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Find video protocols related to scientific articles indexed in Pubmed.
Novel mechanisms of endothelial mechanotransduction.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 10-09-2014
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Atherosclerosis is a focal disease that develops preferentially where nonlaminar, disturbed blood flow occurs, such as branches, bifurcations, and curvatures of large arteries. Endothelial cells sense and respond differently to disturbed flow compared with steady laminar flow. Disturbed flow that occurs in so-called atheroprone areas activates proinflammatory and apoptotic signaling, and this results in endothelial dysfunction and leads to subsequent development of atherosclerosis. In contrast, steady laminar flow as atheroprotective flow promotes expression of many anti-inflammatory genes, such as Kruppel-like factor 2 and endothelial nitric oxide synthase and inhibits endothelial inflammation and athrogenesis. Here we will discuss that disturbed flow and steady laminar flow induce pro- and antiatherogenic events via flow type-specific mechanotransduction pathways. We will focus on 5 mechanosensitive pathways: mitogen-activated protein kinases/extracellular signal-regulated kinase 5/Kruppel-like factor 2 signaling, extracellular signal-regulated kinase/peroxisome proliferator-activated receptor signaling, and mechanosignaling pathways involving SUMOylation, protein kinase C-?, and p90 ribosomal S6 kinase. We think that clarifying regulation mechanisms between these 2 flow types will provide new insights into therapeutic approaches for the prevention and treatment of atherosclerosis.
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Identification of activators of ERK5 transcriptional activity by high-throughput screening and the role of endothelial ERK5 in vasoprotective effects induced by statins and antimalarial agents.
J. Immunol.
PUBLISHED: 09-03-2014
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Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5- and Kruppel-like factor 2/4-independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection.
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Cytotoxic T Lymphocytes Block Tumor Growth Both by Lytic Activity and IFN?-Dependent Cell-Cycle Arrest.
Cancer Immunol Res
PUBLISHED: 08-15-2014
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To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors and B16 melanoma cells as targets. In addition to upregulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell-cycle-promoting genes were downregulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing a fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) and performed CTL therapy in mice bearing B16-fucci tumors. Three days after CTL transfer, we observed diffuse infiltration of CTLs into the tumor with a large number of tumor cells arrested at the G1 phase of the cell cycle, and the presence of spotty apoptotic or necrotic areas. Thus, tumor growth suppression was largely dependent on G1 cell-cycle arrest rather than killing by CTLs. Neutralizing antibody to IFN? prevented both tumor growth inhibition and G1 arrest. The mechanism of G1 arrest involved the downregulation of S-phase kinase-associated protein 2 (Skp2) and the accumulation of its target cyclin-dependent kinase inhibitor p27 in the B16-fucci tumor cells. Because tumor-infiltrating CTLs are far fewer in number than the tumor cells, we propose that CTLs predominantly regulate tumor growth via IFN?-mediated profound cytostatic effects rather than via cytotoxicity. This dominance of G1 arrest over other mechanisms may be widespread but not universal because IFN? sensitivity varied among tumors. Cancer Immunol Res; 1-11. ©2014 AACR.
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Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds.
Plant Cell Rep.
PUBLISHED: 08-15-2014
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A platform of gene silencing by amiRNA had been established in fertile transgenic soybean. We demonstrated that knockdown of storage protein shifted the distribution of nitrogen sources in soybean seeds. Artificial microRNAs (amiRNAs) were designed using the precursor sequence of the endogenous soybean (Glycine max L. Merrill) miRNA gma-miR159a and expressed in transgenic soybean plants to suppress the biosynthesis of 7S globulin, which is one of the major storage proteins. Seed-specific expression of these amiRNAs (amiR-7S) resulted in a strong suppression of 7S globulin subunit genes and decreased accumulation of the 7S globulin subunits in seeds. Thus, the results demonstrate that a platform for gene silencing by amiRNA was first developed in fertile transgenic soybean plants. There was no difference in nitrogen, carbon, and lipid contents between amiR-7S and control seeds. Four protein fractions were collected from defatted mature seeds on the basis of solubility at different pH to examine the distribution of nitrogen sources and compensatory effects. In the whey and lipophilic fractions, nitrogen content was similar in amiR-7S and control seeds. Nitrogen content was significantly decreased in the major soluble protein fraction and increased in the residual fraction (okara) of the amiR-7S seeds. Amino acid analysis revealed that increased nitrogen compounds in okara were proteins or peptides rather than free amino acids. Our study indicates that the decrease in 7S globulin subunits shifts the distribution of nitrogen sources to okara in transgenic soybean seeds.
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Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses.
J. Immunol.
PUBLISHED: 07-14-2014
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Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.
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Decreased levels of inflammatory cytokines in immunoglobulin-resistant Kawasaki disease after plasma exchange.
Cytokine
PUBLISHED: 06-26-2014
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The pathogenesis of coronary artery aneurysm (CAA) formation in Kawasaki disease (KD) remains unknown. However, inflammatory cytokines are thought to play an important role in KD. Patients with intravenous immunoglobulin (IVIG)-resistant KD are more likely to develop CAA. For such refractory patients, steroids and emerging infliximab (IFX) are used; however, further verification is required for their efficacy and safety. Plasma exchange (PE), which removes various inflammatory cytokines, has been used in Japan for over 15years to prevent CAA in IVIG-resistant KD patients. The sequential change in inflammatory cytokines during the time course of PE has yet to be investigated. In this study, we measured plasma levels of 13 cytokines in nine children with IVIG-resistant KD before the start of PE (day 0: D0), as well as at 1 or 2days (D1/2), and 4 or 5days (D4/5) after starting PE. The median age of onset was 8months (range: 3-53months). Before PE, patients were treated with IVIG (median dose: 4g/kg, range: 3-4g/kg). The median starting period of PE was 8days after the onset of fever (range: 6-21days), while its duration was 3days (range: 2-5days). Among the 13 cytokines, interleukin-6, tumor necrosis factor-?, tumor necrosis factor receptor I (TNFR1), TNFR2, granulocyte colony-stimulating factor, and IL-17 were significantly lower at D4/5 compared with D0 and/or D1/2, reflecting the potential central efficacy of PE. While three patients developed moderate CAA, their condition regressed within 1year. The removal of inflammatory cytokines could be the central efficacy of PE against refractory KD.
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Tracking of intertissue migration reveals the origins of tumor-infiltrating monocytes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-13-2014
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Myeloid cells such as monocytes and monocyte-derived macrophages promote tumor progression. Recent reports suggest that extramedullary hematopoiesis sustains a sizable reservoir of tumor-infiltrating monocytes in the spleen. However, the influence of the spleen on tumor development and the extent to which spleen monocytes populate the tumor relative to bone marrow (BM) monocytes remain controversial. Here, we used mice expressing the photoconvertible protein Kikume Green-Red to track the redistribution of monocytes from the BM and spleen, and mice expressing fluorescent ubiquitination-based cell-cycle indicator proteins to monitor active hematopoiesis in these tissues. In mice bearing late-stage tumors, the BM, besides being the major site of monocyte production, supplied the expansion of the spleen reservoir, replacing 9% of spleen monocytes every hour. Deployment of monocytes was equally rapid from the BM and the spleen. However, BM monocytes were younger than those in the spleen and were 2.7 times more likely to migrate into the tumor from the circulation. Partly as a result of this intrinsic difference in migration potential, spleen monocytes made only a minor contribution to the tumor-infiltrating monocyte population. At least 27% of tumor monocytes had traveled from the BM in the last 24 h, compared with only 2% from the spleen. These observations highlight the importance of the BM as the primary hematopoietic tissue and monocyte reservoir in tumor-bearing mice, despite the changes that occur in the spleen monocyte reservoir during tumor development.
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ERK5 activation in macrophages promotes efferocytosis and inhibits atherosclerosis.
Circulation
PUBLISHED: 04-28-2014
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Efferocytosis is a process by which dead and dying cells are removed by phagocytic cells. Efferocytosis by macrophages is thought to curb the progression of atherosclerosis, but the mechanistic insight of this process is lacking.
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Shear stress and atherosclerosis.
Mol. Cells
PUBLISHED: 04-03-2014
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Hemodynamic shear stress, the frictional force acting on vascular endothelial cells, is crucial for endothelial homeostasis under normal physiological conditions. When discussing blood flow effects on various forms of endothelial (dys)function, one considers two flow patterns: steady laminar flow and disturbed flow because endothelial cells respond differently to these flow types both in vivo and in vitro. Laminar flow which exerts steady laminar shear stress is atheroprotective while disturbed flow creates an atheroprone environment. Emerging evidence has provided new insights into the cellular mechanisms of flow-dependent regulation of vascular function that leads to cardiovascular events such as atherosclerosis, atherothrombosis, and myocardial infarction. In order to study effects of shear stress and different types of flow, various models have been used. In this review, we will summarize our current views on how disturbed flow-mediated signaling pathways are involved in the development of atherosclerosis.
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Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod-induced psoriasis.
J. Immunol.
PUBLISHED: 03-24-2014
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Psoriasis is an inflammatory skin disease with accelerated epidermal cell turnover. Neutrophil accumulation in the skin is one of the histological characteristics of psoriasis. However, the precise mechanism and role of neutrophil infiltration remain largely unknown. In this article, we show that orchestrated action of CXCR2 and leukotriene B4 receptor BLT1 plays a key role in neutrophil recruitment during the development of imiquimod (IMQ)-induced psoriatic skin lesions in mice. Depletion of neutrophils with anti-Ly-6G Ab ameliorated the disease severity, along with reduced expression of proinflammatory cytokine IL-1? in the skin. Furthermore, CXCR2 and BLT1 coordinately promote neutrophil infiltration into the skin during the early phase of IMQ-induced inflammation. In vitro, CXCR2 ligands augment leukotriene B4 production by murine neutrophils, which, in turn, amplifies chemokine-mediated neutrophil chemotaxis via BLT1 in autocrine and/or paracrine manners. In agreement with the increased IL-19 expression in IMQ-treated mouse skin, IL-1? markedly upregulated expression of acanthosis-inducing cytokine IL-19 in human keratinocytes. We propose that coordination of chemokines, lipids, and cytokines with multiple positive feedback loops might drive the pathogenesis of psoriasis and, possibly, other inflammatory diseases as well. Interference to this positive feedback or its downstream effectors could be targets of novel anti-inflammatory treatment.
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Inexplicably female-biased sex ratios in Melittobia wasps.
Evolution
PUBLISHED: 01-06-2014
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The sex ratio behavior of parasitoid wasps in the genus Melittobia is scandalous. In contrast to the prediction of Hamilton's local mate competition theory, and the behavior of numerous other species, their extremely female-biased sex ratios (1-5% males) change little in response to the number of females that lay eggs on a patch. We examined the mating structure and fitness consequences of adjusting the sex ratio in M. australica and found that (1) the rate of inbreeding did not differ from that expected with random mating within each patch; (2) the fitness of females that produced less female-biased sex ratios (10 or 20% males) was greater than that of females who produced the sex ratio normally observed in M. australica. These results suggest that neither assortative mating nor asymmetrical competition between males can explain the extreme sex ratios. More generally, the finding that the sex ratios produced by females led to a decrease in their fitness suggests that the existing theory fails to capture a key aspect of the natural history of Melittobia, and emphasizes the importance of examining the fitness consequences of different sex ratio strategies, not only whether observed sex ratios correlate with theoretical predictions.
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Natural variation in the genes responsible for maturity loci E1, E2, E3 and E4 in soybean.
Ann. Bot.
PUBLISHED: 11-26-2013
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Background and AimsThe timing of flowering has a direct impact on successful seed production in plants. Flowering of soybean (Glycine max) is controlled by several E loci, and previous studies identified the genes responsible for the flowering loci E1, E2, E3 and E4. However, natural variation in these genes has not been fully elucidated. The aims of this study were the identification of new alleles, establishment of allele diagnoses, examination of allelic combinations for adaptability, and analysis of the integrated effect of these loci on flowering.MethodsThe sequences of these genes and their flanking regions were determined for 39 accessions by primer walking. Systematic discrimination among alleles was performed using DNA markers. Genotypes at the E1-E4 loci were determined for 63 accessions covering several ecological types using DNA markers and sequencing, and flowering times of these accessions at three sowing times were recorded.Key ResultsA new allele with an insertion of a long interspersed nuclear element (LINE) at the promoter of the E1 locus (e1-re) was identified. Insertion and deletion of 36 bases in the eighth intron (E2-in and E2-dl) were observed at the E2 locus. Systematic discrimination among the alleles at the E1-E3 loci was achieved using PCR-based markers. Allelic combinations at the E1-E4 loci were found to be associated with ecological types, and about 62-66 % of variation of flowering time could be attributed to these loci.ConclusionsThe study advances understanding of the combined roles of the E1-E4 loci in flowering and geographic adaptation, and suggests the existence of unidentified genes for flowering in soybean.
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Insulin activates RSK (p90 ribosomal S6 kinase) to trigger a new negative feedback loop that regulates insulin signaling for glucose metabolism.
J. Biol. Chem.
PUBLISHED: 09-13-2013
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We previously demonstrated that the mTORC1/S6K1 pathway is activated by insulin and nutrient overload (e.g. amino acids (AA)), which leads to the inhibition of the PI3K/Akt pathway via the inhibitory serine phosphorylation of IRS-1, notably on serine 1101 (Ser-1101). However, even in the absence of AA, insulin can still promote IRS-1 Ser-1101 phosphorylation by other kinases that remain to be fully characterized. Here, we describe a new negative regulator of IRS-1, the p90 ribosomal S6 kinase (RSK). Computational analyses revealed that Ser-1101 within IRS-1 falls into the consensus motif of RSK. Moreover, recombinant RSK phosphorylated IRS-1 C-terminal fragment on Ser-1101, which was prevented by mutations of this site or when a kinase-inactive mutant of RSK was used. Using antibodies directed toward the phosphorylation sites located in the activation segment of RSK (Ser-221 or Ser-380), we found that insulin activates RSK in L6 myocytes in the absence of AA overload. Inhibition of RSK using either the pharmacological inhibitor BI-D1870 or after adenoviral expression of a dominant negative RSK1 mutant (RSK1-DN) showed that RSK selectively phosphorylates IRS-1 on Ser-1101. Accordingly, expression of the RSK1-DN mutant in L6 myocytes and FAO hepatic cells improved insulin action on glucose uptake and glucose production, respectively. Furthermore, RSK1 inhibition prevented insulin resistance in L6 myocytes chronically exposed to high glucose and high insulin. These results show that RSK is a novel regulator of insulin signaling and glucose metabolism and a potential mediator of insulin resistance, notably through the negative phosphorylation of IRS-1 on Ser-1101.
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Development of a cryogenic load frame for the neutron diffractometer at Takumi in Japan Proton Accelerator Research Complex.
Rev Sci Instrum
PUBLISHED: 07-05-2013
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To prepare for projects such as the Large Hadron Collider upgrade, International Thermonuclear Experimental Reactor and Demonstration reactor, it is important to form a clear understanding of stress-strain properties of the materials that make up superconducting magnets. Thus, we have been studying the mechanical properties of superconducting wires using neutron diffraction measurements. To simulate operational conditions such as temperature, stress, and strain, we developed a cryogenic load frame for stress-strain measurements of materials using a neutron diffractometer at Japan Proton Accelerator Research Complex (J-PARC) Takumi beam line. The maximum load that can be applied to a sample using an external driving machine is 50 kN. Using a Gifford-MacMahon cryocooler, samples can be measured down to temperatures below 10 K when loaded. In the present paper, we describe the details of the cryogenic load frame with its test results by using type-304 stainless steel wire.
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Cardiac nuclear high mobility group box 1 prevents the development of cardiac hypertrophy and heart failure.
Cardiovasc. Res.
PUBLISHED: 05-25-2013
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High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload.
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Adoptive cytotoxic T lymphocyte therapy triggers a counter-regulatory immunosuppressive mechanism via recruitment of myeloid-derived suppressor cells.
Int. J. Cancer
PUBLISHED: 05-18-2013
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Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T-cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel-1-specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor-bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN-?, exerted anti-tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs). Notably, CD11b(+) Gr1(int) Ly6G(-) Ly6C(+) monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen-specific CD8(+) T cells. The anti-tumor activity of the adoptively-transferred CTLs and the accumulation of MDSCs both depended on IFN-? production on recognition of tumor antigens by the former. In CCR2(-/-) mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter-regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment-induced recruitment of these MDSCs would improve the efficacy of immunotherapy.
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Functional modulations in brain activity for the first and second music: A comparison of high- and low-proficiency bimusicals.
Neuropsychologia
PUBLISHED: 05-15-2013
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Bilingual studies have shown that brain activities for first (L1) and second (L2) languages are influenced by L2 proficiency. Does proficiency with a second musical system (M2) influence bimusical brains in a manner similar to that of bilingual brains? Our magnetoencephalography study assessed the influence of M2 proficiency on the spatial, strength, and temporal properties of brain activity in a musical syntactic-processing task (i.e., tonal processing) involving first (M1) and second (M2) music systems. Two bimusical groups, differing in M2 proficiency (high, low), listened to melodies from both their M1 and M2 musical cultures. All melodies ended with a tonally consistent or inconsistent tone. In both groups, tonal deviations in both M1 and M2 elicited magnetic early right anterior negativities (mERANs) that were generated from brain areas around the inferior frontal gyrus (IFG). We also analyzed the dipole locations, dipole strengths, and peak latencies of mERAN. Results revealed: (a) the distances between dipole locations for M1 and M2 were shorter in the M2 high-proficiency group than in the M2 low-proficiency group; (b) the dipole strengths were greater in the high than the low group; (c) the peak latencies of M2 were shorter in the high than low group. The dipole location results were consistent with those from bilingual studies in that the distances between the (left) IFG peak activations for L1 and L2 syntactic processing shortened as L2 proficiency increased. The parallel results for bimusicals and bilinguals suggest that the functional changes induced by proficiency in a second (linguistic or musical) system are defined by domain-general neural constraints.
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Successful remission induced by plasma exchange combined with leukocytapheresis against refractory systemic juvenile idiopathic arthritis.
Eur. J. Pediatr.
PUBLISHED: 05-10-2013
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Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory cytokine-related disorder associated with overproduction of interleukin (IL)-6, IL-1?, and IL-18. Macrophage activation syndrome (MAS) is a critical and lethal complication of sJIA. Therefore, prompt induction of remission in the active phase of sJIA is important for prevention of MAS. However, treatment of corticosteroid-resistant sJIA is still challenging. We propose a new strategy to induce remission in the active phase of sJIA. A 7-year-old boy with new-onset sJIA was treated with oral prednisolone, methylprednisolone pulse therapy, and parenteral cyclosporine, but he continuously showed remittent high fever, arthralgia, skin rash, and elevation of inflammatory markers, including hyperferritinemia. Because of impending MAS, plasma exchange was started, but he was resistant to plasma exchange. Therefore, we combined leukocytapheresis with PE. After five PE and two leukocytapheresis sessions, he finally achieved remission in accordance with a reduction in inflammatory cytokines. Elevated serum tumor necrosis factor-?, interferon-?, and IL-12 levels returned to normal 10 days after initiating plasma exchange combined with leukocytapheresis, with a reduction in white blood cell count. In conclusion, plasma exchange combined with leukocytapheresis is a new alternative option to induce remission for patients with methylprednisolone pulse therapy- or cyclosporine-refractory sJIA.
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Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemia-reperfusion injury.
J. Mol. Cell. Cardiol.
PUBLISHED: 04-22-2013
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Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (?-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates ?-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.
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Late development of coronary artery abnormalities could be associated with persistence of non-fever symptoms in Kawasaki disease.
Pediatr Rheumatol Online J
PUBLISHED: 04-16-2013
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Persistent fever after intravenous immunoglobulin (IVIG) is considered to be a major criterion of IVIG resistance in Kawasaki disease (KD), and a risk factor for the development of coronary artery abnormalities (CAA). However, the importance of persistent non-fever symptoms after defervescence has not yet been investigated. We examined the relationship between persistent non-fever symptoms and CAA in KD.
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Coordinated changes in DNA methylation in antigen-specific memory CD4 T cells.
J. Immunol.
PUBLISHED: 03-18-2013
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Memory CD4(+) T cells are central regulators of both humoral and cellular immune responses. T cell differentiation results in specific changes in chromatin structure and DNA methylation of cytokine genes. Although the methylation status of a limited number of gene loci in T cells has been examined, the genome-wide DNA methylation status of memory CD4(+) T cells remains unexplored. To further elucidate the molecular signature of memory T cells, we conducted methylome and transcriptome analyses of memory CD4(+) T cells generated using T cells from TCR-transgenic mice. The resulting genome-wide DNA methylation profile revealed 1144 differentially methylated regions (DMRs) across the murine genome during the process of T cell differentiation, 552 of which were associated with gene loci. Interestingly, the majority of these DMRs were located in introns. These DMRs included genes such as CXCR6, Tbox21, Chsy1, and Cish, which are associated with cytokine production, homing to bone marrow, and immune responses. Methylation changes in memory T cells exposed to specific Ag appeared to regulate enhancer activity rather than promoter activity of immunologically relevant genes. In addition, methylation profiles differed between memory T cell subsets, demonstrating a link between T cell methylation status and T cell differentiation. By comparing DMRs between naive and Ag-specific memory T cells, this study provides new insights into the functional status of memory T cells.
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Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean.
BMC Plant Biol.
PUBLISHED: 03-02-2013
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Absence of or low sensitivity to photoperiod is necessary for short-day crops, such as rice and soybean, to adapt to high latitudes. Photoperiod insensitivity in soybeans is controlled by two genetic systems and involves three important maturity genes: E1, a repressor for two soybean orthologs of Arabidopsis FLOWERING LOCUS T (GmFT2a and GmFT5a), and E3 and E4, which are phytochrome A genes. To elucidate the diverse mechanisms underlying photoperiod insensitivity in soybean, we assessed the genotypes of four maturity genes (E1 through E4) in early-flowering photoperiod-insensitive cultivars and their association with post-flowering responses.
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Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.
Am. J. Pathol.
PUBLISHED: 02-15-2013
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Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially in vivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.
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De-SUMOylation enzyme of sentrin/SUMO-specific protease 2 regulates disturbed flow-induced SUMOylation of ERK5 and p53 that leads to endothelial dysfunction and atherosclerosis.
Circ. Res.
PUBLISHED: 02-04-2013
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Disturbed flow induces proinflammatory and apoptotic responses in endothelial cells, causing them to become dysfunctional and subsequently proatherogenic.
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The evolution of male-female sexual dimorphism predates the gender-based divergence of the mating locus gene MAT3/RB.
Mol. Biol. Evol.
PUBLISHED: 01-30-2013
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The molecular bases for the evolution of male-female sexual dimorphism are possible to study in volvocine algae because they encompass the entire range of reproductive morphologies from isogamy to oogamy. In 1978, Charlesworth suggested the model of a gamete size gene becoming linked to the sex-determining or mating type locus (MT) as a mechanism for the evolution of anisogamy. Here, we carried out the first comprehensive study of a candidate MT-linked oogamy gene, MAT3/RB, across the volvocine lineage. We found that evolution of anisogamy/oogamy predates the extremely high male-female divergence of MAT3 that characterizes the Volvox carteri lineage. These data demonstrate very little sex-linked sequence divergence of MAT3 between the two sexes in other volvocine groups, though linkage between MAT3 and the mating locus appears to be conserved. These data implicate genetic determinants other than or in addition to MAT3 in the evolution of anisogamy in volvocine algae.
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B cells regulate antibody responses through the medullary remodeling of inflamed lymph nodes.
Int. Immunol.
PUBLISHED: 12-20-2011
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Lymph node (LN) structure is remodeled during immune responses, a process which is considered to play an important role in the regulation of immune function. To date, little attention has been paid to the remodeling of the medullary region, despite its proposed role as a niche for antibody-producing plasma cells. Here, we show that B cells mediate medullary remodeling of antigen-draining LNs during inflammation. This process occurs with kinetics similar to changes in plasma cell number and is accompanied by stromal renetworking which manifests as the segregation of B cells and plasma cells. Medullary remodeling depends on signaling via the lymphotoxin-? receptor and the presence of B cells but occurs independently of T-dependent humoral responses or other immune cell subsets including T cells, monocytes and neutrophils. Moreover, reconstitution of non-cognate polyclonal B cells in B cell-deficient mice restores not only the medullary remodeling but also the antibody response by separately transferred cognate B cells, suggesting that non-cognate B cells contribute to antibody responses through medullary remodeling. We propose that non-cognate B cells mediate the expansion of the plasma cell niche in LN through medullary remodeling, thereby regulating the size of the LN plasma cell pool.
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Disturbed-flow-mediated vascular reactive oxygen species induce endothelial dysfunction.
Circ. J.
PUBLISHED: 11-10-2011
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Emerging evidence is revealing the different roles of steady laminar flow (s-flow) and disturbed flow (d-flow) in the regulation of the vascular endothelium. s-flow is atheroprotective while d-flow creates an atheroprone environment. Most recently, we found unique atheroprone signals, which involve protein kinase C (PKC)? activation, elicited by d-flow. We and others have defined a novel role for PKC? as a shared mediator for tumor necrosis factor alpha (TNF alpha) and d-flow, which cause pro-inflammatory and pro-apoptotic events in endothelial cells (ECs) in the atheroprone environment. Under such conditions, ONOO(-) formation is increased in a d-flow-mediated PKC?-dependent manner. Here, we propose a new signaling pathway involving d-flow-induced EC inflammation via PKC?-ERK5 interaction-mediated downregulation of KLF2/eNOS stability, which leads to PKC?-mediated p53-SUMOylation and EC apoptosis. In addition, we highlight several mechanisms contributing to endothelial dysfunction, focusing on the relations between flow patterns and activation of reactive oxygen species generating enzymes.
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Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex.
Plant Cell Physiol.
PUBLISHED: 07-29-2011
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Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.
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Neural processing associated with comprehension of an indirect reply during a scenario reading task.
Neuropsychologia
PUBLISHED: 07-27-2011
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In daily communication, we often use indirect speech to convey our intention. However, little is known about the brain mechanisms that underlie the comprehension of indirect speech. In this study, we conducted a functional MRI experiment using a scenario reading task to compare the neural activity induced by an indirect reply (a type of indirect speech) and a literal sentence. Participants read a short scenario consisting of three sentences. The first two sentences explained the situation of the protagonists, whereas the third sentence had an indirect, literal, or unconnected meaning. The indirect reply condition primarily activated the bilateral fronto-temporal networks (Brodmanns Areas (BA) 47 and 21) and the dorso-medial prefrontal cortex (dmPFC). In the literal sentence condition, only the left fronto-temporal network (BA 45 and 21) and the dmPFC (posterior region) were activated. In addition, we found greater activation resulting from comprehension of an indirect reply than from literal sentence comprehension in the dmPFC, the left middle frontal area (BA 9), the bilateral inferior frontal area (BA 9/47), and the right middle temporal area (BA 21). Our findings indicate that the right and left fronto-temporal networks play a crucial role in detecting contextual violations, whereas the medial frontal cortex is important for generating inferences to make sense of remarks within a context.
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Chemokine receptor CXCR3 facilitates CD8(+) T cell differentiation into short-lived effector cells leading to memory degeneration.
J. Exp. Med.
PUBLISHED: 07-25-2011
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Strength of inflammatory stimuli during the early expansion phase plays a crucial role in the effector versus memory cell fate decision of CD8(+) T cells. But it is not known how early lymphocyte distribution after infection has an impact on this process. We demonstrate that the chemokine receptor CXCR3 is involved in promoting CD8(+) T cell commitment to an effector fate rather than a memory fate by regulating T cell recruitment to an antigen/inflammation site. After systemic viral or bacterial infection, the contraction of CXCR3(-/-) antigen-specific CD8(+) T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8(+) T cells. Early after infection, CXCR3(-/-) antigen-specific CD8(+) T cells fail to cluster at the marginal zone in the spleen where inflammatory cytokines such as IL-12 and IFN-? are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3(-/-) CD8(+) T cells exhibit transient expression of CD25 and preferentially differentiate into memory precursor effector cells as compared with wild-type CD8(+) T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8(+) T cells via inhibition of CXCR3-mediated T cell migration to inflamed microenvironments.
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Fast optical channel recovery in field demonstration of 100-Gbit/s Ethernet over OTN using real-time DSP.
Opt Express
PUBLISHED: 07-13-2011
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A field trial of 100-Gbit/s Ethernet over an optical transport network (OTN) is conducted using a real-time digital coherent signal processor. Error free operation with the Q-margin of 3.2 dB is confirmed at a 100 Gbit/s Ethernet analyzer by concatenating a low-density parity-check code with a OTN framer forward error correction, after 80-ch WDM transmission through 6 spans x 70 km of dispersion shifted fiber without inline-dispersion compensation. Also, the recovery time of 12 msec is observed in an optical route switching experiment, which is achieved through fast chromatic dispersion estimation functionality.
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PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation.
J. Cell Biol.
PUBLISHED: 06-01-2011
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Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ? (PKC?) activation and d-flow has been reported, but the exact role of d-flow-mediated PKC? activation in atherosclerosis remains unclear. We tested the hypothesis that PKC? activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKC? activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKC? with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKC?-PIASy interaction and PKC?-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKC?-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.
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p90 ribosomal S6 kinase regulates activity of the renin-angiotensin system: a pathogenic mechanism for ischemia-reperfusion injury.
J. Mol. Cell. Cardiol.
PUBLISHED: 05-03-2011
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Increasing evidence suggests that local renin-angiotensin system (RAS) plays an important role in cardiac diseases. Elevated p90 ribosomal S6 kinase (RSK) activity has been observed in diabetic animal, as well as in human failing hearts. We hypothesize that RSK mediates cardiac dysfunction by up regulating local RAS signaling. In the present study, we show that the prorenin mRNA level was significantly increased (~5.6-fold) in transgenic mouse hearts with cardiac specific expression of RSK (RSK-Tg). The RSK-Tg mice were more vulnerable to ischemia/reperfusion (I/R) injury than non-transgenic littermate controls (NLC). To further understand the direct contribution of cardiac renin to I/R injury, we used a Langendorff system to evaluate the effect of renin inhibition by aliskiren in RSK-Tg mouse hearts. In the vehicle-perfused group, I/R significantly decreased left ventricular developed pressure (LVDP) in RSK-Tg hearts compared to NLC (7% versus 60% of the baseline). However, aliskiren perfusion significantly increased LVDP in RSK-Tg (7% to 61%, p<0.01) but not in NLC hearts (60% to 62%, n.s.). The protective effect of aliskiren in RSK-Tg hearts was further demonstrated with positive (contraction) dp/dt (6.5% to 63%, p<0.01) and rate pressure product (RPP) (5% to 51%, p<0.01). Moreover, aliskiren significantly decreased I/R induced infarction in RSK-Tg (60% to 32%, p<0.01), compared to NLC hearts (37% to 32%, n.s.). These results suggest that RSK plays a crucial role in regulating local cardiac renin, which contributes to I/R induced cardiac injury and dysfunction. Thus, renin inhibition may provide an alternative therapeutic strategy under conditions of increased RAS.
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Genetic variation of ?-tocopherol methyltransferase gene contributes to elevated ?-tocopherol content in soybean seeds.
BMC Plant Biol.
PUBLISHED: 04-12-2011
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Improvement of ?-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the ?-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to ?-tocopherol biosynthesis or through changes to crop management practices. Varieties with high ?-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high ?-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low ?-tocopherol variety Ichihime. In this study, the genetic mechanism of the high ?-tocopherol content trait of KAS was elucidated.
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Increased production of vascular endothelial growth factor-d and lymphangiogenesis in acute Kawasaki disease.
Circ. J.
PUBLISHED: 04-12-2011
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Kawasaki disease (KD) is characterized by systemic vasculitis with tissue edema. During the healing process of inflammation, lymphangiogenesis is essential for reducing tissue edema. One potential responsible candidate for the induction of lymphangiogenesis in the healing process of acute KD is vascular endothelial growth factor-D (VEGF-D).
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Flow shear stress and atherosclerosis: a matter of site specificity.
Antioxid. Redox Signal.
PUBLISHED: 04-08-2011
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It is well accepted that atherosclerosis occurs in a site-specific manner especially at branch points where disturbed blood flow (d-flow) predisposes to the development of plaques. Investigations both in vivo and in vitro have shown that d-flow is pro-atherogenic by promoting oxidative and inflammatory states in the artery wall. In contrast, steady laminar blood flow (s-flow) is atheroprotective by inhibition of oxidative stress and inflammation in the vessel wall. The mechanism for inflammation in endothelial cells (ECs) exposed to d-flow has been well studied and includes redox-dependent activation of apoptosis signal-regulating kinase 1 (ASK1) and Jun NH2-terminal kinase (JNK) that ultimately lead to the expression of adhesive molecules. In contrast, s-flow leads to the activation of the mitogen extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) pathway that prevents pro-inflammatory signaling. Important transcriptional events that reflect the pro-oxidant and pro-inflammatory condition of ECs in d-flow include the activation of activator protein 1 (AP-1) and nuclear factor kappaB (NF?B), whereas in s-flow, activation of Krüppel-like factor 2 (KLF2) and nuclear factor erythroid 2-like 2 (Nrf2) are dominant. Recent studies have shown that protein kinase c zeta (PKC?) is highly activated under d-flow conditions and may represent a molecular switch for EC signaling and gene expression. The targeted modulation of proteins activated in a site-specific manner holds the promise for a new approach to limit atherosclerosis.
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Inclusive fitness theory and eusociality.
Patrick Abbot, Jun Abe, John Alcock, Samuel Alizon, Joao A C Alpedrinha, Malte Andersson, Jean-Baptiste André, Minus van Baalen, François Balloux, Sigal Balshine, Nick Barton, Leo W Beukeboom, Jay M Biernaskie, Trine Bilde, Gerald Borgia, Michael Breed, Sam Brown, Redouan Bshary, Angus Buckling, Nancy T Burley, Max N Burton-Chellew, Michael A Cant, Michel Chapuisat, Eric L Charnov, Tim Clutton-Brock, Andrew Cockburn, Blaine J Cole, Nick Colegrave, Leda Cosmides, Iain D Couzin, Jerry A Coyne, Scott Creel, Bernard Crespi, Robert L Curry, Sasha R X Dall, Troy Day, Janis L Dickinson, Lee Alan Dugatkin, Claire El Mouden, Stephen T Emlen, Jay Evans, Régis Ferrière, Jeremy Field, Susanne Foitzik, Kevin Foster, William A Foster, Charles W Fox, Juergen Gadau, Sylvain Gandon, Andy Gardner, Michael G Gardner, Thomas Getty, Michael A D Goodisman, Alan Grafen, Rick Grosberg, Christina M Grozinger, Pierre-Henri Gouyon, Darryl Gwynne, Paul H Harvey, Ben J Hatchwell, Jürgen Heinze, Heikki Helanterä, Ken R Helms, Kim Hill, Natalie Jiricny, Rufus A Johnstone, Alex Kacelnik, E Toby Kiers, Hanna Kokko, Jan Komdeur, Judith Korb, Daniel Kronauer, Rolf Kümmerli, Laurent Lehmann, Timothy A Linksvayer, Sébastien Lion, Bruce Lyon, James A R Marshall, Richard McElreath, Yannis Michalakis, Richard E Michod, Douglas Mock, Thibaud Monnin, Robert Montgomerie, Allen J Moore, Ulrich G Mueller, Ronald Noë, Samir Okasha, Pekka Pamilo, Geoff A Parker, Jes S Pedersen, Ido Pen, David Pfennig, David C Queller, Daniel J Rankin, Sarah E Reece, Hudson K Reeve, Max Reuter, Gilbert Roberts, Simon K A Robson, Denis Roze, François Rousset, Olav Rueppell, Joel L Sachs, Lorenzo Santorelli, Paul Schmid-Hempel, Michael P Schwarz, Tom Scott-Phillips, Janet Shellmann-Sherman, Paul W Sherman, David M Shuker, Jeff Smith, Joseph C Spagna, Beverly Strassmann, Andrew V Suarez, Liselotte Sundström, Michael Taborsky, Peter Taylor, Graham Thompson, John Tooby, Neil D Tsutsui, Kazuki Tsuji, Stefano Turillazzi, Francisco Úbeda, Edward L Vargo, Bernard Voelkl, Tom Wenseleers, Stuart A West, Mary Jane West-Eberhard, David F Westneat, Diane C Wiernasz, Geoff Wild, Richard Wrangham, Andrew J Young, David W Zeh, Jeanne A Zeh, Andrew Zink.
Nature
PUBLISHED: 03-25-2011
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Arising from M. A. Nowak, C. E. Tarnita & E. O. Wilson 466, 1057-1062 (2010); Nowak et al. reply. Nowak et al. argue that inclusive fitness theory has been of little value in explaining the natural world, and that it has led to negligible progress in explaining the evolution of eusociality. However, we believe that their arguments are based upon a misunderstanding of evolutionary theory and a misrepresentation of the empirical literature. We will focus our comments on three general issues.
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Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-deficient mice.
J. Exp. Med.
PUBLISHED: 12-20-2010
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Cyclophilin A (CyPA; encoded by Ppia) is a ubiquitously expressed protein secreted in response to inflammatory stimuli. CyPA stimulates vascular smooth muscle cell migration and proliferation, endothelial cell adhesion molecule expression, and inflammatory cell chemotaxis. Given these activities, we hypothesized that CyPA would promote atherosclerosis. Apolipoprotein E-deficient (Apoe(-/-)) mice fed a high-cholesterol diet for 16 wk developed more severe atherosclerosis compared with Apoe(-/-)Ppia(-/-) mice. Moreover, CyPA deficiency was associated with decreased low-density lipoprotein uptake, VCAM-1 (vascular cell adhesion molecule 1) expression, apoptosis, and increased eNOS (endothelial nitric oxide synthase) expression. To understand the vascular role of CyPA in atherosclerosis development, bone marrow (BM) cell transplantation was performed. Atherosclerosis was greater in Apoe(-/-) mice compared with Apoe(-/-)Ppia(-/-) mice after reconstitution with CyPA(+/+) BM cells, indicating that vascular-derived CyPA plays a crucial role in the progression of atherosclerosis. These data define a role for CyPA in atherosclerosis and suggest CyPA as a target for cardiovascular therapies.
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MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation.
Blood
PUBLISHED: 12-03-2010
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Actin filament remodeling regulates several endothelial cell (EC) processes such as contraction, migration, adhesion, and shape determination. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-mediated phosphorylation of heat-shock protein 27 kDa (HSP27) promotes actin filament remodeling, but little is known about the regulation of this event in ECs. We found that tumor necrosis factor-? (TNF-?) SUMOylated MK2 at lysine (K)-339 affected EC actin filament organization and migration. Loss of the MK2 SUMOylation site (MK2-K339R) increased MK2 kinase activity and prolonged HSP27 phosphorylation, enhancing its effects on actin filament-dependent events. Both TNF-?-mediated EC elongation and steady laminar shear stress-mediated EC alignment were increased by MK2-K339R. Moreover, kinase-dead dominant-negative MK2 (DN-MK2) inhibited these effects. Cell migration is a dynamic process regulated by actin filament remodeling. Both wild-type MK2 (WT-MK2) and DN-MK2 significantly enhanced TNF-mediated inhibition of EC migration, and MK2-K339R further augmented this effect. Interestingly, the p160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 reversed this effect by MK2-K339R, which strongly suggests that both excessive and insufficient levels of actin filament remodeling can block EC migration. Our study shows that MK2 SUMOylation is a new mechanism for regulating actin filament dynamics in ECs.
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Ca2+/calmodulin-stimulated PDE1 regulates the beta-catenin/TCF signaling through PP2A B56 gamma subunit in proliferating vascular smooth muscle cells.
FEBS J.
PUBLISHED: 11-16-2010
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The phenotypic change of vascular smooth muscle cells (VSMCs), from a contractile phenotype to a synthetic phenotype, is crucial for pathogenic vascular remodeling in vascular diseases such as atherosclerosis and restenosis. Ca(2+)/calmodulin-stimulated phosphodiesterase 1 (PDE1) isozymes, including PDE1A and PDE1C, play integral roles in regulating the proliferation of synthetic VSMCs. However, the underlying molecular mechanism(s) remain unknown. In this study, we explore the role and mechanism of PDE1 isoforms in regulating ?-catenin/T-cell factor (TCF) signaling in VSMCs, a pathway important for vascular remodeling through promoting VSMC growth and survival. We found that inhibition of PDE1 activity markedly attenuated ?-catenin/TCF signaling by downregulating ?-catenin protein. The effect of PDE1 inhibition on ?-catenin protein reduction is exerted via promoting glycogen synthase kinase 3 (GSK3)? activation, ?-catenin phosphorylation and subsequent ?-catenin protein degradation. Moreover, PDE1 inhibition specifically upregulated phosphatase protein phosphatase 2A (PP2A) B56? subunit gene expression, which is responsible for the effects of PDE1 inhibition on GSK3? and ?-catenin/TCF signaling. Furthermore, the effect of PDE1 inhibition on ?-catenin was specifically mediated by PDE1A but not PDE1C isozyme. Interestingly, in synthetic VSMCs, PP2A B56?, phospho-GSK3? and phospho-?-catenin were all found in the nucleus, suggesting that PDE1A regulates nuclear ?-catenin protein stability through the nuclear PP2A-GSK3?-?-catenin signaling axis. Taken together, these findings provide direct evidence for the first time that PP2A B56? is a critical mediator for PDE1A in the regulation of ?-catenin signaling in proliferating VSMCs.
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Two coordinately regulated homologs of FLOWERING LOCUS T are involved in the control of photoperiodic flowering in soybean.
Plant Physiol.
PUBLISHED: 09-23-2010
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FLOWERING LOCUS T (FT) is a key flowering integrator in Arabidopsis (Arabidopsis thaliana), with homologs that encode florigens in many plant species regardless of the type of photoperiodic response. We identified 10 FT homologs, which were arranged as five pairs of linked genes in different homoeologous chromosomal regions, in soybean (Glycine max), a paleopolyploid species. Two of the FT homologs, GmFT2a and GmFT5a, were highly up-regulated under short-day (SD) conditions (inductive for flowering in soybean) and had diurnal expression patterns with the highest expression 4 h after dawn. Under long-day (LD) conditions, expression of GmFT2a and GmFT5a was down-regulated and did not follow a diurnal pattern. Flowering took much longer to initiate under LD than under SD, and only the GmFT5a transcript accumulated late in development under LD. Ectopic expression analysis in Arabidopsis confirmed that both GmFT2a and GmFT5a had the same function as Arabidopsis FT, but the effect of GmFT5a was more prominent. A double-mutant soybean line for two PHYTOCHROME A (PHYA) genes expressed high levels of GmFT2a and GmFT5a under LD, and it flowered slightly earlier under LD than the wild type grown under SD. The expression levels of GmFT2a and GmFT5a were regulated by the PHYA-mediated photoperiodic regulation system, and the GmFT5a expression was also regulated by a photoperiod-independent system in LD. Taken together, our results suggest that GmFT2a and GmFT5a coordinately control flowering and enable the adaptation of soybean to a wide range of photoperiodic environments.
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Novel role of C terminus of Hsc70-interacting protein (CHIP) ubiquitin ligase on inhibiting cardiac apoptosis and dysfunction via regulating ERK5-mediated degradation of inducible cAMP early repressor.
FASEB J.
PUBLISHED: 08-19-2010
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Growing evidence indicates a critical role of ubiquitin-proteosome system in apoptosis regulation. A cardioprotective effect of ubiquitin (Ub) ligase of the C terminus of Hsc70-interacting protein (CHIP) on myocytes has been reported. In the current study, we found that the cardioprotective effect of insulin growth factor-1 (IGF-1) was mediated by ERK5-CHIP signal module via inducible cAMP early repressor (ICER) destabilization. In vitro runoff assay and Ub assay showed ICER as a substrate of CHIP Ub ligase. Both disruption of ERK5-CHIP binding with inhibitory helical linker domain fragment (aa 101-200) of CHIP and the depletion of ERK5 by siRNA inhibited CHIP Ub ligase activity, which suggests an obligatory role of ERK5 on CHIP activation. Depletion of CHIP, using siRNA, inhibited IGF-1-mediated reduction of isoproterenol-mediated ICER induction and apoptosis. In diabetic mice subjected to myocardial infarction, the CHIP Ub ligase activity was decreased, with an increase in ICER expression. These changes were attenuated significantly in a cardiac-specific constitutively active form of MEK5? transgenic mice (CA-MEK5?-Tg) previously shown to have greater functional recovery. Furthermore, pressure overload-mediated ICER induction was enhanced in heterozygous CHIP(+/-) mice. We identified ICER as a novel CHIP substrate and that the ERK5-CHIP complex plays an obligatory role in inhibition of ICER expression, cardiomyocyte apoptosis, and cardiac dysfunction.
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1,5-anhydro-D-fructose and its derivatives: biosynthesis, preparation and potential medical applications.
Planta Med.
PUBLISHED: 07-19-2010
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1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with ?-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.
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Virginity and the clutch size behavior of a parasitoid wasp where mothers mate their sons.
Behav. Ecol.
PUBLISHED: 07-01-2010
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Theoretical and empirical research on the evolution of clutch size has proved to be an extremely productive area of evolutionary biology. A general prediction is that individuals should produce a smaller number of offspring when resources are more limited, such as when multiple individuals compete for the same resources for their development. However, we expect that the opposite prediction arises with virgin females of haplodiploid species, which are subject to extreme local mate competition. We test the key assumption and predictions of this theory with the parasitoid wasp Melittobia australica. Our data demonstrate that there is a trade-off between the size of the first and subsequent clutches and that virgin females adjust their production of sons according to the mating status (mated or not) of cofounding females. We also found that mated females facultatively change their offspring sex ratio in response to the mating status of cofoundresses. We discuss the potential mechanisms used to recognize the mating status and the implications of our results in the context of the extremely female-biased sex ratios observed across Melittobia species..
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PKCzeta decreases eNOS protein stability via inhibitory phosphorylation of ERK5.
Blood
PUBLISHED: 06-10-2010
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PKC? has emerged as a pathologic mediator of endothelial cell dysfunction, based on its essential role in tumor necrosis factor ? (TNF?)-mediated inflammation. In contrast, extracellular signal-regulated kinase 5 (ERK5) function is required for endothelial cell homeostasis as shown by activation of Krüppel-like factor 2 (KLF2), increased endothelial nitric-oxide synthase (eNOS) expression, and inhibition of apoptosis. We hypothesized that protein kinase C ? (PKC?) activation by TNF? would inhibit the ERK5/KLF2/eNOS pathway. TNF? inhibited the steady laminar flow-induced eNOS expression, and this effect was reversed by the dominant-negative form of PKC? (Ad.DN-PKC?). In addition, ERK5 function was inhibited by either TNF? or the transfection of the catalytic domain of PKC?. This inhibition was reversed by PKC? small interfering RNA. PKC? was found to bind to ERK5 under basal conditions with coimmunoprecipitation and the mammalian 2-hybrid assay. Furthermore, PKC? phosphorylates ERK5, and mutation analysis showed that the preferred site is S486. Most importantly, we found that the predominant effect of TNF? stimulation of PKC? was to decrease eNOS protein stability that was recapitulated by transfecting Ad.ERK5S486A mutant. Finally, aortic en face analysis of ERK5/PKC? activity showed high PKC? and ERK5 staining in the athero-prone region. Taken together our results show that PKC? binds and phosphorylates ERK5, thereby decreasing eNOS protein stability and contributing to early events of atherosclerosis.
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Vinpocetine inhibits NF-kappaB-dependent inflammation via an IKK-dependent but PDE-independent mechanism.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-06-2010
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Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-alpha-induced NF-kappaB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-alpha- or LPS-induced up-regulation of proinflammatory mediators, including TNF-alpha, IL-1beta, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-alpha- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-kappaB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca(2+) regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.
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Bone marrow graft-versus-host disease: early destruction of hematopoietic niche after MHC-mismatched hematopoietic stem cell transplantation.
Blood
PUBLISHED: 03-30-2010
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Disrupted hematopoiesis and delayed immune reconstitution are life-threatening complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although graft-versus-host disease (GVHD) is a major risk factor for the bone marrow (BM) insufficiency, how GVHD impairs BM hematopoiesis has been largely unknown. We hypothesized that BM stromal niche could be a target of GVHD. In major histocompatibility complex (MHC)-mismatched murine models of GVHD, we have demonstrated the early destruction of osteoblasts that especially affected B-cell lineages. The defective B lymphopoiesis was due to the impaired ability of BM stroma and osteoblasts to support the hematopoiesis, as evidenced by the failure of GVHD-affected BM to reconstitute the hematopoietic cells. The administration of anti-CD4 monoclonal antibody (mAb) ameliorated these effects and improved B lymphopoiesis while preserving graft-versus-tumor effects. Genetic ablation of Fas-Fas ligand signaling also partially restored B lymphopoiesis. Our present study provided evidence of BM GVHD, with the identification of osteoblasts as the main target for GVHD in BM. Moreover, our data showed the potential for mAb therapies to enhance immune reconstitution in vivo for patients undergoing allo-HSCT.
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[Is the victim of irony identified by echoic mention?].
Shinrigaku Kenkyu
PUBLISHED: 03-19-2010
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Sperber and Wilson (1981) proposed that the speaker who uttered the expectation that is contradicted by irony is identified as the victim of the irony by the echoic mention of his/her utterance. We conducted two experiments to explore whether the victim of irony is identified by echoic mention. Experiment 1 examined the validity of this hypothesis using three conditions. In the Hearer (H) echo condition, the speaker who is echoed is the hearer of the potential irony. In the No-Hearer (NH) echo condition, the speaker who is echoed is not the hearer of the potential irony. The third condition is the no-echo condition. The result supported the hypothesis. In Experiment 2, we manipulated the H echo factor and the NH echo factor in 4 conditions. The results replicated Experiment 1, and showed that the NH is identified as the victim of irony more than the H. These results suggest that echoic mention and also status in the discourse are factors in the comprehension of irony.
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The soybean stem growth habit gene Dt1 is an ortholog of Arabidopsis TERMINAL FLOWER1.
Plant Physiol.
PUBLISHED: 03-10-2010
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Classical genetic analysis has revealed that the determinate habit of soybean (Glycine max) is controlled by a recessive allele at the determinate stem (Dt1) locus. To dissect the molecular basis of the determinate habit, we isolated two orthologs of pea (Pisum sativum) TERMINAL FLOWER1a, GmTFL1a and GmTFL1b, from the soybean genome. Mapping analysis indicated that GmTFL1b is a candidate for Dt1. Despite their high amino acid identity, the two genes had different transcriptional profiles. GmTFL1b was expressed in the root and shoot apical meristems (SAMs), whereas GmTFL1a was mainly expressed in immature seed. The GmTFL1b transcript accumulated in the SAMs during early vegetative growth in both the determinate and indeterminate lines but thereafter was abruptly lost in the determinate line. Introduction of the genomic region of GmTFL1b from the indeterminate line complemented the stem growth habit in the determinate line: more nodes were produced, and flowering in the terminal raceme was delayed. The identity between Dt1 and GmTFL1b was also confirmed with a virus-induced gene silencing experiment. Taken together, our data suggest that Dt1 encodes the GmTFL1b protein and that the stem growth habit is determined by the variation of this gene. The dt1 allele may condition the determinate habit via the earlier loss in GmTFL1b expression concomitant with floral induction, although it functions normally under the noninductive phase of flowering. An association test of DNA polymorphisms with the stem growth habit among 16 cultivars suggested that a single amino acid substitution in exon 4 determines the fate of the SAM after floral induction.
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SUMO--a post-translational modification with therapeutic potential?
Curr Opin Pharmacol
PUBLISHED: 01-14-2010
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Sumoylation is a covalent modification, which is mediated by small ubiquitin-like modifier (SUMO) polypeptides. A growing body of evidence has shown that sumoylation affects the functional properties of many substrates in the regulation of cellular processes. Recent reports indicate the crucial role of sumoylation in human diseases including familial dilated cardiomyopathy, suggesting that targeting of sumoylation would be of considerable interest for novel therapies. Even though hundreds of SUMO substrates have been identified, their pathophysiological roles remain to be determined. Among them, ERK5-sumoylation has recently been linked to diabetes and implicated in endothelial dysfunction and cardiomyocyte apoptosis in vivo. These findings support the idea that ERK5-sumoylation is a novel therapeutic target for the treatment of diabetes-related cardiovascular diseases.
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Targeted deletion of the extracellular signal-regulated protein kinase 5 attenuates hypertrophic response and promotes pressure overload-induced apoptosis in the heart.
Circ. Res.
PUBLISHED: 01-14-2010
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Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood.
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QTL mapping for photoperiod insensitivity of a Japanese soybean landrace Sakamotowase.
J. Hered.
PUBLISHED: 12-03-2009
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The insensitivity of flowering to long daylength is an important characteristic which soybeans have used to adapt to environments at higher latitude. The objective of this study was to map the novel gene(s) for photoperiod insensitivity in the Japanese soybean landrace Sakamotowase. A previous study suggested that Sakamotowase possessed the genotype e1e1e3e3E4E4. The progeny of testcrosses with the Harosoy isoline for e3 (L62-667) produced the roughly expected segregation pattern for the monogenic inheritance, suggesting the major involvement of a single gene in photoperiod insensitivity of Sakamotowase. By mapping analysis for 6 linkage groups (LGs) harboring the known major genes and quantitative trait loci (QTLs) for flowering, we detected a major QTL for the insensitivity near an simple sequence repeat marker (Satt577) in LG C2 and a minor QTL in LG L. Our results therefore suggest that a novel gene for photoperiod insensitivity of Sakamotowase was located in LG C2. It was estimated from the position of the tagging marker that the novel gene may be an allele at the E1 or E7 loci or a novel gene tightly linked to the E1 locus.
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Metabolism of procymidone derivatives in female rats.
J. Agric. Food Chem.
PUBLISHED: 10-29-2009
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PCM-CH2OH [N-(3,5-dichlorophenyl)-1-hydroxymethyl-2-methylcyclopropane-1,2-dicarboximide] and PA-CH2OH [2-carboxyl-N-(3,5-dichlorophenyl)-1-hydroxymethyl-2-methylcyclopropane-1-carboxamide] are metabolites of the fungicide procymidone [N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide] in rat. The distribution and metabolism of PCM-CH2OH and PA-CH2OH were here clarified by analyzing plasma and tissues (liver, kidney, heart, lung, spleen and ovary) of female rats after single subcutaneous administration of [phenyl-14C]PCM-CH2OH and [phenyl-14C]PA-CH2OH at 62.5 mg/kg, respectively. In both rats dosed with PCM-CH2OH and PA-CH2OH, the radioactivity was similarly distributed into plasma and tissues, and PA-CH2OH was detected as the main metabolite in plasma, whereas PCM-CH2OH predominated in tissues except for kidney at 1 h after administration of PA-CH2OH. Furthermore, the cyclization ratio [PCM-CH2OH/(PCM-CH2OH+PA-CH2OH)] increased in tissues of PA-CH2OH dosed rats with passage of time. Both procymidone and PCM-CH2OH have convertible conformations (closed and open ring forms), so influence of pH conditions to their conversion was examined. Both compounds demonstrated closed rings under acidic conditions, and open rings under alkaline conditions. Generally, intracellar pH is kept at approximately neutral, and extracellular pH is kept at 0.6-0.7 units higher in all the animal species, so that our in vitro results supported in vivo findings.
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Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy.
Circ. Res.
PUBLISHED: 09-24-2009
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Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown.
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Neural substrates of irony comprehension: A functional MRI study.
Brain Res.
PUBLISHED: 08-16-2009
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In daily communication, we sometimes use ironic expressions to convey the opposite meaning. To understand these contradictory statements, we have to infer contextual implications and the speakers mental state. However, little is known about how our brains carry out these complex processes. In this study, we investigated the neural substrates involved in irony comprehension using echoic utterance (Sperber and Wilson, 1986, 1995). Participants read a short scenario that consisted of five sentences. The first four sentences explained the situation of the protagonists. The fifth connoted either an ironic, literal, or unconnected meaning. The participants had to press a button to indicate whether or not the final sentence expressed irony. In the ironic sentence condition, the bilateral superior frontal gyrus, middle frontal gyrus, inferior frontal gyrus, medial prefrontal cortex, superior temporal gyrus, inferior parietal lobule, caudate, thalamus, the left insula, and amygdala were activated. In the literal sentence condition, the right superior frontal gyrus, the bilateral middle frontal gyrus, inferior frontal gyrus, medial prefrontal cortex, superior temporal gyrus, inferior parietal lobule, caudate, the left insula, the right thalamus, and the left amygdala were activated. However, in the ironic sentence condition minus the literal sentence condition, we observed higher activation in the right medial prefrontal cortex (BA 10), the right precentral (BA 6), and the left superior temporal sulcus (BA 21). Our results suggest that irony comprehension is strongly related to mentalizing processes and that activation in these regions might be affected by higher-order cognitive operations.
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TR4 nuclear receptor functions as a fatty acid sensor to modulate CD36 expression and foam cell formation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-28-2009
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Testicular orphan nuclear receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily with diverse physiological functions. Using TR4 knockout (TR4(-/-)) mice to study its function in cardiovascular diseases, we found reduced cluster of differentiation (CD)36 expression with reduced foam cell formation in TR4(-/-) mice. Mechanistic dissection suggests that TR4 induces CD36 protein and mRNA expression via a transcriptional regulation. Interestingly, we found this TR4-mediated CD36 transactivation can be further enhanced by polyunsaturated fatty acids (PUFAs), such as omega-3 and -6 fatty acids, and their metabolites such as 15-hydroxyeico-satetraonic acid (15-HETE) and 13-hydroxy octa-deca dieonic acid (13-HODE) and thiazolidinedione (TZD)-rosiglitazone. Both electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that TR4 binds to the TR4 response element located on the CD36 5-promoter region for the induction of CD36 expression. Stably transfected TR4-siRNA or functional TR4 cDNA in the RAW264.7 macrophage cells resulted in either decreased or increased CD36 expression with decreased or increased foam cell formation. Restoring functional CD36 cDNA in the TR4 knockdown macrophage cells reversed the decreased foam cell formation. Together, these results reveal an important signaling pathway controlling CD36-mediated foam cell formation/cardiovascular diseases, and findings that TR4 transactivation can be activated via its ligands/activators, such as PUFA metabolites and TZD, may provide a platform to screen new drug(s) to battle the metabolism syndrome, diabetes, and cardiovascular diseases.
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Down-regulation of flavonoid 3-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence.
J. Plant Physiol.
PUBLISHED: 07-07-2009
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Changes in flavonoid content are often manifested as altered pigmentation in plant tissues. Two loci have been identified as controlling pigmentation in soybean pubescence. Of these, the T locus appears to encode flavonoid 3-hydroxylase (F3H) protein: the T and t alleles are associated with tawny and gray colors, respectively, in pubescence. We previously down-regulated F3H gene expression by virus-induced gene silencing (VIGS) in soybean. Despite this successful VIGS, the tawny pubescence pigmentation proved to be unchanged in greenhouse-grown plants. We hypothesized that the reduced mRNA level of the F3H gene resulting from VIGS remained high enough to induce pigmentation. To verify this hypothesis, in the present study, we performed F3H VIGS on plants grown under controlled conditions, in which the steady-state mRNA level of the F3H gene was reduced to approximately 5% of that of greenhouse-grown plants. This VIGS treatment resulted in the loss of tawny pigmentation in pubescence, suggesting that the sf3h1 gene is involved in the control of pigmentation in pubescence. We detected a marked decrease in target mRNA, an accumulation of short interfering RNAs (siRNAs), and a decrease in quercetin content relative to kaempferol in leaf tissues, indicating that sequence-specific mRNA degradation of the F3H gene was induced. These results suggest that leaf tissues have a threshold mRNA level of the F3H gene, which is associated with the occurrence of tawny pigmentation in pubescence. The estimated threshold mRNA level for pigmentation in pubescence was approximately 3% of the steady-state mRNA level of the F3H gene in greenhouse-grown plants.
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Identification of cleistogamy-associated proteins in flower buds of near-isogenic lines of soybean by differential proteomic analysis.
Peptides
PUBLISHED: 06-13-2009
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Differential proteomics analyses were performed on flower buds of chasmogamous (CH) and cleistogamous (CL) soybean to identify candidate genes that are associated with cleistogamy which is the production of permanently closed flowers. The proteins were extracted from flower buds of CH cv. Toyosuzu and CL cv. Karafuto-1, and separated by two-dimensional polyacrylamide gel electrophoresis. Of the 640 proteins detected on the gel, nine were differentially expressed in Toyosuzu and nine in Karafuto-1. Among these differentially expressed proteins, those associated with cleistogamy were identified using a pair of near-isogenic lines (NILs). beta-Galactosidase and protein disulfide isomerase were expressed differentially between the NILs for cleistogamy. Furthermore, the mRNA expression pattern of protein disulfide isomerase corresponded to the protein level, whereas that of beta-galactosidase was not consistent with the protein level. These results suggest that the protein disulfide isomerase and beta-galactosidase may be useful markers for achieving a better understanding of the molecular-biological mechanisms of cleistogamy in soybean.
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Expression of allene oxide cyclase from Pharbitis nil upon theobroxide treatment.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2009
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In previous reports we have reported that theobroxide induces characteristic accumulation of allene oxide cyclase (AOC; EC 5.3.99.6) protein and jasmonic acid (JA) in Pharbitis nil. In the present study, PnAOC, an AOC gene from Pharbitis nil was cloned. Immunofluorescence assays indicated that the AOC protein is located in the chloroplast of vascular bundles in Pharbitis nil leaves. The PnAOC cDNA sequence lacking the chloroplast signal peptide was successfully expressed in Escherichia coli, and a gas chromatography-mass spectrum assay suggested the relative AOC activity of the recombinant PnAOC protein in comparison with Arabidopsis AOC2. Interestingly, a biphasic expression of PnAOC was induced by theobroxide, which is consistent with the accumulation patterns of AOC protein and JA. All these results indicate that AOC is the primary target of theobroxide regulation and suggest that feedback regulation of PnAOC by JA occurs upon theobroxide treatment in Pharbitis nil.
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Adaptive evolution involving gene duplication and insertion of a novel Ty1/copia-like retrotransposon in soybean.
J. Mol. Evol.
PUBLISHED: 03-31-2009
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Gene duplication is a major force for generating evolutionary novelties that lead to adaptations to environments. We previously identified two paralogs encoding phytochrome A (phyA), GmphyA1 and GmphyA2, in soybean, a paleopolyploid species. GmphyA2 is encoded by the E4 locus responsible for photoperiod sensitivity. In photoperiod insensitive lines, GmphyA2 is inactivated by the insertion of a retrotransposon in exon 1. Here, we describe the detailed characterization of the element and its evolutionary significance inferred from the distribution of the allele that harbors the element. Structural characteristics indicated that the element, designated SORE-1, is a novel Ty1/copia-like retrotransposon in soybean, which was phylogenetically related to the Sto-4, BARE-1, and RIRE1 elements. The element was transcriptionally active, and the transcription was partially repressed by an epigenetic mechanism. Sequences homologous with SORE-1 were detected in a genome sequence database of soybean, most of which appeared silent. GmphyA2 that harbors the SORE-1 insertion was detected only in cultivated soybean lines grown in northern regions of Japan, consistent with the notion that photoperiod insensitivity caused by the dysfunction of GmphyA2 is one of genetic changes that allowed soybean cultivation at high latitudes. Taking into account that genetic redundancy is conferred by the two phyA genes, we propose a novel model for the consequences of gene duplication and transposition of retrotransposons: when the gene is duplicated, retrotransposon insertion that causes the loss of a gene function can lead to adaptive evolution while the organism is sustained by the buffering effect brought about by gene duplication.
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Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms.
Nat. Med.
PUBLISHED: 03-31-2009
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Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe-/- mice, we show that Apoe-/-Ppia-/- mice are completely protected from AngII-induced AAA formation, in contrast to Apoe-/-Ppia+/+ mice. Apoe-/-Ppia-/- mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.
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Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.
Toxicol. Appl. Pharmacol.
PUBLISHED: 03-10-2009
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To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.
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Bcr kinase activation by angiotensin II inhibits peroxisome-proliferator-activated receptor gamma transcriptional activity in vascular smooth muscle cells.
Circ. Res.
PUBLISHED: 02-12-2009
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Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor-mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II-mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)gamma transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARgamma activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II-mediated inhibition of PPARgamma activity significantly, suggesting the critical role of Bcr in Ang II-mediated inhibition of PPARgamma activity. Point-mutation and in vitro kinase analyses showed that PPARgamma was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPARgamma1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARgamma activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II-mediated nuclear factor kappaB activation in VSMCs. DN-PPARgamma reversed DN-Bcr-mediated inhibition of nuclear factor kappaB activation, suggesting that PPARgamma is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPARgamma/nuclear factor kappaB transcriptional activity.
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The Chlamydomonas hatching enzyme, sporangin, is expressed in specific phases of the cell cycle and is localized to the flagella of daughter cells within the sporangial cell wall.
Plant Cell Physiol.
PUBLISHED: 01-28-2009
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The timely breakdown of the extracellular matrix by proteolytic enzymes is essential for development, morphogenesis and cell proliferation in plant and animal cells. Sporangin of the unicellular green alga Chlamydomonas reinhardtii that mediates breakdown of the sporangial cell wall to liberate the daughter cells after cell division is characterized as a subtilase-like serine protease. The sporangin gene is specifically transcribed during S/M phase in a synchronized vegetative cell cycle. In immunoblot analyses using a polyclonal antibody raised against the sporangin polypeptide, the enzyme is synthesized after mitotic cell division and accumulated in the daughter cells before hatching. Immunofluorescence analyses showed that sporangin is localized to the flagella of the daughter cells within the sporangial cell wall, and released into the culture medium. The data suggest that sporangin is released from flagella concurrently with the digestion of sporangial cell wall, and then the daughter cells are hatched from the sporangia in the Chlamydomonas vegetative cell cycle.
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Distinct response in maintenance of human naive and memory B cells via IL-21 receptor and TCL1/Akt pathways.
Cell. Immunol.
PUBLISHED: 01-15-2009
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The molecular mechanisms involving in B-cell survival/proliferation are poorly understood. Here we investigated the molecules affecting the survival of human naïve and memory B cells. Without stimulation, naïve B cells survived longer than memory B cells. Moreover, the viability of memory B cells decreased more rapidly than that of naïve B cells following with Staphylococcus aureus Cowan strain (SAC), anti-immunoglobulin (Ig), or anti-CD40 stimulation, but displayed the same levels of survival following CpG DNA stimulation. We analyzed the transcriptional differences between B-cell subsets by gene expression profiling, and identified 15 genes significantly correlated to survival/proliferation. Among them, IL-21 receptor (IL-21R) and T-cell leukemia 1 (TCL1) proto-oncogene were highly expressed in naïve B cells. IL-21 induced the proliferation of both naïve and memory B cells. Marked phosphorylation of Akt was found in naïve B cells compared with memory B cells. This study suggests that naive and memory B cells are regulated by several distinct molecules, and the IL-21R and TCL1/Akt pathways might play crucial roles in naïve B cells for their maintenance.
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Acute encephalopathy and tubulointerstitial nephritis associated with Yersinia pseudotuberculosis.
Pediatr Int
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We report the case of a 28-month-old boy with encephalopathy and acute tubulointerstitial nephritis possibly associated with Yersinia pseudotuberculosis (Yp) infection. He was transferred to our center because of impairment of renal function and altered consciousness. He had fever for 5 days after recurrent vomiting and diarrhea. Computed tomography scan was normal, but electroencephalogram (EEG) analyses showed generalized slow wave patterns. Continuous hemodialysis was undergone and then his renal function was improved, but altered consciousness persisted. Single photon emission computed tomography (SPECT) revealed abnormally low signals at entire field, which suggested that he was suffered from encephalopathy. Phenobarbital administration and post-encephalopathy rehabilitation were started, and he recovered in fully premorbid state with normal EEG and SPECT findings on the 33rd hospital day. Various bacterial cultures were negative, but both Yp antibody and Yp-derived mitogen (YPM) antibody, the antibody of a specific Yp exotoxin, had an extremely high titer. This is the first report of encephalopathy potentially caused by Yp, indicated by the presence of a high Yp and YPM antibody titer.
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A crucial role for p90RSK-mediated reduction of ERK5 transcriptional activity in endothelial dysfunction and atherosclerosis.
Circulation
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Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis.
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Phosphorylation of protein inhibitor of activated STAT1 (PIAS1) by MAPK-activated protein kinase-2 inhibits endothelial inflammation via increasing both PIAS1 transrepression and SUMO E3 ligase activity.
Arterioscler. Thromb. Vasc. Biol.
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Protein inhibitor of activated signal transducer and activator of transcription-1 (PIAS1) is known to function as small ubiquitin-like modifier (SUMO) E3 ligase as well as transrepressor. The aim of the study is to elucidate the regulatory mechanisms for these 2 different functions, especially with respect to endothelial inflammation.
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Genetic and molecular bases of photoperiod responses of flowering in soybean.
Breed. Sci.
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Flowering is one of the most important processes involved in crop adaptation and productivity. A number of major genes and quantitative trait loci (QTLs) for flowering have been reported in soybean (Glycine max). These genes and QTLs interact with one another and with the environment to greatly influence not only flowering and maturity but also plant morphology, final yield, and stress tolerance. The information available on the soybean genome sequence and on the molecular bases of flowering in Arabidopsis will undoubtedly facilitate the molecular dissection of flowering in soybean. Here, we review the present status of our understanding of the genetic and molecular mechanisms of flowering in soybean. We also discuss our identification of orthologs of Arabidopsis flowering genes from among the 46,367 genes annotated in the publicly available soybean genome database Phytozome Glyma 1.0. We emphasize the usefulness of a combined approach including QTL analysis, fine mapping, and use of candidate gene information from model plant species in genetic and molecular studies of soybean flowering.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.