Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell-surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2-DS domain), and identified the binding site to fibrillar collagen by transferred cross-saturation experiments. The DDR2-DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen-binding site suggests that the DDR2-DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen-binding mode of the DDR2-DS domain.     

Administration of fibroblast growth factor 2 in combination with bone marrow transplantation synergistically improves carbon-tetrachloride-induced liver fibrosis in mice

We previously reported that fibroblast growth factor 2 (FGF2) facilitated the differentiation of transplanted bone marrow cells (BMCs) into hepatocytes. Our earlier study also demonstrated that administration of FGF2 in combination with bone marrow transplantation (BMT) synergistically activated tumor necrosis factor-alpha signaling and significantly improved liver function and prognosis more than BMT alone. However, the way that it affected the extracellular matrix remained unclear. Here, we investigated the effect of FGF2 treatment together with BMT on liver fibrosis in mice treated with carbon tetrachloride (CCl₄). Transplantation of BMCs and concurrent treatment with FGF2 caused a statistically significant reduction in CCl₄-induced liver fibrosis that was accompanied by strong expression of matrix metalloproteinase 9 as compared with FGF2-only treatment or BMT alone. Moreover, in this process, the proliferation of bone-marrow-derived cells was accelerated without causing apoptosis. Thus, the administration of FGF2 in combination with BMT synergistically improves CCl₄-induced liver fibrosis in mice. This treatment has the potential of being an effective therapy for patients with liver cirrhosis. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 16390211 and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5 and H17-Special-015).     

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by ~20°C) than that of the hairpin (by ~10°C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.     

Regulation of DNA nucleases by molecular crowding

Here, we examined the effects of molecular crowding on the function, structure and stability of nucleases. We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases. Moreover, kinetic analysis showed that the maximum velocity for the reaction of DNase I at 25°C was increased from 0.1 to 2.7μM/min by molecular crowding with 20% (w/v) PEG, whereas that of exonuclease I at 37°C decreased from 2.2 to 0.4μM/min. In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I. These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

Novel membrane-localizing TEMPO derivatives for measurement of cellular oxidative stress at the cell membrane

Oxidative stress affecting lipid membranes is considered to be closely related to cardiovascular disease and brain ischemia. In this study, we designed and synthesized membrane-localizing TEMPO derivatives and demonstrated that one of these synthesized probes, compound 1, localized and detected oxidative stress in the cell membrane in an endotoxic model of a mouse macrophage-like cell line. Compound 1 is therefore a potentially useful probe for evaluating oxidative stress at the cell membrane.     

LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins

Neurons are polarized cells that contain distinct sets of proteins in their axons and dendrites. Synaptic vesicles (SV) and many SV proteins are exclusively localized in the presynaptic regions but not in dendrites. Despite their fundamental importance, the mechanisms underlying the polarized localization of SV proteins remain unclear. The transparent nematode Caenorhabditis elegans can be used to examine sorting and transport of SV proteins in vivo. Here, we identify a novel protein kinase LRK-1, a C. elegans homolog of the familial Parkinsonism gene PARK8/LRRK2 that is required for polarized localization of SV proteins. In lrk-1 deletion mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons. This aberrant localization of SV proteins in the dendrites is dependent on the AP-1 mu1 clathrin adaptor UNC-101, which is involved in polarized dendritic transport, but not on UNC-104 kinesin, which is required for the transport of SV to presynaptic regions. The LRK-1 proteins are localized in the Golgi apparatus. These results suggest that the LRK-1 protein kinase determines polarized sorting of SV proteins to the axons by excluding SV proteins from the dendrite-specific transport machinery in the Golgi.     

Synthesis and matrix metalloproteinase (MMP)-12 inhibitory activity of ageladine A and its analogs

Ageladine A (1) and its analogs 2-10 were expeditiously synthesized by featuring the biosynthetic route proposed for 1 (for 1-10) and by employing 2-(N-t-butoxycarbonylamino)imidazol-4-carbaldehyde as the starting material (for 1-8). From MMP-12 inhibitory activity assay, it appeared evident that the two bromine atoms and the three NH groups (1-NH, 14-NH, and 15-NH2) were indispensable for 1 to exhibit excellent activity.