母子分離ストレスが報酬探索行動に及ぼす影響と側坐核におけるドーパミンD1受容体のDNAのメチル化機構を介した発現変化について

Early-life stress has long-lasting effects on the stress response, emotions, and behavior throughout an individual’s life. Clinical reports have demonstrated that child abuse victims exhibit impairments in reward-associated behavior; yet, the mechanism for this effect remains unclear. Maternal separation (MS) or MS coupled with social isolation (SI) (MS + SI) is widely used as a model for early-life stress in rodent studies. We employed mice subjected to MS + SI to clarify the long-term effect of early-life stress on reward-seeking involving palatable foods by a conditioned place-preference (CPP) paradigm. Prior MS + SI experience decreased exploration time in a chocolate-paired compartment in adult female mice, but not in male mice. We then focused on the mesolimbic dopamine pathway associated with reward-seeking behavior and measured both mRNA and protein levels of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) and dopamine D1 and D2 receptors in the nucleus accumbens (NAc). MS + SI female mice had significantly lower D1 receptor mRNA and protein levels than controls, whereas the expression of TH and the D2 receptor was similar in the 2 groups. All mRNA and protein levels were unchanged in MS + SI male mice. When attempting to elucidate the mechanism underlying downregulation of the D1 receptor in the NAc of MS + SI females, we found hypermethylation of the Drd1a promoter region. These results suggest that early-life stress affects reward-seeking behavior in female mice, which may be associated with the downregulation of D1 receptor in the NAc via epigenetic modification of its promoter region.博士（医学）・甲第672号・平成29年6月28日Copyright © 2017 Elsevier B.V. All rights reserved

Chemical Visualization of an Attractant Peptide, LURE

The pollen tube attractant peptide LUREs of Torenia fournieri are diffusible peptides that attract pollen tubes in vitro. Here, we report a method enabling the direct visualization of a LURE peptide without inhibiting its attraction activity by conjugating it with the Alexa Fluor 488 fluorescent dye. After purifying and refolding the recombinant LURE2 with a polyhistidine tag, its amino groups were targeted for conjugation with the Alexa Fluor dye. Labeling of LURE2 was confirmed by its fluorescence and mass spectrometry. In our in vitro assay using gelatin beads, Alexa Fluor 488-labeled LURE2 appeared to have the same activity as unlabeled LURE2. Using the labeled LURE2, the relationship between the spatiotemporal change of distribution and activity of LURE2 was examined. LURE2 attracted pollen tubes when embedded in gelatin beads, but hardly at all when in agarose beads. Direct visualization suggested that the significant difference between these conditions was the retention of LURE2 in the gelatin bead, which might delay diffusion of LURE2 from the bead. Direct visualization of LURE peptide may open the way to studying the spatiotemporal dynamics of LURE in pollen tube attraction

網膜へのレーザー凝固により惹起される脈絡膜新生血管はヘッジホッグシグナル系因子を発現する。

Choroidal neovascularization is one of the major pathological changes in age-related macular degeneration, which causes devastating blindness in the elderly population. The molecular mechanism of choroidal neovascularization has been under extensive investigation, but is still an open question. We focused on sonic hedgehog signaling, which is implicated in angiogenesis in various organs. Laser-induced injuries to the mouse retina were made to cause choroidal neovascularization. We examined gene expression of sonic hedgehog, its receptors (patched1, smoothened, cell adhesion molecule down-regulated by oncogenes (Cdon) and biregional Cdon-binding protein (Boc)) and downstream transcription factors (Gli1-3) using real-time RT-PCR. At seven days after injury, mRNAs for Patched1 and Gli1 were upregulated in response to injury, but displayed no upregulation in control retinas. Immunohistochemistry revealed that Patched1 and Gli1 proteins were localized to CD31-positive endothelial cells that cluster between the wounded retina and the pigment epithelium layer. Treatment with the hedgehog signaling inhibitor cyclopamine did not significantly decrease the size of the neovascularization areas, but the hedgehog agonist purmorphamine made the areas significantly larger than those in untreated retina. These results suggest that the hedgehog-signaling cascade may be a therapeutic target for age-related macular degeneration.博士（医学）・甲第653号・平成28年7月8日Copyright © 2016 The Japan Society of Histochemistry and Cytochemistry（日本組織細胞化学会）J-STAGEへのリンク：http://doi.org/10.1267/ahc.1503

High Excitation Molecular Gas in the Galactic Center Loops; 12CO(J =2-1 and J =3-2) Observations

We have carried out 12CO(J =2-1) and 12CO(J =3-2) observations at spatial resolutions of 1.0-3.8 pc toward the entirety of loops 1 and 2 and part of loop 3 in the Galactic center with NANTEN2 and ASTE. These new results revealed detailed distributions of the molecular gas and the line intensity ratio of the two transitions, R3-2/2-1. In the three loops, R3-2/2-1 is in a range from 0.1 to 2.5 with a peak at ~ 0.7 while that in the disk molecular gas is in a range from 0.1 to 1.2 with a peak at 0.4. This supports that the loops are more highly excited than the disk molecular gas. An LVG analysis of three transitions, 12CO J =3-2 and 2-1 and 13CO J =2-1, toward six positions in loops 1 and 2 shows density and temperature are in a range 102.2 - 104.7 cm-3 and 15-100 K or higher, respectively. Three regions extended by 50-100 pc in the loops tend to have higher excitation conditions as characterized by R3-2/2-1 greater than 1.2. The highest ratio of 2.5 is found in the most developed foot points between loops 1 and 2. This is interpreted that the foot points indicate strongly shocked conditions as inferred from their large linewidths of 50-100 km s-1, confirming the suggestion by Torii et al. (2010b). The other two regions outside the foot points suggest that the molecular gas is heated up by some additional heating mechanisms possibly including magnetic reconnection. A detailed analysis of four foot points have shown a U shape, an L shape or a mirrored-L shape in the b-v distribution. It is shown that a simple kinematical model which incorporates global rotation and expansion of the loops is able to explain these characteristic shapes.Comment: 59 pages, accepted to PAS

Temperature and Density in the Foot Points of the Molecular Loops in the Galactic Center; Analysis of Multi-J Transitions of 12CO(J=1-0, 3-2, 4-3, 7-6), 13CO(J=1-0) and C18O(J=1-0)

Fukui et al. (2006) discovered two molecular loops in the Galactic center and argued that the foot points of the molecular loops, two bright spots at both loops ends, represent the gas accumulated by the falling motion along the loops, subsequent to magnetic flotation by the Parker instability. We have carried out sensitive CO observations of the foot points toward l=356 deg at a few pc resolution in the six rotational transitions of CO; 12CO(J=1-0, 3-2, 4-3, 7-6), 13CO(J=1-0) and C18O(J=1-0). The high resolution image of 12CO (J=3-2) has revealed the detailed distribution of the high excitation gas including U shapes, the outer boundary of which shows sharp intensity jumps accompanying strong velocity gradients. An analysis of the multi-J CO transitions shows that the temperature is in a range from 30-100 K and density is around 10^3-10^4 cm^-3, confirming that the foot points have high temperature and density although there is no prominent radiative heating source such as high mass stars in or around the loops. We argue that the high temperature is likely due to the shock heating under C-shock condition caused by the magnetic flotation. We made a comparison of the gas distribution with theoretical numerical simulations and note that the U shape is consistent with numerical simulations. We also find that the region of highest temperature of ~100 K or higher inside the U shape corresponds to the spur having an upward flow, additionally heated up either by magnetic reconnection or bouncing in the interaction with the narrow neck at the bottom of the U shape. We note these new findings further reinforce the magnetic floatation interpretation.Comment: 40 pages, 23 figures, accepted by PASJ on Vol.62 No.

Dysbindin Regulates the Transcriptional Level of Myristoylated Alanine-Rich Protein Kinase C Substrate via the Interaction with NF-YB in Mice Brain

BACKGROUND: An accumulating body of evidence suggests that Dtnbp1 (Dysbindin) is a key susceptibility gene for schizophrenia. Using the yeast-two-hybrid screening system, we examined the candidate proteins interacting with Dysbindin and revealed one of these candidates to be the transcription factor NF-YB. METHODS: We employed an immunoprecipitation (IP) assay to demonstrate the Dysbindin-NF-YB interaction. DNA chips were used to screen for altered expression of genes in cells in which Dysbindin or NF-YB was down regulated, while Chromatin IP and Reporter assays were used to confirm the involvement of these genes in transcription of Myristoylated alanine-rich protein kinase C substrate (MARCKS). The sdy mutant mice with a deletion in Dysbindin, which exhibit behavioral abnormalities, and wild-type DBA2J mice were used to investigate MARCKS expression. RESULTS: We revealed an interaction between Dysbindin and NF-YB. DNA chips showed that MARCKS expression was increased in both Dysbindin knockdown cells and NF-YB knockdown cells, and Chromatin IP revealed interaction of these proteins at the MARCKS promoter region. Reporter assay results suggested functional involvement of the interaction between Dysbindin and NF-YB in MARCKS transcription levels, via the CCAAT motif which is a NF-YB binding sequence. MARCKS expression was increased in sdy mutant mice when compared to wild-type mice. CONCLUSIONS: These findings suggest that abnormal expression of MARCKS via dysfunction of Dysbindin might cause impairment of neural transmission and abnormal synaptogenesis. Our results should provide new insights into the mechanisms of neuronal development and the pathogenesis of schizophrenia