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Prevention of immune rejection without immunosuppression is the ultimate goal of transplant immunobiology. One way to achieve this in cellular transplantation, such as with islet transplantation, is to create a favorable local environment at the transplant site. In the current study, we found that C57BL/6 mice with streptozotocin-induced diabetes remained normoglycemic for >1 year after transplantation of BALB/c islets without immunosuppression when the inguinal subcutaneous white adipose tissue (ISWAT) was the site of transplantation and when the site was pretreated with basic fibroblast growth factor. Mechanistically, mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment was found to produce transforming growth factor-β (TGF-β), and prevention of islet allograft rejection could be achieved by cotransplantation with syngeneic MSCs isolated from the ISWAT after the treatment, which was abolished by anti–TGF-β antibody treatment. Importantly, TGF-β–producing cells remained present at the site of cotransplantation up to the end of observation period at 240 days after transplantation. These findings indicate that prevention of islet allograft rejection without immunosuppression is feasible with the use of syngeneic TGF-β–producing MSCs expanded in the ISWAT after the treatment with bFGF, providing a novel strategy for prevention of islet allograft rejection without immunosuppression.

Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with ∼730 mU mg−1 protein of specific activity, and the yield was ∼420 mU l−1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.

Prevention of immune rejection without immunosuppression is the ultimate goal of transplant immunobiology. One way to achieve this in cellular transplantation, such as with islet transplantation, is to create a favorable local environment at the transplant site. In the present study, we found that streptozotocin (STZ)-induced diabetic C57BL/6 mice remained normoglycemic for more than one year after transplantation of BALB/c islets without immunosuppression when the inguinal subcutaneous white adipose tissue (ISWAT) was the site of transplantation and when the site was pretreated with basic fibroblast growth factor. Mechanistically, mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment was found to produce TGFb and prevention of islet allograft rejection could be achieved by co-transplantation with syngeneic MSCs isolated from the ISWAT after the treatment, which was abolished by anti-TGFb antibody treatment. Importantly, TGFb-producing cells remained present at the site of co-transplantation for up to the end of observation period at 240 days after transplantation. These findings indicate that prevention of islet allograft rejection without immunosuppression is feasible with the use of syngeneic TGFb-producing MSCs expanded in the ISWAT after the treatment with bFGF, providing a novel strategy for prevention of islet allograft rejection without immunosuppression.

The Na+/Ca2+ exchanger type-1 (NCX1) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na+ and Ca2+. Vascular smooth muscle NCX1 plays an important role in intracellular Ca2+ homeostasis and Ca2+ signaling. We found that NCX1 was upregulated in the pulmonary arteries of mice exposed to chronic hypoxia (10% O2 for 4 weeks). Hence, we investigated the pathophysiological role of NCX1 in hypoxia-induced pulmonary arterial hypertension (PAH), using NCX1-heterozygous (NCX1+/−) mice, in which NCX1 expression is reduced by half, and SEA0400, a specific NCX1 inhibitor. NCX1+/− mice exhibited attenuation of hypoxia-induced PAH and right ventricular (RV) hypertrophy compared with wild-type mice. Furthermore, continuous administration of SEA0400 (0.5 mg/kg/day for 4 weeks) to wild-type mice by osmotic pumps significantly suppressed hypoxia-induced PAH and pulmonary vessel muscularization, with a slight reduction in RV hypertrophy. These findings indicate that the upregulation of NCX1 contributes to the development of hypoxia-induced PAH, suggesting that NCX1 inhibition might be a novel approach for the treatment of PAH.

We herein designed and synthesized amino-phosphine ligands 1a–c, which bear a P-chirogenic group, (R)-PtBuMe, and an N-group (NHR) on the pyridylene backbone, as a novel class of chiral ligands pos...

Fil: Numaga-Tomita, Takuro. National Institutes of Natural Sciences. National Institute for Physiological Sciences; Japón Fil: Numaga-Tomita, Takuro. National Institutes of Natural Sciences. Exploratory Research Center on Life and Living Systems; Japón Fil: Numaga-Tomita, Takuro. The Graduate University for Advanced Studies. School of Life Science. SOKENDAI; Japón Fil: Numaga-Tomita, Takuro. Shinshu University School of Medicine; Japón Fil: Shimauchi, Tsukasa. National Institutes of Natural Sciences. National Institute for Physiological Sciences; Japón Fil: Shimauchi, Tsukasa. National Institutes of Natural Sciences. Exploratory Research Center on Life and Living Systems; Japón Fil: Shimauchi, Tsukasa. Kyushu University. Graduate School of Pharmaceutical Sciences; Japón Fil: Shimauchi, Tsukasa. Kyushu University. Graduate School of Medical Sciences; Japón Fil: Kato, Yuri. Kyushu University. Graduate School of Pharmaceutical Sciences; Japón Fil: Nishiyama, Kazuhiro. Kyushu University. Graduate School of Pharmaceutical Sciences; Japón Fil: Nishimura, Akiyuki. National Institutes of Natural Sciences. National Institute for Physiological Sciences; Japón Fil: Nishimura, Akiyuki. National Institutes of Natural Sciences. Exploratory Research Center on Life and Living Systems; Japón Fil: Nishimura, Akiyuki. The Graduate University for Advanced Studies. School of Life Science. SOKENDAI; Japón Fil: Sakata, Kosuke. Kyushu University. Graduate School of Pharmaceutical Sciences; Japón Fil: Inada, Hiroyuki. National Institutes of Natural Sciences. National Institute for Physiological Sciences; Japón Fil: Kita, Satomi. Fukuoka University. Faculty of Medicine; Japón Fil: Kita, Satomi. Tokushima Bunri University. Faculty of Pharmaceutical Sciences; Japón Fil: Iwamoto, Takahiro. Fukuoka University. Faculty of Medicine; Japón Fil: Nabekura, Junichi. National Institutes of Natural Sciences. National Institute for Physiological Sciences; Japón Fil: Birnbaumer, Lutz. Research Triangle Park. National Institutes of Health. National Institute of Environmental Health Sciences; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Mori, Yasuo. Kyoto University. Graduate School of Engineering; Japón Fil: Nishida, Motohiro. National Institutes of Natural Sciences. National Institute for Physiological Sciences; Japón Fil: Nishida, Motohiro. National Institutes of Natural Sciences. Exploratory Research Center on Life and Living Systems; Japón Fil: Nishida, Motohiro. The Graduate University for Advanced Studies. School of Life Science. SOKENDAI; Japón Fil: Nishida, Motohiro. Kyushu University. Graduate School of Pharmaceutical Sciences; Japón Abstract: Background and Purpose: Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. Experimental Approach: Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. Key Results: Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. Conclusion and Implications: Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation.

A combination of iron(III) fluoride and 1,3-bis(2,6-diisopropylphenyl)imidazolin-2-ylidene (SIPr) catalyzes the high-yielding cross coupling of an electron-rich aryl chloride with an alkyl Grignard...

The first iron-catalysed enantioselective Suzuki-Miyaura coupling reaction has been developed. In the presence of catalytic amounts of FeCl2 and (R,R)-QuinoxP*, lithium arylborates are cross-coupled with tert-butyl α-bromopropionate in an enantioconvergent manner, enabling facile access to various optically active α-arylpropionic acids including several nonsteroidal anti-inflammatory drugs (NSAIDs) of commercial importance. (R,R)-QuinoxP* is specifically able to induce chirality when compared to analogous P-chiral ligands that give racemic products, highlighting the critical importance of transmetalation in the present asymmetric cross-coupling system.

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis. The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.

In this study, we assessed the regulation of transient receptor potential vanilloid 4 (TRPV4) promoting lymphangio/angiogenesis to improve the ischemic hindlimb animal model, and revealed that (1) a TRPV4 agonist improved the blood flow of ischemic hindlimbs by inducing both angiogenesis and lymphangiogenesis; (2) excessive TRPV4 expression was detected on lymphatic endothelial cells (LECs) in the ischemic hindlimb; and (3) hypoxic conditions promoted Ca2+ influx into LECs via TRPV4. It is considered that the upregulation of both lymphatic and blood vessels by activating TRPV4 would be a promising therapeutic strategy for peripheral artery disease.

The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.

Na+/Ca2+ exchangers (NCXs) are mainly expressed in the plasma membrane and exchange one Ca2+ for three Na+, depending on the electrochemical gradients across the plasma membrane. NCXs have three isoforms, NCX1–3, encoded by distinct genes in mammals. Here, we report that heterozygous mice lacking NCX1 (NCX1+/−) exhibit impaired amygdala-dependent cued fear memory. NCX1+/− mice showed significant impairment in fear-related behaviors measured with the elevated-plus maze, light-dark, open-field, and marble-burying tasks. In addition, NCX1+/− mice showed abnormality in cued fear memory but not in contextual fear memory in a fear-conditioning task. In immunohistochemical analyses, NCX1+/− mice had significantly increased number of c-Fos-positive cells in the lateral amygdala (LA) but not in the central amygdala following fear-related tone stimuli. c-Fos expression peaked at 1 h. In concordance with the aberrant fear-related behaviors in NCX1+/− mice, enhanced long-term potentiation was also observed in the LA of these mice. Furthermore, enhancement of CaMKII or CaMKIV activity in the LA was observed in NCX1+/− mice by immunoblot analyses. In contrast, CaMKII+/− but not CaMKIV−/− mice insufficiently exhibited tone-induced cued fear memory and there was no increase in the number of c-Fos-positive cells in the LA. Altogether, the increased CaMKII activity and consequent c-Fos expression likely account for the dysregulation of amygdala-dependent cued fear memory in NCX1+/− mice.

Na+/Ca2+ exchangers (NCXs) are expressed primarily in the plasma membrane of most cell types, where they mediate electrogenic exchange of one Ca2+ for three Na+ ions, depending on Ca2+ and Na+ electrochemical gradients across the membrane. Three mammalian NCX isoforms (NCX1, NCX2, and NCX3) are each encoded by a distinct gene. Here, we report that NCX2 and NCX3 protein and mRNA levels are relatively reduced in hippocampal CA1 of APP23 and APP-KI mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. Moreover, relative to controls, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in NCX2+/- mouse hippocampus but increased in hippocampus of NCX3+/- mice. NCX2 or NCX3 heterozygotes displayed impaired maintenance of hippocampal LTP, a phenotype that in NCX2+/- mice was correlated with elevated calcineurin activity and rescued by treatment with the calcineurin (CaN) inhibitor FK506. Likewise, FK506 treatment significantly restored impaired hippocampal LTP in APP-KI mice. Moreover, Ca2+ clearance after depolarization following high frequency stimulation was slightly delayed in hippocampal CA1 regions of NCX2+/- mice. Electron microscopy revealed relatively decreased synaptic density in CA1 of NCX2+/- mice, while the number of spines with perforated synapses in CA1 significantly increased in NCX3+/- mice. We conclude that memory impairment seen in NCX2+/- and NCX3+/- mice reflect dysregulated hippocampal CaMKII activity, which alters dendritic spine morphology, findings with implications for memory deficits seen in Alzheimer's disease model mice.

Background and Aims Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. Methods Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. Results Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. Conclusions Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.

Na+/Ca2+ exchanger (NCX) is mainly expressed in the plasma membrane and mediates electrogenical exchange of one Ca2+ for three Na+, depending on the electrochemical gradients across the plasma membrane. NCX has three different isoforms (NCX1, NCX2, NCX3) encoded by distinct genes in mammals. Here, we report that NCX2 and NCX3 protein levels are relatively reduced in hippocampal CA1 of Alzheimer's disease model mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. In immunoblot analyses, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in hippocampal CA1 of NCX2+/- mice compared to wild-type mice. By contrast, NCX2+/- mice was correlated with elevated calcineurin (CaN) activity and rescued by treatment with the calcineurin inhibitor FK506. Taken together, the imbalance of CaMKII and CaN activities with concomitant LTP impairment likely accounts for the learning disability observed in NCX2+/- mice.

Reported is the anti-selective carbosilylation of internal alkynes with silylborane and alkyl halides using a FeBr2 /DPPE catalyst system. The iron catalyst allows simultaneous introduction of a carbon electrophile and a silicon nucleophile to simple internal alkynes, including diaryl-, dialkyl-, and aryl/alkyl-substituted alkynes, in a highly stereoselective manner. Alkyl halides are applicable as the electrophiles, thereby enabling the synthesis of a variety of tetrasubstituted alkenylsilanes. In addition, syn-selective carbosilylation was achieved through stereoswitching, by using a silylborane having oxygen functionality on the silyl group. This novel iron-catalyzed carbosilylation is a useful tool for expedient synthesis of stereodefined multisubstituted olefins, a fundamental structural motif in organic chemistry.

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31+F4/80+ hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31+F4/80+ cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31+F4/80+ cells transdifferentiated into NG2/PDGFRβ/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.