthermore, we demonstrate that stage-specific NF-κB activation, mediated by Nkx3.2, regulates chondrocyte viability during cartilage maturation.

ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

BackgroundThe ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9).ResultsWe found that a variant of ASB9 that lacks the SOCS box (ASB9ΔSOCS) was naturally detected in human cell lines but not in peripheral blood mononuclear cells or normal hepatocytes. We also identified ubiquitous mitochondrial creatine kinase (uMtCK) as a new target of ASB9 in human embryonic kidney 293 (HEK293) cells. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of uMtCK in a SOCS box-independent manner. The overexpression of ASB9, but not ASB9ΔSOCS, induces ubiquitination of uMtCK. ASB9 and ASB9ΔSOCS can interact and colocalise with uMtCK in the mitochondria. However, only expression of ASB9 induced abnormal mitochondrial structure and a decrease of mitochondrial membrane potential. Furthermore, the creatine kinase activities and cell growth were significantly reduced by ASB9 but not by ASB9ΔSOCS.ConclusionsASB9 interacts with the creatine kinase system and negatively regulates cell growth. The differential expression and function of ASB9 and ASB9ΔSOCS may be a key factor in the growth of human cell lines and primary cells.

Indian Hedgehog signalling triggers Nkx3.2 protein degradation during chondrocyte maturation

The Ihh (Indian Hedgehog) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 (NK3 homeobox 2) is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in the present study, we investigated whether Nkx3.2, an early-stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. We show that Ihh signalling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (low-density-lipoprotein-receptor-related protein) (Wnt co-receptor) and Sfrp (secreted frizzled-related protein) (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocytes. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signalling by deletion of either Ihh or smoothened. Thus these results suggest that Ihh/Wnt5a signalling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis.

2,4-Dinitrofluorobenzene Modifies Cellular Proteins and Induces Macrophage Inflammatory Protein-2 Gene Expression via Reactive Oxygen Species Production in RAW 264.7 Cells

The skin sensitizer 2,4-dinitrofluorobenzene (DNFB) provokes delayed hypersensitivity responses as a result of topical application to the skin. Here, we demonstrate that DNFB modifies proteins in RAW 264.7 cells and skin tissues in NC/Nga mice; we also show the functional involvement of DNFB-induced modification of cellular proteins in the DNFB-induced macrophage inflammatory protein (MIP)-2 gene expression in RAW 264.7 cells. In addition, we demonstrate that DNFB strongly induces reactive oxygen species (ROS) production. Our RT-PCR analysis and reporter gene assays reveal that the DNFB-induced intracellular ROS production is necessary for MIP-2 gene expression by DNFB. We observed that the vitamin C and chemical oxidant scavenger N-acetyl-cysteine have an inhibitory effect on the generation of ROS, the activation of MAP kinase pathways, and the MIP-2 gene expression in DNFB-treated RAW 264.7 cells. These results provide insight into the mechanisms involved in DNFB-induced contact hypersensitivity.

Solution structure of TA1092, a ribosomal protein S24e from Thermoplasma acidophilum

Introduction. The ribosome is a large ribonucleoprotein complex that catalyzes protein synthesis. In archaea, the ribosome is composed of a small 30S subunit and a large 50S subunit. The 30S subunit of the archaea ribosome is composed of a 16S rRNA and 28 ribosomal proteins. Genes that encode ribosomal proteins consist of multiple processed pseudogenes, which are dispersed through the genome. Recent advances in ribosome structure suggest close molecular interactions between rRNA and ribosomal protein, within ribosomal subunits. However, the structural role of ribosomal components during various stages of translation remains to be characterized. Here we report the solution structure of TA1092, a member of the S24e protein family from Thermoplasma acidophilum. Members of the S24e protein family are components of the 30S subunit in both archaea and eukaryotes. Because the detailed structure of ribosomal protein S24e is not yet available for model organisms, the structure of TA1092 will expand our knowledge and understanding of 30S ribosomal protein structure–function for different species.

Improved Time-Series Photometry and Calibration Method for Non-Crowded Fields: MMT Megacam and HAT-South Experiences

We present a new photometric reduction method for precise time-series photometry of non-crowded fields that does not need to involve relatively complicated and CPU intensive techniques such as point-spread-function (PSF) fitting or difference image analysis. This method, which combines multi-aperture index photometry and a spatio-temporal de-trending algorithm, gives much superior performance in data recovery and light-curve precision. In practice, the brutal filtering that is often applied to remove outlying data points can result in the loss of vital data, with seriously negative impacts on short-term variations such as flares. Our method utilizes nearly 100% of available data and reduces the rms scatter to several times smaller than that for archived light curves for brighter stars. We outline the details of our new method, and apply it to cases of sample data from the MMT survey of the M37 field, and the HAT-South survey.

Reciprocal control of excitatory synapse numbers by Wnt and Wnt inhibitor PRR7 secreted on exosomes

Secreted Wnts play crucial roles in synaptogenesis and synapse maintenance, but endogenous factors promoting synapse elimination in central neurons remain unknown. Here we show that proline-rich 7 (PRR7) induces specific removal of excitatory synapses and acts as a Wnt inhibitor. Remarkably, transmembrane protein PRR7 is activity-dependently released by neurons via exosomes. Exosomal PRR7 is uptaken by neurons through membrane fusion and eliminates excitatory synapses in neighboring neurons. Conversely, PRR7 knockdown in sparse neurons greatly increases excitatory synapse numbers in all surrounding neurons. These non-cell autonomous effects of PRR7 are effectively negated by augmentation or blockade of Wnt signaling. PRR7 exerts its effect by blocking the exosomal secretion of Wnts, activation of GSK3β, and promoting proteasomal degradation of PSD proteins. These data uncover a proximity-dependent, reciprocal mechanism for the regulation of excitatory synapse numbers in local neurons and demonstrate the significance of exosomes in inter-neuronal signaling in the vertebrate brain.Wnts are important for synapse formation and maintenance. Here, the authors show that proline-rich 7 (PRR7) is a Wnt inhibitor that is secreted via exosomes to regulate excitatory synapse numbers.

De-Trending Time Series Data for Variability Surveys

We present an algorithm for the removal of trends in time series data. The trends could be caused by various systematic and random noise sources such as cloud passages, change of airmass or CCD noise. In order to determine the trends, we select template stars based on a hierarchical clustering algorithm. The hierarchy tree is constructed using the similarity matrix of light curves of stars whose elements are the Pearson correlation values. A new bottom-up merging algorithm is developed to extract clusters of template stars that are highly correlated among themselves, and may thus be used to identify the trends. We then use the multiple linear regression method to de-trend all individual light curves based on these determined trends. Experimental results with simulated light curves which contain artificial trends and events are presented. We also applied our algorithm to TAOS (Taiwan-American Occultation Survey) wide field data observed with a 0.5m f/1.9 telescope equipped with 2k by 2k CCD. With our approach, we successfully removed trends and increased signal to noise in TAOS light curves.