The dynamic properties of water confined within nanospaces are of interest given that such water plays important roles in geological and biological systems. The enthalpy-relaxation properties of ordinary and heavy water confined within silica-gel voids of 1.1, 6, 12, and 52 nm in average diameter were examined by adiabatic calorimetry. Most of the water was found to crystallize within the pores above about 2 nm in diameter but to remain in the liquid state down to 80 K within the pores less than about 1.6 nm in diameter. Only one glass transition was observed, at T(g) = 119, 124, and 132 K for ordinary water and T(g) = 125, 130, and 139 K for heavy water, in the 6-, 12-, and 52-nm diameter pores, respectively. On the other hand, two glass transitions were observed at T(g) = 115 and 160 K for ordinary water and T(g) = 118 and 165 K for heavy water in the 1.1-nm pores. Interfacial water molecules on the pore wall, which remain in the noncrystalline state in each case, were interpreted to be responsible for the glass transitions in the region 115-139 K, and internal water molecules, surrounded only by water molecules in the liquid state, are responsible for those at 160 or 165 K in the case of the 1.1-nm pores. It is suggested that the glass transition of bulk supercooled water takes place potentially at 160 K or above due to the development of an energetically more stable hydrogen-bonding network of water molecules at low temperatures.

The retinoic acid-related orphan nuclear receptor ␥t (ROR␥t)/ROR␥2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of ROR␥t and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the ROR␥t ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited ROR␥t activity and decreased IL-17 production but not ROR␣ activity. Digoxin inhibited IL-17 production in CD4 ؉ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that ROR␥t is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel ROR␥t antagonists.

Osteoclasts and osteoblasts are responsible for strict bone maintenance with a balance between bone formation and resorption by interacting with each other. Recently, it has been revealed that osteoblasts/stromal cells regulate differentiation of osteoclasts/hematopoietic cells by two factors, the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) on the plasma membrane, and secreted osteoprotegerin (OPG). However, no factors have yet been reported by which osteoclasts/hematopoietic cells regulate osteoblasts/stromal cells. To elucidate the possibility of signal transduction from osteoclasts to osteoblasts, we studied the conditioned medium of mouse osteoclast-like myeloma cell line RAW264.7 treated with RANKL. We found that this medium contains a factor that inhibits differentiation of mouse osteoblast precursor-like cell line MC3T3-E1 to osteoblasts induced by bone morphogenetic protein 4 (BMP-4) and named this factor osteoblastogenesis inhibitory factor (OBIF). OBIF was purified by successive three-step chromatography by heparin affinity, anion exchange, and reversed-phase columns. Osteoblastogenesis inhibitory activity made one peak in each chromatography step, showing the factor is a single entity. Active fractions were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bands of proteins were excised, digested by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Consequently, we have identified this factor to be platelet-derived growth factor BB (PDGF BB) homodimer. Furthermore, this identification of PDGF BB as OBIF was confirmed by neutralization of the inhibitory activity of the medium with anti-PDGF antibody. These results show, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.

The specialized role of mouse Gr-1high monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during sub-clinical endotoxemia in mice, and investigated if marginated monocytes contribute to lung injury in response to further septic stimuli. Sub-clinical low-dose i.v. LPS induced a rapid (within 2h), large scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these pre-marginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the pre-administration of low-dose LPS. The LPS-zymosan induced permeability increases were effectively abrogated by pre-treatment (30 min before zymosan challenge) with the platelet-activating factor (PAF) antagonist WEB 2086, in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of PAF/ceramide mediated pathways in this model. Depletion of monocytes (at 18h post clodronate-liposome treatment) significantly attenuated the LPS-zymosan induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48h post clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during sub-clinical endotoxemia primes the lungs toward further septic stimuli, and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.