GNAS and KRAS Mutations are Common in Intraductal Papillary Neoplasms of the Bile Duct

Intraductal papillary neoplasms of the bile duct (IPNB) shows favorable prognosis and is regarded as a biliary counterpart of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Although activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of IPMNs of the pancreas, there have been few studies on GNAS mutations in IPNBs. This study investigates the status of GNAS and KRAS mutations and their association with clinicopathological factors in IPNBs. We examined the status of GNAS mutation at codon 201 and KRAS mutation at codon 12&13, degree of mucin production and immunohistochemical expressions of MUC mucin core proteins in 29 patients (M/F = 15/14) with IPNB in intrahepatic and perihilar bile ducts (perihilar IPNB) and 6 patients (M/F = 5/1) with IPNB in distal bile ducts (distal IPNB). GNAS mutations and KRAS mutations were detected in 50% and 46.2% of IPNBs, respectively. There was no significant correlation between the status of GNAS mutation and clinicopathological factors in IPNBs, whereas, the status of KRAS mutation was significantly inversely correlated with the degree of MUC2 expression in IPNBs (p<0.05). All IPNBs with GNAS mutation only showed high-mucin production. Degree of mucin production was significantly higher in perihilar IPNBs than distal IPNBs (p<0.05). MUC2 and MUC5AC expression was significantly higher in IPNBs with high-mucin production than those with low-mucin production (p<0.01 and p<0.05, respectively). In conclusions, this study firstly disclosed frequent GNAS mutations in IPNBs, similarly to IPMNs. This may suggest a common histopathogenesis of IPNBs and IPMNs. The status of KRAS mutations was inversely correlated to MUC2 expression and this may suggest heterogeneous properties of IPNBs. IPNBs with high-mucin production are characterized by perihilar location and high expression of MUC2 and MUC5AC, irrespective of the status of GNAS and KRAS mutations.     

Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: A culture study

Cellular senescence is reportedly involved in cholangiopathy in primary biliary cirrhosis and oxidative stress is proposed as a pathogenetic factor in biliary epithelial cells (BECs). This study investigated the involvement of proinflammatory cytokines (IFN-β, IFN-γ and TNF-α) and ataxia telangiectasia-mutated (ATM)/p53/ p21^WAF1/Cip1 pathway with respect to oxidative stress in cellular senescence of BECs. H₂O₂ treatment (oxidative stress) induced phosphorylation (activation) of ATM and p53 and also p21^WAF1/Cip1 expression in BECs. Treatment with inflammatory cytokines generated reactive oxygen species (ROS) in cultured BECs followed by activation of the ATM/p53/p21^WAF1/Cip1 pathway and the induction of cellular senescence. Pre-treatment with ATM inhibitor (2-aminopurine) and antioxidant (N-acetylcysteine) significantly blocked the cellular senescence of BECs induced by oxidative stress or inflammatory cytokines. In conclusion, proinflamamtory cytokines induce ROS generation and activate the ATM/p53/p21^WAF1/Cip1 pathway, followed by biliary epithelial senescence. This senescent process may be involved in the development of destructive cholangiopathy in humans.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.     

Pathway for the movement of water and cryoprotectants in bovine oocytes and embryos

The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.     

pH-responsive switching of near-infrared absorption of a diradical complex of Pt and 3,4-diaminobenzoate formed in aqueous solutions

In alkaline aqueous solutions, 3,4-diaminobenzoate (H₂(²L^PDA)⁻) reacts with Pt^II to form a 1:2 (Pt:L) complex that intensely absorbs near-infrared (NIR) light at 713 nm (ε = 8.0 × 10⁴ M⁻¹ cm⁻¹). The absorption disappeared at pH < 3 (in DMSO), showing pH-responsive switching of the NIR absorption. By comparing the NIR-absorbing behavior of this complex to that of a complex, [Pt^II(¹L^ISQ)₂]²⁻, containing the analogous phenylenediamine ligand [(¹L^ISQ)²⁻ = o-diiminobenzosemiquinonate radical], the complex can be formulated as [Pt^II(²L^ISQ)₂]²⁻. The assignment of the entity was consistent with the redox and spectroelectrochemical behaviors and electronic spin resonance (ESR) spectroscopy. First, one-electron oxidation of [Pt^II(²L^ISQ)₂]²⁻ formed an ESR-silent complex assignable to the dimeric complex [{Pt^II(²L^ISQ)(²L^IBQ)}₂]²⁻ [(²L^IBQ) = o-iminobenzoquinone form] in which the two radical centers at were antiferromagnetically coupled. Second, the one-electron reduced complex of [Pt^II(²L^ISQ)₂]²⁻ exhibited an ESR signal attributed to [Pt^II(²L^ISQ)(²L^PDA)]³⁻; 34% of the electronic spin was located at the Pt^II center rather than on the moiety. The pH-responsive switching-off of the NIR absorption was thus rationally explained by oxidation of [Pt^II(²L^ISQ)₂]²⁻ to [{Pt^II(²L^ISQ)(²L^IBQ)}₂]²⁻ by the increase of the rest potential of the solution in the lower pH region.     

Evolution of motor innervation to vertebrate fins and limbs

The evolution and diversification of vertebrate behaviors associated with locomotion depend highly on the functional transformation of paired appendages. Although the evolution of fins into limbs has long been a focus of interest to scientists, the evolution of neural control during this transition has not received much attention. Recent studies have provided significant progress in the understanding of the genetic and developmental bases of the evolution of fin/limb motor circuitry in vertebrates. Here we compare the organization of the motor neurons in the spinal cord of various vertebrates. We also discuss recent advances in our understanding of these events and how they can provide a mechanistic explanation for the evolution of fin/limb motor circuitry in vertebrates.     

Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.     

Regulation of PIP5K activity by Arf6 and its physiological significance

The phospholipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4-phosphate to generate the pleiotropic phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2) ]. To date, three mammalian PIP5K isozymes, α, β, and γ, and several splicing variants of the γ isozyme have been identified. These PIP5K isozymes and PIP5Kγ variants play critical roles in various cellular functions through their product PI(4,5)P(2) . The small GTPase Arf6 is one of the key activators of PIP5K. Increasing evidence suggests that PIP5K functions as a downstream effector of Arf6 to regulate a wide variety of cellular functions, such as exocytosis, endocytosis, endosomal recycling, membrane ruffle formation, immune response, and bacterial invasion. In this review, we place our focus on the recent advances in Arf6/PIP5K signaling and its linkage to cellular functions.     

Sphingosylphosphorylcholine and lysosulfatide have inverse regulatory functions in monocytic cell differentiation into macrophages

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G_i/o, whereas the SPC-induced calcium influx was dependent on G_q. Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G_i/o and G_q. These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms.