CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2

The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr(401) in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr(401) region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly(45) was critical for binding to the EPOR but not to JAK2, while Leu(22) was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr(401), reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.     

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Functional polymorphisms of HSPA5: possible association with bipolar disorder

Altered endoplasmic reticulum stress (ER) response signaling is suggested in bipolar disorder. Previously, we preliminarily reported the genetic association of HSPA5 (GRP78/BiP) with bipolar disorder. Here, we extended our analysis by increasing the number of Japanese case-control samples and NIMH Genetics Initiative bipolar trio samples (NIMH trios), and also analyzed schizophrenia samples. In Japanese, nominally significant association of one haplotype was observed in extended samples of bipolar disorder but not in schizophrenia. In NIMH trios, no association was found in total samples. However, an exploratory analysis suggested that the other haplotype was significantly over-transmitted to probands only from the paternal side. The associated haplotype in Japanese or NIMH pedigrees shared three common polymorphisms in the promotor, which was found to alter promotor activity. These findings suggested promotor polymorphisms of HSPA5 may affect the interindividual variability of ER stress response and may confer a genetic risk factor for bipolar disorder.     

Variation in mutation rates caused by RB69pol fidelity mutants can be rationalized on the basis of their kinetic behavior and crystal structures

We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3′-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations.     

Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

TGF-β subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-β signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-β signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2^fl/fl;Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-β2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-β signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.     

The kinase inhibitor fasudil (HA-1077) reduces intimal hyperplasia through inhibiting migration and enhancing cell loss of vascular smooth muscle cells.

Smooth muscle cell (SMC) migration plays an important role in restenosis after angioplasty. Myosin phosphorylation is necessary for cell migration. Fasudil is an inhibitor of protein kinases, including myosin light chain kinase and Rho associated kinase, thereby inhibiting myosin phosphorylation, and it has been clinically used to prevent vasospasm following subarachnoid hemorrage. Based on these findings, we examined the anti-migrative action of fasudil. In SMC (SM-3), fasudil (1-100 microM) inhibited SMC migration in a dose-dependent manner (p < 0.001). Fasudil suppressed actin stress fiber formation dose dependently. In rabbit carotid artery, fasudil (10 mg/kg/day) markedly reduced intimal hyperplasia 14 days following balloon injury. Cell kinetic study showed that fasudil did not affect proliferation but enhanced cell loss in the media after injury. We concluded that fasudil reduced neointimal formation after balloon injury through both inhibiting migration and enhancing cell loss of medial SMC.     

Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Exogenous cytosine deaminase gene expression in Bifidobacterium breve I-53-8w for tumor-targeting enzyme/prodrug therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.     

Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris

The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6M urea and purified by urea-nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6M urea could be refolded rapidly by dilution with 50mM Tris-HCl (pH 7.8)-10mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1muM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.