Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (Lc), lamellar gel (L beta'), ripple gel (P beta') and liquid crystal (L alpha), in turn. The Lc phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L beta I) phase appeared above the critical interdigitation pressure (CIP) between the L beta' and P beta' phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F'497/F'430 value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L beta'/L beta I phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L beta I phase in the SPPC bilayer has a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.     

Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.     

TGF-beta mediated Dlx5 signaling plays a crucial role in osteo-chondroprogenitor cell lineage determination during mandible development

Transforming growth factor-β (TGF-β) signaling is crucial for mandible development. During its development, the majority of the mandible is formed through intramembranous ossification whereas the proximal region of the mandible undergoes endochondral ossification. Our previous work has shown that TGF-β signaling is required for the proliferation of cranial neural crest (CNC)-derived ectomesenchyme in the mandibular primordium where intramembranous ossification takes place. Here we show that conditional inactivation of Tgfbr2 in CNC cells results in accelerated osteoprogenitor differentiation and perturbed chondrogenesis in the proximal region of the mandible. Specifically, the appearance of chondrocytes in Tgfbr2^fl/fl;Wnt1-Cre mice is delayed and they are smaller in size in the condylar process and completely missing in the angular process. TGF-β signaling controls Sox9 expression in the proximal region, because Sox9 expression is delayed in condylar processes and missing in angular process in Tgfbr2^fl/fl;Wnt1-Cre mice. Moreover, exogenous TGF-β can induce Sox9 expression in the mandibular arch. In the angular processes of Tgfbr2^fl/fl;Wnt1-Cre mice, osteoblast differentiation is accelerated and Dlx5 expression is elevated. Significantly, deletion of Dlx5 in Tgfbr2^fl/fl;Wnt1-Cre mice results in the rescue of cartilage formation in the angular processes. Finally, TGF-β signaling-mediated Scleraxis expression is required for tendonogenesis in the developing skeletal muscle. Thus, CNC-derived cells in the proximal region of mandible have a cell intrinsic requirement for TGF-β signaling.     

Crucial role of peroxiredoxin III in placental antioxidant defense of mice

We observed frequent stillbirth in peroxiredoxin III (PrxIII) knockout maternal mice. Quantitative real time PCR (qRT-PCR) and Western-blot analysis revealed increased oxidative stress in placentas that were deficient in PrxIII. We did not find significant difference between PrxIII knockout maternal mice and wild-type littermates in hematological parameters, fetal number, and embryonic development. Nevertheless, we noticed enhanced expression of PrxI in erythrocytes of pregnant knockout mice. Our results provided in vivo evidence that PrxIII played a crucial role in placental antioxidant defense. Up-regulation of PrxI might provide a compensation that protected erythrocytes against oxidative damage.     

Stimulation of fetal hypothalamus induces uterine contractions in pregnant rats at term

The fetal brain is thought to have a role in the onset and progression of labor. Evidence also exists for fetal oxytocin release just before and during parturition. The present study examined whether activation of the fetal brain could induce uterine myometrial contractions through oxytocin receptors in the dam. Under urethane anesthesia, electrical stimulation of the hypothalamus of fetal rats that were still connected with the dams by an intact umbilical cord induced uterine contractions in term pregnant rats. Intraperitoneal injections of synthetic oxytocin in fetuses induced uterine contractions in the dams similar to those induced by electrical stimulation of the fetal hypothalamus. Maternal intravenous injections of an oxytocin antagonist immediately attenuated uterine contractions induced by fetal oxytocin injections and electrical stimulation of the fetal hypothalamus. These findings suggest the possibility that oxytocin released from the fetal hypothalamus is involved in parturition.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

Asymmetry in membrane protein sequence and structure: glycine outside rule

The growth in the number of known membrane protein structures enables us to conduct new statistical analyses that were not possible before. Using the recently developed Orientation of Protein in Membrane database that has explicit orientation information, here we address the asymmetry in sequence and structure of transmembrane helical proteins. We found that the Gly is markedly more populated at the outer side, the noncytoplasmic side, rather than the inner side, and that Lys, Arg, Leu, and Ile are biased toward the inner side. The asymmetry in Gly distribution is partly attributed to the fact that the coil is more populated in the outer side. Moreover, the frequency of Gly in the coil, but not in the helix, is weakly biased toward the outer side. The asymmetry in Gly distribution is confirmed by a genome-wide sequence-based analysis of membrane proteins accompanied by transmembrane helix prediction.     

Effects of retinoic acids on the dendritic morphology of cultured hippocampal neurons

Vitamin A-derived retinoic acids (RAs) are known to exert a variety of biological actions, including modulatory effects on cell differentiation and apoptosis. A recent study has demonstrated that 13- cis -RA and all- trans -RA suppressed neurogenesis in the dentate gyrus of the hippocampus in adult mice. The present experiments were performed to see whether 13- cis -RA and all- trans -RA could alter the dendritic morphology of cultured hippocampal neurons via RA receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). High doses of 13- cis -RA and all- trans -RA exerted a negative effect on the cultured hippocampal neurons, while a low dose of 13- cis -RA but not all- trans -RA caused a positive effect. The negative changes induced by 13- cis -RA and all- trans -RA were antagonized by RXR antagonists and RAR antagonists, respectively. The positive changes induced by a low dose of 13- cis -RA were blocked by both RXR antagonists and RAR antagonists. These results suggest that RAs at high concentrations cause a negative effect on the dendritic morphology of cultured hippocampal neurons through RA receptors, while RAs at low concentrations exert a positive influence on cultured hippocampal neurons.     

Genetic association study on in and around the APOE in late-onset Alzheimer disease in Japanese

The ?4 allele of APOE is a well-characterized genetic risk factor for late-onset Alzheimer disease (LOAD). Nevertheless, using high-density single nucleotide polymorphisms (SNPs), there have only been a few studies involving genetic association and linkage disequilibrium (LD) analyses of in and around the APOE. Here, we report fine mapping of a genomic region (about 200 kb) including the APOE in Japanese using 260 SNPs (mean intermaker distance, 0.77 kb). A case-control study demonstrated that 36 of these SNPs exhibited significance after adjustment for multiple testing. These SNPs are located in a genomic region including four genes, PVRL2, TOMM40, APOE and APOC1. Recombination rate estimation revealed that the associated region is firmly sandwiched between two recombination hotspots. Strong LD between these SNPs was observed (mean |D′| = 0.914). These data suggest that the three genes other than APOE, i.e. PVRL2, TOMM40 and APOC1, could also yield a predisposition to LOAD.