Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

Asymmetric intercellular communication between bone cells: Propagation of the calcium signaling

Bone functional adaptation by remodeling is achieved by harmonized activities of bone cells in which osteocytes in the bone matrix are believed to play critical roles in sensing mechanical stimuli and transmitting signals to osteoclasts/osteoblasts on the bone surface in order to regulate their bone remodeling activities through the lacuno-canalicular network with many slender osteocytic processes. In this study, we investigated the intercellular communication between bone cells, particularly focusing on its directionality, through in vitro observations of the calcium signaling response to mechanical stimulus and its propagation to neighboring cells (NCs). Direct mechanical stimulus was applied to isolated bone cells from chick calvariae, osteocytes (Ocys) and bone surface cells (BSCs) mainly containing osteoblasts, and the percentage of calcium signaling propagation from the stimulated cell to NCs was analyzed. The results revealed that, regardless of the type of stimulated cell, the signaling propagated to BSCs with a significantly higher percentage, implying that calcium signaling propagation between bone cells strongly depends on the type of receiver cell and not the transmitter cell. In addition, in terms of mutual communication between Ocys and BSCs, the percentage of propagation from Ocys to BSCs is significantly higher than that in the opposite direction, suggesting that the calcium signaling mainly propagates asymmetrically with a bias from Ocys in bone matrix to BSCs on bone surfaces. This asymmetric communication between Ocys and BSCs suggests that osteocytic mechanosensing and cellular communications, which significantly affect bone surface remodeling activities to achieve functional adaptation, seem to be well coordinated and active at the location of biologically suitable and mechanically sensitive regions close to the bone surfaces.     

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD₂)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys⁶⁵ residue and its network of hydrogen bonds with the polar residues Ser⁴⁵, Thr⁶⁷, and Ser⁸¹, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys⁶⁵ residue is activated by its interaction with Ser⁴⁵ and Thr⁶⁷ and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.Prostaglandin (PG)⁶ D synthase (PGDS; PGH₂ d-isomerase (EC 5.3.99.2)) (1, 2) produces PGD₂, having 9α-hydroxy and 11-keto groups, from PGH₂, which bears the chemically labile 9,11-endoperoxide group and is produced as a common intermediate of all prostanoids by the action of cyclooxygenase (PGH₂ synthase). Two distinct types of PGDS have evolved from phylogenetically distinct protein families (2, 3). One is hematopoietic PGDS (H-PGDS), which belongs to the σ class of GSH S-transferases (4, 5), and the other is lipocalin type PGDS (L-PGDS), a member of the lipocalin family (6, 7). L-PGDS is the only enzyme in the lipocalin family and is identical to β-trace, a major protein in human cerebrospinal fluid (8, 9). Although H-PGDS and L-PGDS catalyze the same reaction, their amino acid sequences and tertiary structures are quite different from each other, indicating that these enzymes are a new example of functional convergence (2, 3).L-PGDS is expressed in the heart, central nervous system, and male genital organs of various mammals and is involved in various physiological and pathological functions (reviewed in Refs. 6 and 7). In the brain, L-PGDS produces PGD₂, which is involved in the regulation of pain and non-rapid eye movement sleep, as was shown in studies using gene knock-out mice (10, 11) and human enzyme transgenic mice (12). L-PGDS is regulated by SOX9 and is involved in the differentiation of male genital organs (13–15). This enzyme is also expressed in adipocytes (16), vascular smooth muscle cells (17), and myocardial cells (18, 19) and is involved in adipocyte differentiation, the progression of arteriosclerosis (20), and the protection against hypoxemia (18) or ischemia/reperfusion injury (19). L-PGDS binds various lipophilic compounds, such as retinoids (21), bilirubin, biliverdin (22), gangliosides (23), and amyloid-β peptides (24, 25), with high affinity, acting as an extracellular transporter of these compounds and serving as an endogenous amyloid-β chaperone to prevent amyloid deposition in vivo (24).Although many biochemical and physiological studies suggest important roles of PGD₂ and L-PGDS/β-trace in the regulation of sleep and other biological functions, the crystal structure of L-PGDS has not been resolved. In this study, we determined the crystal structures of two different forms of the Δ^1–24-C65A mutant of mouse L-PGDS in both open and closed conformations. L-PGDS was shown to possess a typical lipocalin fold, the β-barrel, which is a unique structural component specific to L-PGDS and comprises a mobile E-F loop and a large central cavity with two compartments. By performing site-directed mutagenesis of Δ^1–24-L-PGDS and the Δ^1–24-C65A mutant, we found that the Cys⁶⁵ surrounded by the hydroxyl side chains of Ser⁴⁵, Thr⁶⁷, and Ser⁸¹ was activated to contribute to the catalysis by L-PGDS.     

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas.     

Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.