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Sample records for 3a 4a 5a

  1. Myosin5a Tail Associates Directly with Rab3A-containing Compartments in Neurons*

    PubMed Central

    Wöllert, Torsten; Patel, Anamika; Lee, Ying-Lung; Provance, D. William; Vought, Valarie E.; Cosgrove, Michael S.; Mercer, John A.; Langford, George M.

    2011-01-01

    Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles. PMID:21349835

  2. Myosin5a tail associates directly with Rab3A-containing compartments in neurons.

    PubMed

    Wöllert, Torsten; Patel, Anamika; Lee, Ying-Lung; Provance, D William; Vought, Valarie E; Cosgrove, Michael S; Mercer, John A; Langford, George M

    2011-04-22

    Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles. PMID:21349835

  3. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    SciTech Connect

    Sakisaka, Yukihiko; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

    2015-08-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.

  4. Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses

    PubMed Central

    Holmström, Fredrik; Chen, Margaret; Balasiddaiah, Anangi; Sällberg, Matti; Ahlén, Gustaf; Frelin, Lars

    2016-01-01

    The hepatitis C virus nonstructural (NS) 3/4A and NS5A proteins are major targets for the new direct-acting antiviral compounds. Both viral proteins have been suggested as modulators of the response to the host cell. We have shown that NS3/4A- and NS5A-specific T cell receptors confer different effector functions, and that killing of NS3/4A-expressing hepatocytes is highly dependent on IFN-γ. We here characterize the functional differences in the T cell responses to NS3/4A and NS5A. NS3/4A- and NS5A-specific T cells could be induced at various frequencies in wild-type-, NS3/4A-, and NS5A-transgenic mice. Priming of NS5A-specific T cells required a high DNA dose, and was unlike NS3/4A dependent on both CD4+ and CD8+ T cells, but less influenced by CD25+/GITR+ regulatory T cells. The presence of IL-12 greatly improved specific CD8+ T cell priming by NS3/4A but not by NS5A, suggesting a less dependence of IFN-γ for NS5A. This notion was supported by the observation that NS5A-specific T cells could eliminate NS5A-expressing hepatocytes also in the absence of IFN-γ-receptor-2. This supports that NS3/4A- and NS5A-specific T cells become activated and eliminate antigen expressing, or infected hepatocytes, by distinct mechanisms, and that NS5A-specific T cells show an overall less dependence of IFN-γ. PMID:27141891

  5. Conserved co-regulation and promoter sharing of hoxb3a and hoxb4a in zebrafish.

    PubMed

    Hadrys, Thorsten; Punnamoottil, Beena; Pieper, Mareike; Kikuta, Hiroshi; Pezeron, Guillaume; Becker, Thomas S; Prince, Victoria; Baker, Robert; Rinkwitz, Silke

    2006-09-01

    The expression of zebrafish hoxb3a and hoxb4a has been found to be mediated through five transcripts, hoxb3a transcripts I-III and hoxb4a transcripts I-II, driven by four promoters. A "master" promoter, located about 2 kb downstream of hoxb5a, controls transcription of a pre-mRNA comprising exon sequences of both genes. This unique gene structure is proposed to provide a novel mechanism to ensure overlapping, tissue-specific expression of both genes in the posterior hindbrain and spinal cord. Transgenic approaches were used to analyze the functions of zebrafish hoxb3a/hoxb4a promoters and enhancer sequences containing regions of homology that were previously identified by comparative genomics. Two neural enhancers were shown to establish specific anterior expression borders within the hindbrain and mediate expression in defined neuronal populations derived from hindbrain rhombomeres (r) 5 to 8, suggesting a late role of the genes in neuronal cell lineage specification. Species comparison showed that the zebrafish hoxb3a r5 and r6 enhancer corresponded to a sequence within the mouse HoxA cluster controlling activity of Hoxa3 in r5 and r6, whereas a homologous region within the HoxB cluster activated Hoxb3 expression but limited to r5. We conclude that the similarity of hoxb3a/Hoxa3 regulatory mechanisms reflect the shared descent of both genes from a single ancestral paralog group 3 gene. PMID:16860306

  6. Derivation of ligands for the complement C3a receptor from the C-terminus of C5a

    PubMed Central

    Halai, Reena; Bellows-Peterson, Meghan L; Branchett, Will; Smadbeck, James; Kieslich, Chris A; Croker, Daniel E; Cooper, Matthew A; Morikis, Dimitrios; Woodruff, Trent M; Floudas, Christodoulos A; Monk, Peter N

    2014-01-01

    The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury. Complement C3a and C5a are key mediators produced by this cascade, and their dysregulation has been linked to a plethora of inflammatory and autoimmune diseases. Consequently, this has stimulated interest in the development of ligands for the receptors for these complement peptides, C3a receptor, and C5a1 (C5aR/CD88). In this study we used computational methods to design novel C5a1 receptor ligands. However, functional screening in human monocyte-derived macrophages using the xCELLigence label-free platform demonstrated altered specificity of our ligands. No agonist/antagonist activity was observed at C5a1, but we instead saw that the ligands were able to partially agonize the closely related complement receptor C3a receptor. This was verified in the presence of C3a receptor antagonist SB 290157 and in a stable cell line expressing either C5a1 or C3a receptor alone. C3a agonism has been suggested to be a potential treatment of acute neutrophil-driven traumatic pathologies, and may have great potential as a therapeutic avenue in this arena. PMID:25446428

  7. Distinct Patterns of Wnt3a and Wnt5a Signaling Pathway in the Lung from Rats with Endotoxic Shock.

    PubMed

    Hii, Hiong-Ping; Liao, Mei-Hui; Chen, Shiu-Jen; Wu, Chin-Chen; Shih, Chih-Chin

    2015-01-01

    Septic shock is a syndrome with severe hypotension and multiple organ dysfunction caused by an imbalance between pro-inflammatory and anti-inflammatory response. The most common risk factor of acute lung injury is severe sepsis. Patients with sepsis-related acute respiratory distress syndrome have higher mortality. Recent studies reveal regulatory roles of Wnt3a and Wnt5a signaling in inflammatory processes. Wnt3a signaling has been implicated in anti-inflammatory effects, whereas Wnt5a signaling has been postulated to have pro-inflammatory properties. However, the balance between Wnt3a and Wnt5a signaling pathway in the lung of rats with endotoxic shock has not been determined. Thus, we investigated the major components of Wnt3a and Wnt5a signaling pathway in the lung of endotoxemic rats. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS, 10 mg/kg). The changes of hemodynamics, biochemical variables, and arterial blood gas were examined during the experimental period. At 6 h after saline or LPS, animals were sacrificed, and lungs were obtained for analyzing superoxide production, water accumulation, histologic assessment, and protein expressions of Wnt3a and Wnt5a signaling pathway. Animals that received LPS showed circulatory failure, multiple organ dysfunction, metabolic acidosis, hyperventilation, lung edema, and high mortality. The lung from rats with endotoxic shock exhibited significant decreases in the levels of Wnt3a, Fzd1, Dsh1, phosphorylated GSK-3β at Ser9, and β-catenin. In contrast, the expressions of Wnt5a, Fzd5, and CaMKII were up-regulated in the lung of endotoxemic rats. These findings indicate the major components of Wnt3a and Wnt5a signaling in the lung are disturbed under endotoxic insult. PMID:26218875

  8. Differential effects of complement activation products c3a and c5a on cardiovascular function in hypertensive pregnant rats.

    PubMed

    Lillegard, Kathryn E; Loeks-Johnson, Alex C; Opacich, Jonathan W; Peterson, Jenna M; Bauer, Ashley J; Elmquist, Barbara J; Regal, Ronald R; Gilbert, Jeffrey S; Regal, Jean F

    2014-11-01

    Early-onset pre-eclampsia is characterized by decreased placental perfusion, new-onset hypertension, angiogenic imbalance, and endothelial dysfunction associated with excessive activation of the innate immune complement system. Although our previous studies demonstrated that inhibition of complement activation attenuates placental ischemia-induced hypertension using the rat reduced uterine perfusion pressure (RUPP) model, the important product(s) of complement activation has yet to be identified. We hypothesized that antagonism of receptors for complement activation products C3a and C5a would improve vascular function and attenuate RUPP hypertension. On gestational day (GD) 14, rats underwent sham surgery or vascular clip placement on ovarian arteries and abdominal aorta (RUPP). Rats were treated once daily with the C5a receptor antagonist (C5aRA), PMX51 (acetyl-F-[Orn-P-(D-Cha)-WR]), the C3a receptor antagonist (C3aRA), SB290157 (N(2)-[(2,2-diphenylethoxy)acetyl]-l-arginine), or vehicle from GD 14-18. Both the C3aRA and C5aRA attenuated placental ischemia-induced hypertension without affecting the decreased fetal weight or decreased concentration of free circulating vascular endothelial growth factor (VEGF) also present in this model. The C5aRA, but not the C3aRA, attenuated placental ischemia-induced increase in heart rate and impaired endothelial-dependent relaxation. The C3aRA abrogated the acute pressor response to C3a peptide injection, but it also unexpectedly attenuated the placental ischemia-induced increase in C3a, suggesting nonreceptor-mediated effects. Overall, these results indicate that both C3a and C5a are important products of complement activation that mediate the hypertension regardless of the reduction in free plasma VEGF. The mechanism by which C3a contributes to placental ischemia-induced hypertension appears to be distinct from that of C5a, and management of pregnancy-induced hypertension is likely to require a broad anti

  9. [3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors.

    PubMed

    Yu, Hongyi; Moore, Michael L; Erhard, Karl; Hardwicke, Mary Ann; Lin, Hong; Luengo, Juan I; McSurdy-Freed, Jeanelle; Plant, Ramona; Qu, Junya; Raha, Kaushik; Rominger, Cynthia M; Schaber, Michael D; Spengler, Michael D; Rivero, Ralph A

    2013-02-14

    A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. PMID:24900655

  10. [3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors

    PubMed Central

    2013-01-01

    A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. PMID:24900655

  11. Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family.

    PubMed

    McPhee, I; Pooley, L; Lobban, M; Bolger, G; Houslay, M D

    1995-09-15

    COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE4A5). This led to the expression of a novel, cyclic AMP-specific, rolipram-inhibited phosphodiesterase activity. In such transfected cells a novel approximately 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant. RD1 (RNPDE4A1A). RPDE-6 activity and immunoreactivity was found distributed between both pellet (approximately 25%) and cytosol (approximately 75%) fractions of transfected COS-7 cells. Soluble and pellet RPDE-6 activities exhibited similar low Km values for cyclic AMP (approximately 2.4 microM) and were both inhibited by low concentrations of rolipram, with IC50 values for the soluble activity being lower (approximately 0.16 microM) than for the pellet activity (approximately 1.2 microM). Pellet RPDE-6 was resistant to release by either high NaCl concentrations or the detergent Triton X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an approximately 79 kDa species reflecting RD1 and an approximately 109 kDa species that co-migrated with the immunoreactive species seen in COS cells transfected to express RPDE-6. The approximately 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-transfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immuno-precipitated by these antisera from brain cytosol had the characteristics of COS cell-expressed RPDE-6 with KmcyclicAMP approximately 3.7 microM and IC50rolipram approximately 0.12 microM. The distribution of PDE activity immunoprecipitated from the cytosol of various brain regions paralleled that seen for

  12. TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

    PubMed Central

    He, Xiaoqing; Wang, Hai; Jin, Tao; Xu, Yongqing; Mei, Liangbin; Yang, Jun

    2016-01-01

    Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity. PMID:26930594

  13. Linkage and association of haplotypes at the APOA1/C3/A4/A5 genecluster to familial combined hyperlipidemia

    SciTech Connect

    Eichenbaum-Voline, Sophie; Olivier, Michael; Jones, Emma L.; Naoumova, Rossitza P.; Jones, Bethan; Gau, Brian; Seed, Mary; Betteridge,D. John; Galton, David J.; Rubin, Edward M.; Scott, James; Shoulders,Carol C.; Pennacchio, Len A.

    2002-09-15

    Combined hyperlipidemia (CHL) is a common disorder of lipidmetabolism that leads to an increased risk of cardiovascular disease. Thelipid profile of CHL is characterised by high levels of atherogeniclipoproteins and low levels of high-density-lipoprotein-cholesterol.Apolipoprotein (APO) A5 is a newly discovered gene involved in lipidmetabolism located within 30kbp of the APOA1/C3/A4 gene cluster. Previousstudies have indicated that sequence variants in this cluster areassociated with increased plasma lipid levels. To establish whethervariation at the APOA5 gene contributes to the transmission of CHL, weperformed linkage and linkage disequilibrium (LD) tests on a large cohortof families (n=128) with familial CHL (FCHL). The linkage data producedevidence for linkage of the APOA1/C3/A4/A5 genomic interval to FCHL (NPL= 1.7, P = 0.042). The LD studies substantiated these data. Twoindependent rare alleles, APOA5c.56G and APOC3c.386G of this gene clusterwere over-transmitted in FCHL (P = 0.004 and 0.007, respectively), andthis was associated with a reduced transmission of the most commonAPOA1/C3/A4/A5 haplotype (frequency 0.4425) to affected subjects (P =0.013). The APOA5c.56G allele was associated with increased plasmatriglyceride levels in FCHL probands, whereas the second, andindependent, APOC3c.386G allele was associated with increased plasmatriglyceride levels in FCHL pedigree founders. Thus, this allele (or anallele in LD) may mark a quantitative trait associated with FCHL, as wellas representing a disease susceptibility locus for the condition. Thisstudy establishes that sequence variation in the APOA1/C3/A4/A5 genecluster contributes to the transmission of FCHL in a substantialproportion of affected families, and that these sequence variants mayalso contribute to the lipid abnormalities of the metabolic syndrome,which is present in up to 40 percent of persons with cardiovasculardisease.

  14. New technology and cost reductions in the phase 4A2 and 5A2 PVMaT programs of ASE Americas

    SciTech Connect

    Kardauskas, M.; Kalejs, J.; Cao, J.; Ebers, S.; Gonsiorawski, R.; Piwczyk, B.; Rosenblum, M.; Southimath, S.

    1999-03-01

    The Phase 4A2 portion of the PVMaT work at ASE Americas is drawing to a close, and the Phase 5A2 work is beginning. Most of the improvements planned for Phase 4A2 have now been completed, including improvements in EFG crystal growth, cell manufacturing, and module design and production. A few of the original goals were not achieved, but these were replaced with new programs which have been very successful. Module cost reductions of about 15{percent} have been achieved at this point in the program, and the final 4A2 goal of a 25{percent} cost reduction will be met. The work now beginning in Phase 5A2 is expected to contribute further cost reduction into the new millenium. {copyright} {ital 1999 American Institute of Physics.}

  15. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

    PubMed

    Gu, Hongmei; Fisher, Amanda J; Mickler, Elizabeth A; Duerson, Frank; Cummings, Oscar W; Peters-Golden, Marc; Twigg, Homer L; Woodruff, Trent M; Wilkes, David S; Vittal, Ragini

    2016-06-01

    Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with

  16. NS5A sequence heterogeneity of hepatitis C virus genotype 4a predicts clinical outcome of pegylated-interferon-ribavirin therapy in Egyptian patients.

    PubMed

    El-Shamy, Ahmed; Shoji, Ikuo; El-Akel, Wafaa; Bilasy, Shymaa E; Deng, Lin; El-Raziky, Maissa; Jiang, Da-peng; Esmat, Gamal; Hotta, Hak

    2012-12-01

    Hepatitis C virus genotype 4 (HCV-4) is the cause of approximately 20% of the 180 million cases of chronic hepatitis C in the world. HCV-4 infection is common in the Middle East and Africa, with an extraordinarily high prevalence in Egypt. Viral genetic polymorphisms, especially within core and NS5A regions, have been implicated in influencing the response to pegylated-interferon and ribavirin (PEG-IFN/RBV) combination therapy in HCV-1 infection. However, this has not been confirmed in HCV-4 infection. Here, we investigated the impact of heterogeneity of NS5A and core proteins of HCV-4, mostly subtype HCV-4a, on the clinical outcomes of 43 Egyptian patients treated with PEG-IFN/RBV. Sliding window analysis over the carboxy terminus of NS5A protein identified the IFN/RBV resistance-determining region (IRRDR) as the most prominent region associated with sustained virological response (SVR). Indeed, 21 (84%) of 25 patients with SVR, but only 5 (28%) of 18 patients with non-SVR, were infected with HCV having IRRDR with 4 or more mutations (IRRDR ≥ 4) (P = 0.0004). Multivariate analysis identified IRRDR ≥ 4 as an independent SVR predictor. The positive predictive value of IRRDR ≥ 4 for SVR was 81% (21/26; P = 0.002), while its negative predictive value for non-SVR was 76% (13/17; P = 0.02). On the other hand, there was no significant correlation between core protein polymorphisms, either at residue 70 or at residue 91, and treatment outcome. In conclusion, the present results demonstrate for the first time that IRRDR ≥ 4, a viral genetic heterogeneity, would be a useful predictive marker for SVR in HCV-4 infection when treated with PEG-IFN/RBV. PMID:22993188

  17. Complement anaphylatoxin receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis.

    PubMed

    Zhang, Lingjun; Bell, Brent A; Yu, Minzhong; Chan, Chi-Chao; Peachey, Neal S; Fung, John; Zhang, Xiaoming; Caspi, Rachel R; Lin, Feng

    2016-03-01

    Recent studies have suggested that reagents inhibiting complement activation could be effective in treating T cell mediated autoimmune diseases such as autoimmune uveitis. However, the precise role of the complement anaphylatoxin receptors (C3a and C5a receptors) in the pathogenesis of autoimmune uveitis remains elusive and controversial. We induced experimental autoimmune uveitis in mice deficient or sufficient in both C3a and C5a receptors and rigorously compared their retinal phenotype using various imaging techniques, including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy, spectral domain optical coherence tomography, topical endoscopic fundus imaging, and histopathological analysis. We also assessed retinal function using electroretinography. Moreover, we performed Ag-specific T cell recall assays and T cell adoptive transfer experiments to compare pathogenic T cell activity between wild-type and knockout mice with experimental autoimmune uveitis. These experiments showed that C3a receptor/C5a receptor-deficient mice developed much less severe uveitis than did control mice using all retinal examination methods and that these mice had reduced pathogenic T cell responses. Our data demonstrate that both complement anaphylatoxin receptors are important for the development of experimental autoimmune uveitis, suggesting that targeting these receptors could be a valid approach for treating patients with autoimmune uveitis. PMID:26394814

  18. Long-term sublingual immunotherapy for Japanese cedar pollinosis and the levels of IL-17A and complement components 3a and 5a.

    PubMed

    Sakashita, Masafumi; Yamada, Takechiyo; Imoto, Yoshimasa; Hirota, Tomomitsu; Tamari, Mayumi; Ito, Yumi; Kubo, Seita; Osawa, Yoko; Takahashi, Noboru; Fujieda, Shigeharu

    2015-09-01

    Allergen-specific immunotherapy is the only treatment that can alter the natural course of allergic disease. We performed long-term sublingual immunotherapy (SLIT) for patients with seasonal allergic rhinitis caused by Japanese cedar pollen (SAR-JCP), screened molecules as candidate biomarkers, and investigated serum IL-17A and complement components 3a (C3a) and C5a in order to evaluate whether these molecules show changes correlated to symptom scores. In this study, we found that the long-term SLIT reduced the serum levels of IL-17A and C3a and C5a. The levels of C3a in the patients significantly decreased from year 1 compared with those at the baseline, and their levels of IL-17A significantly decreased from year 2 compared with those at baseline. The levels of IL-17A, C3a, and C5a at year 4 of SLIT were significantly lower than not only those at baseline, but also those at year 1. A significant positive correlation was found between the symptom medication scores and the levels of IL-17A at year 4. The symptom medication scores in the group in which IL-17A levels decreased at year 4 were significantly lower than those in the group without such a decrease. The serum level of IL-17A might prove useful as a biological parameter to ascertain the effectiveness of SLIT for patients with SAR-JCP. It is necessary to produce new therapeutics for non-responders in whom serum IL-17A levels are still higher against long-term SLIT. PMID:25934649

  19. The APOA1/C3/A4/A5 cluster and markers of allostatic load in the Boston Puerto Rican Health Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The APOA1/C3/A4/A5 cluster encodes key regulators of plasma lipids. Interactions between dietary factors and single nucleotide polymorphisms (SNPs) in the cluster have been reported. Allostatic load, or physiological dysregulation in response to stress, has been implicated in shaping health disparit...

  20. Effects of variations in the APOA1/C3/A4/A5 gene cluster on different parameters of postprandial lipid metabolism in healthy young men

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The APOA1/C3/A4/A5 gene cluster encodes important regulators of fasting lipids, but the majority of lipid metabolism takes place in the postprandial state, and knowledge about gene regulation in this state is scarce. With the aim of characterizing possible regulators of lipid metabolism...

  1. Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

    PubMed Central

    Farnsworth, C C; Seabra, M C; Ericsson, L H; Gelb, M H; Glomset, J A

    1994-01-01

    Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified. PMID:7991565

  2. Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

    PubMed

    Farnsworth, C C; Seabra, M C; Ericsson, L H; Gelb, M H; Glomset, J A

    1994-12-01

    Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified. PMID:7991565

  3. Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons

    SciTech Connect

    Wang, Qian-fei; Liu, Xin; O'Connell, Jeff; Peng, Ze; Krauss, Ronald M.; Rainwater, David L.; VandeBerg, John L.; Rubin, Edward M.; Cheng, Jan-Fang; Pennacchio, Len A.

    2003-09-15

    Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.

  4. Fidelity of binding of the guanidinium nucleic acid (DNG) d(Tg)4-T-azido with short strand DNA oligomers (A5G3A5, GA4G3A4G, G2A3G3A3G2, G2A2G5A2G2). A kinetic and thermodynamic study.

    PubMed

    Blaskó, A; Minyat, E E; Dempcy, R O; Bruice, T C

    1997-06-24

    Short strand DNA oligomers (A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2) and the guanidinium (g) linked thymidyl nucleoside d(Tg)4-T-azido associate as triplexes. The melting temperatures, Tm, the association and dissociation kinetic and thermodynamic parameters and activation energies for the triplexes were determined by UV thermal analysis. The hypochromic shift and Tm for triplex formation increases with increase in concentration and decreases with the number of mismatches. The melting temperatures are between 35 and 55 degrees C in the range of ionic strength of 0.06-0.24 and decrease with increase in ionic strength at 100 deg/(ionic strength unit). The melting and cooling curves exhibit hysteresis behavior in the temperature range 5-95 degrees C at 0.2 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (k(on), E(on)) and dissociation (k(off), E(off)) processes were obtained. The second-order rate constants, k(on), for the triplex formation at 288 K are between 10 and 500 M(-2) s(-1). Values of k(on) increase with the decrease in the ionic strength. The first order rate constants for the dissociation, k(off), at 288 K are between 10(-6) and 40 x 10(-6) s(-1) and increase with increase in ionic strength. The energies of activation for the association and dissociation processes are in the range -22 to -9 kcal/mol and 8 to 29 kcal/mol, respectively. At 6.3 x 10(-5) M/base and at the physiological ionic strength (0.15-0.30) and below, the triplex structures formed with d(Tg)4-T-azido and A5G3A5 and GA4G3A4G have well-defined Tm values. The melting curves with G2A3G3A3G2 and G2A2G5A2G2 are very shallow with small hypochromic shifts denoting negligible binding at physiological ionic strength. Therefore, with the increase in the G content (mismatched base pairs) at a certain concentration (e.g., 6.3 x 10(-5) M/base), discrimination (change in fidelity) occurs in the formation and strength of binding of d(Tg)4-T

  5. The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

    NASA Astrophysics Data System (ADS)

    Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

    2014-03-01

    CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

  6. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

    PubMed

    Lahser, Frederick C; Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y M; Asante-Appiah, Ernest

    2016-05-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  7. Synthesis of [1,2,4]-triazolo[1,5- a]pyrimidines by Dimroth rearrangement of [1,2,4]-triazolo[4,3- a]pyrimidines: A theoretical and NMR study

    NASA Astrophysics Data System (ADS)

    Salgado, Antonio; Varela, Carmen; García Collazo, Ana María; García, Fernando; Pevarello, Paolo; Alkorta, Ibon; Elguero, José

    2011-02-01

    Novel [1,2,4]-triazolo-[1,5- a]pyrimidine derivatives were prepared by oxidative cyclization of suitable N-benzylidene- N'-pyrimidin-2-yl hydrazine precursors, followed by a Dimroth rearrangement. Reaction of 6-bromo-[1,2,4]-triazolo-[4,3- a]pyrimidines with aliphatic amines under microwave irradiation gave the unexpected 5-amino compounds from an ANRORC-type mechanism. Full NMR and HRMS characterization was done for all the obtained compounds. DFT calculations of absolute shielding permitted to predict 1H, 13C and 15N chemical shifts, which were in good agreement with the experimental ones. Theoretical studies at the B3LYP/6-311++G(d,p) level corroborated that [1,2,4]-triazolo-[1,5- a]pyrimidines were more stable than their [4,3- a] counterparts.

  8. Correlation between mutations in the core and NS5A genes of hepatitis C virus genotypes 1a, 1b, 3a, 3b, 6f and the response to pegylated interferon and ribavirin combination therapy.

    PubMed

    Kumthip, K; Pantip, C; Chusri, P; Thongsawat, S; O'Brien, A; Nelson, K E; Maneekarn, N

    2011-04-01

    Several studies have reported correlation between mutations in core and NS5A proteins of hepatitis C virus (HCV) and response to interferon (IFN) therapy. In particular, mutations in NS5A protein have been shown to correlate with responsiveness to IFN treatment of HCV-1b in Japanese patients. This study investigated whether amino acid (aa) mutations in the core and NS5A proteins of HCV-1a, 1b, 3a, 3b and 6f correlated with the response to pegylated interferon (Peg-IFN) plus ribavirin (RBV) therapy in Thai patients. The entire sequences of core and NS5A of HCV from 76 HCV-infected patients were analysed in comparison with corresponding reference sequences. The data revealed that the number of aa mutations in full-length NS5A, its C-terminus, IFN sensitivity-determining region, variable region 3 (V3) and V3 plus flanking region of HCV-1b NS5A protein were significantly higher in responders than in the treatment failure group (P = 0.010, 0.031, 0.046, 0.020 and 0.006, respectively). Similar results were found in a putative protein kinase R binding domain region in HCV-6f NS5A protein (P = 0.022). Moreover, specific aa substitutions in NS5A that appeared to be associated with responders or the treatment failure group were observed at positions 78 and 305 for HCV-1b (P = 0.028), 64 and 52 for HCV-1a (P = 0.033) and 6f (P = 0.045). Nevertheless, analysis of aa sequences of core protein revealed highly conserved sequences among HCV genotypes and no significant differences between the viruses from responders and the treatment failure group. Our findings indicate that mutations in aa residues of NS5A of HCV-1a, 1b and 6f correlated well with responsiveness to Peg-IFN and RBV combination therapy. PMID:20955493

  9. Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine

    PubMed Central

    Latimer, Brian; Toporovski, Roberta; Yan, Jian; Pankhong, Panyupa; Morrow, Matthew P; Khan, Amir S; Sardesai, Niranjan Y; Welles, Seth L; Jacobson, Jeffrey M; Weiner, David B; Kutzler, Michele A

    2014-01-01

    Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells

  10. Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.

    PubMed

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J; Liu, Yi; Burlingame, A L; Correia, Maria Almira

    2015-02-01

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  11. Fabrication and luminescence properties of one-dimensional ZnAl{sub 2}O{sub 4} and ZnAl{sub 2}O{sub 4}: A{sup 3+} (A = Cr, Eu, Tb) microfibers by electrospinning method

    SciTech Connect

    Peng, Chong; Li, Guogang; Geng, Dongling; Shang, Mengmeng; Hou, Zhiyao; Lin, Jun

    2012-11-15

    Graphical abstract: Different color emission can be realized in one-dimensional ZnAl{sub 2}O{sub 4}: A{sup 3+} (A = Cr, Eu, Tb) microfibers fabricated by electronspinning method by changing the doping ions. Highlights: ► ZnAl{sub 2}O{sub 4}: A{sup 3+} (A = Cr, Eu, Tb) microfibers were obtained by electronspinning method. ► Luminescence properties were due to the transitions of Cr{sup 3+}, Eu{sup 3+} and Tb{sup 3+} ions. ► A new band was observed around 600 nm in CL spectra of ZnAl{sub 2}O{sub 4}: Cr{sup 3+} microfibers. -- Abstract: One-dimensional ZnAl{sub 2}O{sub 4} and ZnAl{sub 2}O{sub 4}: A{sup 3+} (A = Cr, Eu, Tb) microfibers were fabricated by electrospinning method. X-ray diffraction pattern confirmed that the fibers were composed of the cubic ZnAl{sub 2}O{sub 4} phase. Scanning electron microscopy and transmission electron microscopy results indicated that the ZnAl{sub 2}O{sub 4} microfibers annealed at 1000 °C were composed of some smaller nanoparticles and the diameters of the microfibers were in the range of 140–230 nm. Under the excitation of ultraviolet and low-voltage electron beams (3–5 kV), the ZnAl{sub 2}O{sub 4}: A{sup 3+} (A = Cr, Eu, Tb) samples showed the emission of the host and the doping ions. The evolution of the optical properties of microfibers with increasing the doping concentration was investigated in detail. Green and red emissions can be realized in ZnAl{sub 2}O{sub 4} microfibers by changing the doping ions. The ZnAl{sub 2}O{sub 4}: A{sup 3+} (A = Cr, Eu, Tb) microfibers have potential applications in full-color field emission displays.

  12. Immunization with a Recombinant Expression Vector Encoding NS3/NS4A of Hepatitis C Virus Genotype 3a Elicits Cell-Mediated Immune Responses in C57BL/6 Mice.

    PubMed

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab; Kalani, Mehdi; Pouladfar, Gholamreza; Dianatpour, Mehdi; Ziyaeyan, Mazyar

    2016-04-01

    Today, hepatitis C virus (HCV) infection is considered as one of the most significant international health concerns. Although novel therapeutic regimens against the infection have shown satisfactory results, no approved vaccine exists yet. This study aimed to evaluate the immunogenicity of a DNA vaccine candidate for HCV-3a, based on nonstructural proteins NS3/NS4A, in C57BL/6 mice. Immunogenicity effect of pDisplay-NS3/NS4A was analyzed through immunization with 100 and 200 μg concentrations of the construct with complete Freund's adjuvant, monophosphoryl lipid A (MPL), or without adjuvant. The frequencies of different splenic mononuclear cells were measured using the Mouse Th1/Th2/Th17 Phenotyping Kit. Moreover, the number of T-CD8(+) cells was determined using conjugated anti-CD8a and anti-CD3e antibodies by flow cytometry. As observed, the frequencies of Th1, T-CD8(+), and Th2 cells increased in all the experimental groups, compared with the controls. The highest levels of the respective cells were seen in the group immunized with 200 μg of the construct with MPL. Also, there were positive correlations between the frequency of Th1 cells and those of Th2 and T-CD8(+) cells in all the immunized groups, but were significant in those receiving adjuvants. The frequency of Th17 cells did not statistically change among the groups. Taken together, our findings revealed that the constructed DNA vaccine encoding HCV-3a NS3/NS4A gene induces the cell-mediated immune responses significantly. However, its coadministration with adjuvants exhibits more efficient results than the recombinant plasmid alone. Further study is currently underway to evaluate the specific immune responses and recognize the responsible antigenic epitopes. PMID:26909520

  13. β-Catenin-dependent pathway activation by both promiscuous "canonical" WNT3a-, and specific "noncanonical" WNT4- and WNT5a-FZD receptor combinations with strong differences in LRP5 and LRP6 dependency.

    PubMed

    Ring, Larisa; Neth, Peter; Weber, Christian; Steffens, Sabine; Faussner, Alexander

    2014-02-01

    The WNT/β-catenin signalling cascade is the best-investigated frizzled receptor (FZD) pathway, however, whether and how specific combinations of WNT/FZD and co-receptors LRP5 and LRP6 differentially affect this pathway are not well understood. This is mostly due to the fact that there are 19 WNTs, 10 FZDs and at least two co-receptors. In our attempt to identify the signalling capabilities of specific WNT/FZD/LRP combinations we made use of our previously reported TCF/LEF Gaussia luciferase reporter gene HEK293 cell line (Ring et al., 2011). Generation of WNT/FZD fusion constructs - but not their separate transfection - without or with additional isogenic overexpression of LRP5 and LRP6 in our reporter cells permitted the investigation of specific WNT/FZD/LRP combinations. The canonical WNT3a in fusion to almost all FZDs was able to induce β-catenin-dependent signalling with strong dependency on LRP6 but not LRP5. Interestingly, noncanonical WNT ligands, WNT4 and WNT5a, were also able to act "canonically" but only in fusion with specific FZDs and with selective dependence on LRP5 or LRP6. These data and extension of this experimental setup to the poorly characterized other WNTs should facilitate deeper insight into the complex WNT/FZD signalling system and its function. PMID:24269653

  14. Development of a new fluorescent probe: 1,3,5,7-tetramethyl-8-(4'-aminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza- s-indacence for the determination of trace nitrite

    NASA Astrophysics Data System (ADS)

    Li, Mengling; Wang, Hong; Zhang, Xian; Zhang, Hua-shan

    2004-03-01

    A new fluorescent probe, 1,3,5,7-tetramethyl-8-(4'-aminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza- s-indacence (TMABODIPY) has been developed for the determination of trace nitrite in terms of the reaction of nitrite with TMABODIPY first in acidic solution and then in alkaline solution to form diazotate, a stable and highly fluorescent reagent. The method offered the advantage of specificity, sensitivity and simplicity. The linear calibration range for nitrite was 8-300 nmol l -1 s with a 3 σ detection limit of 0.65 nmol l -1. The proposed method has been applied to monitor the trace nitrite in drinking water and vegetable without extraction.

  15. Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies ▿ †

    PubMed Central

    Gottwein, Judith M.; Scheel, Troels K. H.; Callendret, Benoit; Li, Yi-Ping; Eccleston, Heather B.; Engle, Ronald E.; Govindarajan, Sugantha; Satterfield, William; Purcell, Robert H.; Walker, Christopher M.; Bukh, Jens

    2010-01-01

    Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of ∼5.5 log10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes. PMID:20200247

  16. First highly efficient and photostable E and C derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as dye lasers in the liquid phase, thin films, and solid-state rods.

    PubMed

    Duran-Sampedro, Gonzalo; Esnal, Ixone; Agarrabeitia, Antonia R; Bañuelos Prieto, Jorge; Cerdán, Luis; García-Moreno, Inmaculada; Costela, Angel; Lopez-Arbeloa, Iñigo; Ortiz, María J

    2014-02-24

    A new library of E- and C-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivatives has been synthesized through a straightforward protocol from commercially available BODIPY complexes, and a systematic study of the photophysical properties and laser behavior related to the electronic properties of the B-substituent group (alkynyl, cyano, vinyl, aryl, and alkyl) has been carried out. The replacement of fluorine atoms by electron-withdrawing groups enhances the fluorescence response of the dye, whereas electron-donor groups diminish the fluorescence efficiency. As a consequence, these compounds exhibit enhanced laser action with respect to their parent dyes, both in liquid solution and in the solid phase, with lasing efficiencies under transversal pumping up to 73 % in liquid solution and 53 % in a solid matrix. The new dyes also showed enhanced photostability. In a solid matrix, the derivative of commercial dye PM597 that incorporated cyano groups at the boron center exhibited a very high lasing stability, with the laser emission remaining at the initial level after 100 000 pump pulses in the same position of the sample at a 10 Hz repetition rate. Distributed feedback laser emission was demonstrated with organic films that incorporated parent dye PM597 and its cyano derivative. The films were deposited onto quartz substrates engraved with appropriate periodical structures. The C derivative exhibited a laser threshold lower than that of the parent dye as well as lasing intensities up to three orders of magnitude higher. PMID:24453119

  17. Evidence of CH{sub 2}O (a-tilde{sup 3}A{sub 2}) and C{sub 2}H{sub 4} (a-tilde{sup 3}B{sub 1u}) produced from photodissociation of 1,3-trimethylene oxide at 193 nm

    SciTech Connect

    Lee, S.-H.; Ong, C.-S.; Lee, Yuan T.

    2006-02-21

    We investigated the dissociative ionization of formaldehyde (CH{sub 2}O) and ethene (C{sub 2}H{sub 4}) produced from photolysis of 1,3-trimethylene oxide at 193 nm using a molecular-beam apparatus and vacuum-ultraviolet radiation from an undulator for direct ionization. The CH{sub 2}O (C{sub 2}H{sub 4}) product suffers from severe dissociative ionization to HCO{sup +} (C{sub 2}H{sub 3}{sup +} and C{sub 2}H{sub 2}{sup +}) even though photoionization energy is as small as 9.8 eV. Branching ratios of fragmentation of CH{sub 2}O and C{sub 2}H{sub 4} following ionization are revealed as a function of kinetic energy of products using ionizing photons from 9.8 to 14.8 eV. Except several exceptions, branching ratios of daughter ions increase with increasing photon energy but decrease with increasing kinetic energy. The title reaction produces CH{sub 2}O and C{sub 2}H{sub 4} mostly on electronic ground states but a few likely on triplet states; C{sub 2}H{sub 4} (a-tilde{sup 3}B{sub 1u}) seems to have a yield greater than CH{sub 2}O (a-tilde{sup 3}A{sub 2}). The distinct features observed at small kinetic energies of daughter ions are attributed to dissociative ionization of photoproducts CH{sub 2}O (a-tilde{sup 3}A{sub 2}) and C{sub 2}H{sub 4} (a-tilde{sup 3}B{sub 1u}). The observation of triplet products indicates that intersystem crossing occurs prior to fragmentation of 1,3-trimethylene oxide.

  18. Sulfotransferase 4A1.

    PubMed

    Minchin, Rodney F; Lewis, Aaron; Mitchell, Deanne; Kadlubar, Fred F; McManus, Michael E

    2008-01-01

    In this review, we highlight the physical and enzymatic properties of the novel human sulfotransferase, SULT4A1. The gene is most highly expressed in selective regions of the brain, although work to date has failed to identify any specific endogenous substrate for the enzyme. SULT4A1 shares low homology with other human sulfotransferases. Nevertheless, it is highly conserved between species. Despite the low homology, it is structurally very similar to other cytosolic sulfotransferases with a conserved substrate binding domain, dimerization site and partial cofactor binding sites. However, the catalytic cavity is much smaller, and it has been suggested that the cofactor may not be accommodated within it. A recent link between variability in the 5'UTR of the SULT4A1 gene and schizophrenia has heightened interest in the endogenous function of the enzyme and its possible role in human disease. PMID:18248844

  19. SLC4A Transporters

    PubMed Central

    Choi, Inyeong

    2016-01-01

    SLC4A gene family proteins include bicarbonate transporters that move HCO3− across the plasma membrane and regulate intracellular pH and transepithelial movement of acid–base equivalents. These transporters are Cl/HCO3 exchangers, electrogenic Na/HCO3 cotransporters, electroneutral Na/HCO3 cotransporters, and Na+-driven Cl/HCO3 exchanger. Studies of the bicarbonate transporters in vitro and in vivo have demonstrated their physiological importance for acid–base homeostasis at the cellular and systemic levels. Recent advances in structure/function analysis have also provided valuable information on domains or motifs critical for regulation, ion translocation, and protein topology. This chapter focuses on the molecular mechanisms of ion transport along with associated structural aspects from mutagenesis of particular residues and from chimeric constructs. Structure/function studies have helped to understand the mechanism by which ion substrates are moved via the transporters. This chapter also describes some insights into the structure of SLC4A1 (AE1) and SLC4A4 (NBCe1) transporters. Finally, as some SLC4A transporters exist in concert with other proteins in the cells, the structural features associated with protein–protein interactions are briefly discussed. PMID:23177984

  20. Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species

    USGS Publications Warehouse

    Boshra, Hani; Li, Jun; Peters, Rodney; Hansen, John; Matlapudi, Anjan; Sunyer, J. Oriol

    2004-01-01

    C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

  1. Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid.

    PubMed

    Guthrie, H D; Welch, G R

    2007-06-01

    Part of the reduction in boar sperm motility and fertility associated with hypothermic liquid storage and cryopreservation may be due to membrane lipid peroxidation. Lipid peroxidation was monitored by the shift from red to green fluorescence emission of the lipophilic probe 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C(11)BODIPY(581/591) (BODIPY), as measured by fluorescence-activated flow cytometry in live sperm (negative for propidium iodide). Experiments were conducted with Percoll-washed sperm to determine the specificity of BODIPY oxidation in the presence of different reactive oxygen species generators and metal chelators. Compared with no FeSO(4) and Na ascorbate, the combination of FeSO(4) and Na ascorbate (FeAc) increased (P < 0.01) the percentage of sperm containing oxidized BODIPY from 70% and increased (P < 0.05) BOD-IPY fluorescence intensity/cell by 5- to 10-fold after a 30-min incubation. Motility was depressed (P < 0.05) after exposure to FeAc, but viability was not affected. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeAc, because menadione and H(2)O(2) had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H(2)O(2); FeAc had no effect. The presence of the metal chelators EDTA or deferoxamine mesylate at 3 and 9 muM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17 degrees C for 1 and 5 d and the effect of freeze-thawing on basal and FeAc-induced BODIPY oxidation. Basal BODIPY oxidation (no FeAc) was low in liquid stored and thawed viable sperm (1.3 and 3.4%, respectively). Although the incidence of basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were susceptible to FeAc-induced lipid peroxidation. PMID:17296775

  2. PROCEEDINGS: 1993 SO2 CONTROL SYMPOSIUM - VOLUME 2. SESSIONS 4A, 4B, AND 5A

    EPA Science Inventory

    The report documents more than 100 presentations at the 1993 SO2 Control Symposium in Boston, MA, August 24-27, 1993. The presentations covered a wide range of topics: industry's strategies for dealing with Clean Air Act Amendments of 1990, including Phase I strategies, the emiss...

  3. Ryan XV-5A model

    NASA Technical Reports Server (NTRS)

    1962-01-01

    The duct-fan method of propulsion which would enable aircraft to take off helicopter-like was tested with models like this one, the Ryan XV- 5A, which was built and flown at NASA Ames. Photograph published in Winds of Change, 75th Anniversary NASA publication (page 84) and Crafting Flight (page106), by James Schultz.

  4. Douglas OA-4A Dolphin

    NASA Technical Reports Server (NTRS)

    1938-01-01

    Douglas OA-4A Dolphin: This twin-engine Douglas OA-4A Dolphin was unusual in comparison with other OA-4s in that it employed a nose wheel instead of a tail wheel during its NACA testing at Langley. Here is is seen in the NACA hangar in September 1938.

  5. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  6. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  7. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  8. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  9. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  10. Allosterism in human complement component 5a ((h)C5a): a damper of C5a receptor (C5aR) signaling.

    PubMed

    Rana, Soumendra; Sahoo, Amita Rani; Majhi, Bharat Kumar

    2016-06-01

    The phenomena of allosterism continues to advance the field of drug discovery, by illuminating gainful insights for many key processes, related to the structure-function relationships in proteins and enzymes, including the transmembrane G-protein coupled receptors (GPCRs), both in normal as well as in the disease states. However, allosterism is completely unexplored in the native protein ligands, especially when a small covalent change significantly modulates the pharmacology of the protein ligands toward the signaling axes of the GPCRs. One such example is the human C5a ((h)C5a), the potent cationic anaphylatoxin that engages C5aR and C5L2 to elicit numerous immunological and non-immunological responses in humans. From the recently available structure-function data, it is clear that unlike the mouse C5a ((m)C5a), the (h)C5a displays conformational heterogeneity. However, the molecular basis of such conformational heterogeneity, otherwise allosterism in (h)C5a and its precise contribution toward the overall C5aR signaling is not known. This study attempts to decipher the functional role of allosterism in (h)C5a, by exploring the inherent conformational dynamics in (m)C5a, (h)C5a and in its point mutants, including the proteolytic mutant des-Arg(74)-(h)C5a. Prima facie, the comparative molecular dynamics study, over total 500 ns, identifies Arg(74)-Tyr(23) and Arg(37)-Phe(51) "cation-π" pairs as the molecular "allosteric switches" on (h)C5a that potentially functions as a damper of C5aR signaling. PMID:26212097

  11. Synergistic Activity of Combined NS5A Inhibitors.

    PubMed

    O'Boyle, Donald R; Nower, Peter T; Gao, Min; Fridell, Robert; Wang, Chunfu; Hewawasam, Piyasena; Lopez, Omar; Tu, Yong; Meanwell, Nicholas A; Belema, Makonen; Roberts, Susan B; Cockett, Mark; Sun, Jin-Hua

    2015-01-01

    Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potential in vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O'Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245-248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV-NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV-NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function. PMID:26711745

  12. Wnt5a Signaling in Cancer.

    PubMed

    Asem, Marwa S; Buechler, Steven; Wates, Rebecca Burkhalter; Miller, Daniel L; Stack, M Sharon

    2016-01-01

    Wnt5a is involved in activating several non-canonical WNT signaling pathways, through binding to different members of the Frizzled- and Ror-family receptors. Wnt5a signaling is critical for regulating normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However, the aberrant activation or inhibition of Wnt5a signaling is emerging as an important event in cancer progression, exerting both oncogenic and tumor suppressive effects. Recent studies show the involvement of Wnt5a in regulating cancer cell invasion, metastasis, metabolism and inflammation. In this article, we review findings regarding the molecular mechanisms and roles of Wnt5a signaling in various cancer types, and highlight Wnt5a in ovarian cancer. PMID:27571105

  13. Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

    PubMed Central

    Wose Kinge, Constance N.; Espiritu, Christine; Prabdial-Sing, Nishi; Sithebe, Nomathamsaqa Patricia

    2014-01-01

    Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds. PMID:24982066

  14. Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin.

    PubMed Central

    Nishimura, Kazuhiro; Ohki, Yuji; Fukuchi-Shimogori, Tomomi; Sakata, Kaori; Saiga, Kan; Beppu, Takanobu; Shirahata, Akira; Kashiwagi, Keiko; Igarashi, Kazuei

    2002-01-01

    The mechanism of inhibition of cell growth by deoxyspergualin was studied using mouse mammary carcinoma FM3A cells. Results of studies using deoxyspergualin analogues showed that both the guanidinoheptanate amide and glyoxyspermidine moieties of deoxyspergualin were necessary to cause inhibition of cell growth. When deoxyspergualin was added to the medium, there was a strong inhibition of cell growth and formation of active eukaryotic translation initiation factor 5A (eIF5A) at the third day of culture. There was also a marked decrease in cellular putrescine content and a small decrease in spermidine content. Accumulation of decapped mRNA, which is typically associated with eIF5A deficiency in yeast, was also observed. The inhibition of cell growth and the formation of active eIF5A was not reversed by addition of spermidine. The activity of deoxyhypusine synthase, the first enzyme in the formation of active eIF5A, was inhibited by deoxyspergualin in a cell-free system. These results, taken together, indicate that inhibition of active eIF5A formation is strongly involved in the inhibition of cell growth by deoxyspergualin. PMID:11964177

  15. New insights for C5a and C5a receptors in sepsis

    PubMed Central

    Yan, Chunguang; Gao, Hongwei

    2012-01-01

    The complement system plays a central role in inflammation and immunity. Among the complement activation products, C5a is one of the most potent inflammatory peptides with a broad spectrum of functions. There is strong evidence for complement activation including elevated plasma level of C5a in humans and animals with sepsis. C5a exerts its effects through the C5a receptors. Of the two receptors that bind C5a, the C5aR (CD88) is known to mediate signaling activity, whereas the function of another C5a binding receptor, C5L2, remains largely unknown. Here, we review the critical role of C5a in sepsis and summarize evidence indicating that both C5aR and C5L2 act as regulating receptors for C5a during sepsis. PMID:23233853

  16. Discovery of ABT-267, a pan-genotypic inhibitor of HCV NS5A.

    PubMed

    DeGoey, David A; Randolph, John T; Liu, Dachun; Pratt, John; Hutchins, Charles; Donner, Pamela; Krueger, A Chris; Matulenko, Mark; Patel, Sachin; Motter, Christopher E; Nelson, Lissa; Keddy, Ryan; Tufano, Michael; Caspi, Daniel D; Krishnan, Preethi; Mistry, Neeta; Koev, Gennadiy; Reisch, Thomas J; Mondal, Rubina; Pilot-Matias, Tami; Gao, Yi; Beno, David W A; Maring, Clarence J; Molla, Akhter; Dumas, Emily; Campbell, Andrew; Williams, Laura; Collins, Christine; Wagner, Rolf; Kati, Warren M

    2014-03-13

    We describe here N-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2S,5S stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2R,5R analogues. Furthermore, the attachment of substituents at the 4-position of the central N-phenyl group resulted in compounds with improved potency. Substitution with tert-butyl, as in compound 38 (ABT-267), provided compounds with low-picomolar EC50 values and superior pharmacokinetics. It was discovered that compound 38 was a pan-genotypic HCV inhibitor, with an EC50 range of 1.7-19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. Compound 38 decreased HCV RNA up to 3.10 log10 IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects and is currently in phase 3 clinical trials in combination with an NS3 protease inhibitor with ritonavir (r) (ABT-450/r) and an NS5B non-nucleoside polymerase inhibitor (ABT-333), with and without ribavirin. PMID:24400777

  17. Diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication.

    PubMed

    Marriott, Andrew S; Copeland, Nikki A; Cunningham, Ryan; Wilkinson, Mark C; McLennan, Alexander G; Jones, Nigel J

    2015-09-01

    The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA. PMID:26204256

  18. 15 CFR 4a.3 - Classification levels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 15 Commerce and Foreign Trade 1 2013-01-01 2013-01-01 false Classification levels. 4a.3 Section 4a.3 Commerce and Foreign Trade Office of the Secretary of Commerce CLASSIFICATION, DECLASSIFICATION, AND PUBLIC AVAILABILITY OF NATIONAL SECURITY INFORMATION § 4a.3 Classification levels. Information may be classified as national security...

  19. 15 CFR 4a.4 - Classification authority.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 15 Commerce and Foreign Trade 1 2013-01-01 2013-01-01 false Classification authority. 4a.4 Section 4a.4 Commerce and Foreign Trade Office of the Secretary of Commerce CLASSIFICATION, DECLASSIFICATION, AND PUBLIC AVAILABILITY OF NATIONAL SECURITY INFORMATION § 4a.4 Classification authority. Authority to originally classify information as Secret...

  20. 15 CFR 4a.4 - Classification authority.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Classification authority. 4a.4 Section 4a.4 Commerce and Foreign Trade Office of the Secretary of Commerce CLASSIFICATION, DECLASSIFICATION, AND PUBLIC AVAILABILITY OF NATIONAL SECURITY INFORMATION § 4a.4 Classification authority. Authority...

  1. A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase.

    PubMed

    Torada, Atsushi; Koike, Michiya; Ogawa, Taiichi; Takenouchi, Yu; Tadamura, Kazuki; Wu, Jianzhong; Matsumoto, Takashi; Kawaura, Kanako; Ogihara, Yasunari

    2016-03-21

    Seed germination under the appropriate environmental conditions is important both for plant species survival and for successful agriculture. Seed dormancy, which controls germination time, is one of the adaptation mechanisms and domestication traits [1]. Seed dormancy is generally defined as the absence of germination of a viable seed under conditions that are favorable for germination [2]. The seed dormancy of cultivated plants has generally been reduced during domestication [3]. Bread wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Weak dormancy may be an advantage for the productivity due to uniform emergence and a disadvantage for the risks of pre-harvest sprouting (PHS), which decreases grain quality and yield [4]. A number of quantitative trait loci (QTLs) controlling natural variation of seed dormancy have been identified on various chromosomes [5]. A major QTL for seed dormancy has been consistently detected on chromosome 4A [6-13]. The QTL was designated as a major gene, Phs1, which could be precisely mapped within a 2.6 cM region [14]. Here, we identified a mitogen-activated protein kinase kinase 3 (MKK3) gene (designated TaMKK3-A) by a map-based approach as a candidate gene for the seed dormancy locus Phs1 on chromosome 4A in bread wheat. Complementation analysis showed that transformation of a dormant wheat cultivar with the TaMKK3-A allele from a nondormant cultivar clearly reduced seed dormancy. Cultivars differing in dormancy had a single nonsynonymous amino acid substitution in the kinase domain of the predicted MKK3 protein sequence, which may be associated with the length of seed dormancy. PMID:26948878

  2. Structural and functional characterization of human and murine C5a anaphylatoxins

    PubMed Central

    Schatz-Jakobsen, Janus Asbjørn; Yatime, Laure; Larsen, Casper; Petersen, Steen Vang; Klos, Andreas; Andersen, Gregers Rom

    2014-01-01

    Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8Δ71–73, and of murine C5a and C5a-desArg are reported. Whereas A8Δ71–73 adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species. PMID:24914981

  3. Zeolite 5A Catalyzed Etherification of Diphenylmethanol

    ERIC Educational Resources Information Center

    Cooke, Jason; Henderson, Eric J.; Lightbody, Owen C.

    2009-01-01

    An experiment for the synthetic undergraduate laboratory is described in which zeolite 5A catalyzes the room temperature dehydration of diphenylmethanol, (C[subscript 6]H[subscript 5])[subscript 2]CHOH, producing 1,1,1',1'-tetraphenyldimethyl ether, (C[subscript 6]H[subscript 5])[subscript 2]CHOCH(C[subscript 6]H[subscript 5])[subscript 2]. The…

  4. Synthesis and antibacterial activity of some novel 4-oxopyrido[2,3-a]phenothiazines.

    PubMed

    Al-Sinjilawi, Hadeel T; El-Abadelah, Mustafa M; Mubarak, Mohammad S; Al-Aboudi, Amal; Abadleh, Mohammed M; Mahasneh, Adel M; Ahmad, Asaad K M A

    2014-11-01

    A series of substituted 4-oxopyrido[2,3-a]phenothiazine-3-carboxylic acids (6a-d) were prepared via cyclization of the corresponding ethyl 7-(arylthioxy)-8-nitro(or azido)-4-oxoquinoline-3-carboxylates (3a-d/4a-d), followed by hydrolysis of the resultant esters (5a-d). Among these tetracyclics, compound 6a with unsubstituted terminal benzo-ring D was the most active against representative Gram-positive and Gram-negative bacterial strains. These compounds were also active against methicillin-resistant Staphylococcus aureus (MRSA), with very low toxicity to normal cells. Virtual screening using ligand-protein docking modeling predicted that the compounds 6a-d are potential inhibitors of the topoisomerase IV enzyme and that hydrophobic interactions and hydrogen bonds are the major molecular interactions between these compounds and the residues of the active site of topoisomerase IV. PMID:25220428

  5. Structural and functional characterization of human and murine C5a anaphylatoxins

    SciTech Connect

    Schatz-Jakobsen, Janus Asbjørn; Yatime, Laure Larsen, Casper; Petersen, Steen Vang; Klos, Andreas; Andersen, Gregers Rom

    2014-06-01

    The structure of the human C5aR antagonist, C5a-A8, reveals a three-helix bundle conformation similar to that observed for human C5a-desArg, whereas murine C5a and C5a-desArg both form the canonical four-helix bundle. These conformational differences are discussed in light of the differential C5aR activation properties observed for the human and murine complement anaphylatoxins across species. Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8{sup Δ71–73}, and of murine C5a and C5a-desArg are reported. Whereas A8{sup Δ71–73} adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

  6. Atrial Fibrillation and SCN5A Variants

    PubMed Central

    Savio-Galimberti, Eleonora; Darbar, Dawood

    2014-01-01

    Although atrial fibrillation (AF) is clinically and genetically a highly heterogeneous disease, recent studies suggest that the arrhythmia may arise because of interactions between genetic and acquired risk factors – the so called “double-hit” hypothesis. Genome-wide association studies have identified common AF susceptibility loci, and linkage analysis and candidate gene approaches have identified mutations in genes that encode for cardiac ion channels and signaling proteins; however, most of the heritability of AF still remains unexplained. The voltage-dependent cardiac sodium channel, encoded by SCN5A, conducts the main cardiac inward sodium current (INa) and is responsible for the upstroke of the atrial action potential. Mutations in SCN5A, which encodes the α-subunit of the NaV1.5 channel, have been linked with increased susceptibility to not only AF but also ventricular arrhythmias (long QT syndrome, Brugada syndrome), progressive cardiac conduction disease, and overlap syndromes with mixed arrhythmia phenotypes. Over the last decade, functional characterization of SCN5A mutations by expressing the channel in heterologous expression systems and applying cellular electrophysiological techniques has not only advanced our understanding of molecular mechanisms of AF but also potentially identified a mechanism-based approach to treating this common and morbid condition. PMID:25484998

  7. 5 A, version modifée

    PubMed Central

    Vallis, Michael; Piccinini-Vallis, Helena; Sharma, Arya M.; Freedhoff, Yoni

    2013-01-01

    Objectif Adapter le modèle 5 A pour offrir aux professionnels des soins primaires un cadre de counseling sur l'obésité. Sources des données Une recension systématique des ouvrages spécialisés a été effectuée dans MEDLINE à l'aide des expressions de recherche 5 A's (49 articles recensés, tous pertinents) et 5 A's et primary care (8 articles recensés, tous redondants). On a aussi fait une recherche dans les sites web du National Institute of Health et de l'Organisation mondiale de la Santé. Message principal L'approche 5 A (autorisation, analyse, avis, accord et aide ou ask, assess, advise, agree et assist en anglais), élaborée pour la cessation du tabagisme, peut être adaptée au counseling sur l'obésité. Demander l'autorisation de discuter du poids; ne pas porter de jugement et explorer la volonté du patient de changer. Analyser l'indice de masse corporelle, la circonférence de la taille et le stade de l'obésité; explorer les déclencheurs et les complications de l'excès de poids. Donner son avis sur les risques de l'obésité pour la santé, les bienfaits d'une modeste perte de poids, la nécessité d'une stratégie à long terme et les options de traitements. Se mettre d'accord sur des attentes, des objectifs et des changements comportementaux réalistes pour perdre du poids et sur les détails précis du plan de traitement. Assister dans l'identifcation et l'atténuation des obstacles; offrir des ressources, aider à trouver et à consulter les services appropriés et organiser un suivi périodique. Conclusion Les 5 A représentent une stratégie d'intervention comportementale fondée sur des données probantes qui a le potentiel d'améliorer le taux de réussite de la gestion du poids en soins primaires.

  8. 15 CFR 4a.3 - Classification levels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Classification levels. 4a.3 Section 4a.3 Commerce and Foreign Trade Office of the Secretary of Commerce CLASSIFICATION, DECLASSIFICATION... E.O. 12958. The levels established by E.O. 12958 (Top Secret, Secret, and Confidential) are the...

  9. Increased local concentration of complement C5a contributes to incisional pain in mice

    PubMed Central

    2011-01-01

    Background In our previous study, we demonstrated that local injection of complement C5a and C3a produce mechanical and heat hyperalgesia, and that C5a and C3a activate and sensitize cutaneous nociceptors in normal skin, suggesting a contribution of complement fragments to acute pain. Other studies also have shown that the complement system can be activated by surgical incision, and the systemic blockade of C5a receptor (C5aR) reduces incision-induced pain and inflammation. In this study, we further examined the possible contribution of wound area C5a to incisional pain. Methods Using of a hind paw incisional model, the effects of a selective C5aR antagonist, PMX53, on nociceptive behaviors were measured after incision in vivo. mRNA levels of C5 and C5aR in skin, dorsal root ganglia (DRG) and spinal cord, and C5a protein levels in the skin were quantified after incision. The responses of nociceptors to C5a were also evaluated using the in vitro skin-nerve preparation. Results Local administration of PMX53 suppressed heat hyperalgesia and mechanical allodynia induced by C5a injection or after hind paw incision in vivo. mRNA levels of C5 and C5aR in the skin, but not DRG and spinal cord, were dramatically increased after incision. C5a protein in the skin was also increased after incision. In vitro C5a did not increase the prevalence of fibers with ongoing activity in afferents from incised versus control, unincised skin. C5a sensitized C-fiber afferent responses to heat; however, this was less evident in afferents adjacent to the incision. PMX53 blocked sensitization of C-fiber afferents to heat by C5a but did not by itself influence ongoing activity or heat sensitivity in afferents innervating control or incised skin. The magnitude of mechanical responses was also not affected by C5a in any nociceptive fibers innervating incised or unincised skin. Conclusions This study demonstrates that high locally generated C5a levels are present in wounds for at least 72 hours

  10. MCNP4A: Features and philosophy

    SciTech Connect

    Hendricks, J.S.

    1993-05-01

    This paper describes MCNP, states its philosophy, introduces a number of new features becoming available with version MCNP4A, and answers a number of questions asked by participants in the workshop. MCNP is a general-purpose three-dimensional neutron, photon and electron transport code. Its philosophy is ``Quality, Value and New Features.`` Quality is exemplified by new software quality assurance practices and a program of benchmarking against experiments. Value includes a strong emphasis on documentation and code portability. New features are the third priority. MCNP4A is now available at Los Alamos. New features in MCNP4A include enhanced statistical analysis, distributed processor multitasking, new photon libraries, ENDF/B-VI capabilities, X-Windows graphics, dynamic memory allocation, expanded criticality output, periodic boundaries, plotting of particle tracks via SABRINA, and many other improvements. 23 refs.

  11. EE-3A Logging Report

    SciTech Connect

    Anderson, David W.

    1993-12-15

    Two logs of EE-3A were performed during the last couple of weeks. The first of which, was a Temperature/Casing-Collar Locator (CCL) log, which took place on Friday, December 10th., 1993. The second log was a Caliper log which was done in cooperation with the Dia-Log Company, of Odessa, TX. on Monday, December, 13th., 1993.

  12. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  13. Differential effects of the complement peptides, C5a and C5a des Arg on human basophil and lung mast cell histamine release.

    PubMed Central

    Schulman, E S; Post, T J; Henson, P M; Giclas, P C

    1988-01-01

    The ability of purified anaphylatoxins to induce human lung mast cell mediator release was investigated. In eight anti-IgE responsive (histamine release = 22 +/- 5%, mean +/- SEM) mast cell preparations of 1-96% purity, C5a and C5a des Arg (0.55 pg/ml to 55 micrograms/ml), failed to elicit or potentiate histamine release; lung fragments were similarly unresponsive. The related peptide C3a was also inactive. All anaphylatoxins failed to induce mast cell leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) release. LTC4 release was also negligible from basophils where C5a was a potent histamine release stimulus. Supernatants from C5a-challenged mast cells remained fully active on basophils, excluding carboxypeptidase inactivation of C5a as an explanation for the lung mast cell results. In contrast to lung, skin mast cells were C5a-responsive (histamine release = 8 +/- 1%, at 55 micrograms/ml, n = 2). We conclude that C5a, though devoid of activity on the human lung mast cell, is a human basophil and skin mast cell secretagogue. These findings demonstrate significant organ-specific heterogeneity in mast cell responsiveness. PMID:2449462

  14. Wnt5a Supports Osteogenic Lineage Decisions in Embryonic Stem Cells.

    PubMed

    Keller, Kevin C; Ding, Huawen; Tieu, Rudy; Sparks, Nicole R L; Ehnes, Devon D; Zur Nieden, Nicole I

    2016-07-01

    The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: β-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of β-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine. PMID:26956615

  15. In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

    PubMed Central

    Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O.; Delaney, William

    2016-01-01

    Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. PMID:26824950

  16. Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

    PubMed Central

    Pintor, J.; Torres, M.; Castro, E.; Miras-Portugal, M. T.

    1991-01-01

    1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site. PMID:1912985

  17. Homology modelling of the human eukaryotic initiation factor 5A (eIF-5A).

    PubMed

    Facchiano, A M; Stiuso, P; Chiusano, M L; Caraglia, M; Giuberti, G; Marra, M; Abbruzzese, A; Colonna, G

    2001-11-01

    Homology modelling of the human eIF-5A protein has been performed by using a multiple predictions strategy. As the sequence identity between the target and the template proteins is nearly 30%, which is lower than the commonly used threshold to apply with confidence the homology modelling method, we developed a specific predictive scheme by combining different sequence analyses and predictions, as well as model validation by comparison to structural experimental information. The target sequence has been used to find homologues within sequence databases and a multiple alignment has been created. Secondary structure for each single protein has been predicted and compared on the basis of the multiple sequence alignment, in order to evaluate and adjust carefully any gap. Therefore, comparative modelling has been applied to create the model of the protein on the basis of the optimized sequence alignment. The quality of the model has been checked by computational methods and the structural features have been compared to experimental information, giving us a good validation of the reliability of the model and its correspondence to the protein structure in solution. Last, the model was deposited in the Protein Data Bank to be accessible for studies on the structure-function relationships of the human eIF-5A. PMID:11742107

  18. Protein Tyrosine Phosphatase 4A3 (PTP4A3) Is Required for Xenopus laevis Cranial Neural Crest Migration In Vivo

    PubMed Central

    Maacha, Selma; Planque, Nathalie; Laurent, Cécile; Pegoraro, Caterina; Anezo, Océane; Maczkowiak, Frédérique; Monsoro-Burq, Anne H.; Saule, Simon

    2013-01-01

    Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development. PMID:24376839

  19. Water Masers Associated with IRAS 4A

    NASA Astrophysics Data System (ADS)

    Marvel, K. B.; Claussen, M.; Wootten, A.; Wilking, B.

    2000-05-01

    Observations of water masers near Young Stellar Objects (YSOs) are capable of revealing gas kinematics within a few AU of stars undergoing formation. Low mass YSOs are especially interesting as they are analogues of our own Sun. Several sources have been mapped in recent years and have shown proper motions indicative of bipolar outflowing material. Velocities are typically about 75 km/s and are clearly bipolar. Occasionally, bowshock structures have been observed (Imai et al. 1999, Claussen et al. 1999), showing the interaction between the jet and the ambient medium and perhaps indicating that the bipolar outflow occurs episodically. I present images and proper motions for the water masers associated with IRAS 4A. They are consistent with other sources of this class.

  20. Dataset of STAT5A status in breast cancer

    PubMed Central

    Mukhopadhyay, Utpal K.; Cass, Jamaica; Raptis, Leda; Craig, Andrew W.; Bourdeau, Véronique; Varma, Sonal; Gupta, Sandip Sen; Elliott, Bruce E.; Ferbeyre, Gerardo

    2016-01-01

    We analysed STAT5A gene expression in breast cancer using the Oncomine database. We exemplify four representative studies showing that STAT5A is generally downregulated in breast cancer. PMID:27014737

  1. Cytochrome P450 CYP3A in marsupials: cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Crittenden, Tamara A; Marchant, Ceilidh L; Stupans, Ieva; McKinnon, Ross A

    2011-11-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610 bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP

  2. Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species

    USGS Publications Warehouse

    Boshra, Hani; Wang, Tiehui; Hove-Madsen, Leif; Hansen, John D.; Li, Jun; Matlapudi, Anjun; Secombes, Christopher J.; Tort, Lluis; Sunyer, J. Oriol

    2005-01-01

    Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

  3. Alternative promoters of gene MAGE4a

    SciTech Connect

    De Plaen, E.; Naerhuyzen, B.; De Smet, C.

    1997-03-01

    Gene MAGE-4 (HGMW-approved symbol MAGE4) is expressed in several types of tumors, but not in normal tissues, except testis and placenta. The 5{prime} end of this gene contains eight homologous exons spread over a 5.8-kb region. These exons are alternatively spliced to a unique second exon and a unique third exon, which encodes a protein of 317 amino acids. The analysis of transcripts found in testis, placenta, and a sarcoma cell line showed that each of the alternative first exons is used in at least one of these tissues. Various regions of the promoter of the fifth alternative exon (1.5) were cloned in a luciferase reporter plasmid, and the constructs were transfected in a sarcoma cell line that expresses MAGE-4. Two Ets motifs located between positions -70 and -29 relative to the transcription start site were found to drive 55% of the promoter activity. A region containing an Sp1 consensus binding site located upstream of the two Ets motifs was found to be responsible for 44% of the transcriptional activity. MAGE-4a promoters 1.4 and 1.6, which also contain the Sp1 and the two Ets binding motifs, supported a level of transcription comparable to that of promoter 1.5, whereas promoter 1.1, which contains only one Ets binding site, was sixfold less active. In line with observations made with gene MAGE-1 (HGMW-approved symbol MAGE1), we found that promoter 1.5 stimulated a high level of transcription in a melanoma cell line that does not express MAGE-4. This suggests that the tumor-specific expression of MAGE genes is not determined by the presence of specific transcription factors. 26 refs., 7 figs., 2 tabs.

  4. Methyl 5-phenyl-1,2,3,4,4a,5,5a,13c-octahydro-6H-benzo[f]chromeno[3,4-b]indolizine-5a-carboxyl­ate

    PubMed Central

    Kamala, E. Theboral Sugi; Nirmala, S.; Sudha, L.; Kathiravan, S.; Raghunathan, R.

    2009-01-01

    In the title compound, C27H27NO3, the pyrrolidine ring exhibits a twist conformation and the piperidine ring exhibits a chair conformation. The pyrrolidine ring makes dihedral angles of 54.47 (5), 51.50 (5) and 73.37 (6)° with the napthalene ring system and the tetra­hydro­pyran and phenyl rings, respectively. The structure is stabilized by intra­molecular C—H⋯O and C—H⋯N inter­actions. PMID:21583607

  5. Effective suppression of C5a-induced proinflammatory response using anti-human C5a repebody.

    PubMed

    Hwang, Da-Eun; Choi, Jung-Min; Yang, Chul-Su; Lee, Joong-Jae; Heu, Woosung; Jo, Eun-Kyeong; Kim, Hak-Sung

    2016-09-01

    The strongest anaphylatoxin, C5a, plays a critical role in the proinflammatory responses, causing the pathogenesis of a number of inflammatory diseases including sepsis, asthma, and rheumatoid arthritis. Inhibitors of C5a thus have great potential as therapeutics for various inflammatory disorders. Herein, we present the development of a high-affinity repebody against human C5a (hC5a), which effectively suppresses the proinflammatory response. A repebody scaffold composed of leucine-rich repeat (LRR) modules was previously developed as an alternative protein scaffold. A repebody specifically binding to hC5a was selected through a phage display, and its affinity was increased up to 5 nM using modular engineering. The repebody was shown to effectively inhibit the production of C5a-induced proinflammatory cytokines by human monocytes. To obtain insight into a mode of action by the repebody, we determined its crystal structure in complex with hC5a. A structural analysis revealed that the repebody binds to the D1 and D3 regions of hC5a, overlapping several epitope residues with the hC5a receptor (hC5aR). It is thus likely that the repebody suppresses the hC5a-mediated immune response in monocytes by blocking the binding of hC5a to its receptor. The anti-hC5a repebody can be developed as a potential therapeutic for C5a-involved inflammatory diseases. PMID:27416759

  6. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    SciTech Connect

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A.

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  7. 18 CFR 3a.1 - Purpose.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Purpose. 3a.1 Section 3a.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.1 Purpose. This part 3a describes...

  8. Inhibition of C5a receptor alleviates experimental CNS lupus

    PubMed Central

    Jacob, Alexander; Hack, Bradley; Bai, Tao; Brorson, James R.; Quigg, Richard J.; Alexander, Jessy J.

    2010-01-01

    To investigate the role of C5a generated on complement activation in brain, the lupus model, MRL/lpr mice were treated with C5a receptor(R) antagonist (ant). Neutrophil infiltration, ICAM, TNF-α and iNOS mRNA expression, neuronal apoptosis and the expression of p-JNK, pSTAT1 and p-Erk were reduced and p-Akt increased on C5aR inhibition in MRL/lpr brains. MRL/lpr serum caused increased apoptosis in neurons showing that lupus had a direct effect on these cells. C5aRant pretreatment prevented the lupus serum induced loss of neuronal cells. Our findings demonstrate for the first time that C5a/C5aR signaling plays an important role in the pathogenesis of CNS lupus. PMID:20207017

  9. Lack of exonic sulfotransferase 4A1 mutations in controls and schizophrenia cases.

    PubMed

    Lewis, Aaron G; Minchin, Rodney F

    2009-02-01

    Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase expressed almost exclusively in the brain. The gene is located on chromosome 22q13.3, a region implicated in predisposition to schizophrenia. Recently, a variable microsatellite region located upstream of SULT4A1 was found to be associated with an increase in schizophrenia risk. We hypothesised that if functional dysregulation of SULT4A1 was involved in the aetiology of schizophrenia, then genetic variants in the coding sequence of SULT4A1 might be identified in cases compared with controls. To test this, we carried out a mutation analysis of the coding region (exons 2-7) in 71 Australian schizophrenia cases and 69 controls. We found no mutations, either synonymous or nonsynonymous, in either cohort. However, intronic variants (IVS5+12 C>T and IVS5+28 G>C) were identified, the frequency of which was not statistically different between cases and controls. The lack of polymorphisms in the coding region of the SULT4A1 gene is highly unusual and, along with its high conservation between species, suggests that SULT4A1 may have an important function in vivo. However, our findings do not support the hypothesis that germline mutations in the coding region of SULT4A1 contribute to susceptibility to schizophrenia. PMID:19125109

  10. Effects of the C5a anaphylatoxin and its relationship to cyclo-oxygenase metabolites in rabbit vascular strips.

    PubMed Central

    Hugli, T. E.; Marceau, F.

    1985-01-01

    Strips of rabbit blood vessels were suspended in vitro and responses to complement peptides C3a and C5a were recorded isotonically. Human C3a (up to 1.5 microM) was inactive on rabbit vascular strips. Human C5a (2.9-59 nM) decreased spontaneous activity of the rabbit portal vein under resting baseline tension. The C5a relaxed strips of portal vein and pulmonary artery that were precontracted with noradrenaline (NA, 200 nM). On the portal vein, C5a-induced relaxation was preceded by a transient contractile phase which decreased with repeated applications of C5a. The magnitude of C5a-induced relaxation of both vessels increased with repeated stimulation by C5a. Maximal levels of relaxation for the third application of C5a at 59 nM averaged 44% and 17% of the NA-induced contraction plateau in portal vein and pulmonary artery, respectively. Strips of rabbit aorta responded minimally to C5a. Indomethacin (5.6 microM) significantly inhibited C5a-induced relaxation of the portal vein and pulmonary artery but had no effect on the early contractile response of the portal vein. Mepyramine (10 microM) failed to modify the C5a response from either vessel, but it reduced the contractile phase of the C5a response on the portal vein when applied in conjunction with indomethacin. The drug SKF 88046, an end organ antagonist of thromboxane (TX) A2 and some contractile prostaglandins, reduced the contractile phase and increased relaxation of the portal vein to C5a but did not modify the response of the pulmonary artery. Radioimmunoassays for 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and TXB2 were performed on the fluid bathing rabbit isolated blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3921089

  11. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Definition of Underserved Rural Community. 5a.3... PROVISIONS RURAL PHYSICIAN TRAINING GRANT PROGRAM § 5a.3 Definition of Underserved Rural Community. Underserved Rural Community means a community: (a) Located in: (1) A non-Metropolitan County or...

  12. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Definition of Underserved Rural Community. 5a.3... PROVISIONS RURAL PHYSICIAN TRAINING GRANT PROGRAM § 5a.3 Definition of Underserved Rural Community. Underserved Rural Community means a community: (a) Located in: (1) A non-Metropolitan County or...

  13. A review of the Model 5A uranium hexafluoride cylinder

    SciTech Connect

    Dorning, R.E. II

    1989-05-23

    Both the Model 5A (Monel 400) and 5A (Monel 400) Modified five-inch cylinders have been used at the Portsmouth GDP to withdraw, store, and ship highly enriched uranium hexafluoride. As a result of a generic cracking problem with Monel 400 valve-boss material, a cylinder modification was implemented in the mid 1970s. This modification resulted in the violation of the ASME ''Code'' stamp status of the Model 5A Modified cylinder. Hydrostatic testing-to- rupture data indicated that the Model 5A Modified cylinders had ruptured strengths equivalent to that of the original Model 5A cylinders. An independent consultant reviewed the available information and confirmed that the Model 5A Modified cylinders ''will with proper maintenance continue to perform satisfactorily for many additional years of service.'' Based on the test data and consultant's review, DOE approved continued use of the 5A Modified cylinder and also requested procurement of replacement 5B cylinders be expedited. Currently, the 5A modified cylinders are in the production, storage, shipment cycle, and a sufficient number of 5B cylinders has been ordered to accommodate the projected product shipping requirements for the Navy flow. 3 tabs.

  14. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  15. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  16. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  17. 42 CFR 5a.3 - Definition of Underserved Rural Community.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Definition of Underserved Rural Community. 5a.3 Section 5a.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL... Professions Shortage Area, (under section 332(a)(1)(A) of the Public Health Service Act) or (2)...

  18. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  19. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  20. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  1. 42 CFR 5a.1 - Statutory basis and purpose.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Statutory basis and purpose. 5a.1 Section 5a.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL... the Public Health Service Act. These provisions define “underserved rural community” for purposes...

  2. Live Imaging of Xwnt5A-ROR2 Complexes

    PubMed Central

    Rahm, Karolin; Klessing, Tina; Nienhaus, Gerd Ulrich; Wedlich, Doris; Gradl, Dietmar

    2014-01-01

    Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner. PMID:25313906

  3. 18 CFR 3a.41 - Access requirements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Access requirements. 3a.41 Section 3a.41 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Access to Classified Materials § 3a.41 Access requirements. (a) The Personnel...

  4. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  5. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  6. 22 CFR 3a.1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Definitions. 3a.1 Section 3a.1 Foreign Relations DEPARTMENT OF STATE GENERAL ACCEPTANCE OF EMPLOYMENT FROM FOREIGN GOVERNMENTS BY MEMBERS OF THE UNIFORMED SERVICES § 3a.1 Definitions. For purposes of this part— (a) Applicant means any person...

  7. Pharmacological characterization of antagonists of the C5a receptor

    PubMed Central

    Paczkowski, Natalii J; Finch, Angela M; Whitmore, Jacqueline B; Short, Anna J; Wong, Allan K; Monk, Peter N; Cain, Stuart A; Fairlie, David P; Taylor, Stephen M

    1999-01-01

    Potent and highly selective small molecule antagonists have recently been developed by us for C5a receptors (C5aR) on human polymorphonuclear leukocytes (PMN). In this study we compared a new cyclic antagonist, F-[OPdChaWR], with an acyclic derivative, MeFKPdChaWr, for their capacities to bind to C5aR on human PMN and human umbilical artery membranes. We also compared their inhibition of myeloperoxidase (MPO) secretion from human PMNs and their inhibition of human umbilical artery contraction induced by human recombinant C5a.In both PMNs and umbilical artery, the cyclic and acyclic C5a antagonists displayed insurmountable antagonism against C5a. There were differences in selectivities for the C5aR with F-[OPdChaWR] (pKb 8.64±0.21) being 30 times more potent than MeFKPdChaWr (pKb 7.16±0.11, P<0.05) in PMNs, but of similar potency (pKb 8.19±0.38 vs pKb 8.28±0.29, respectively) in umbilical artery. This trend was also reflected in their relative binding affinities, both antagonists having similar affinities (−logIC50 values) for C5aR in umbilical artery membranes (F-[OPdChaWR], 7.00±0.46; MeFKPdChaWr, 7.23±0.17), whereas in PMN membranes the C5aR affinity of the cycle F-[OPdChaWR] (7.05±0.06) was four times higher than that of acyclic MeFKPdChaWr (6.43±0.24, P<0.05).In summary, the results reveal that these antagonists are insurmountable in nature against C5a for C5aR on at least two human cell types, and the differences in relative receptor binding affinities and antagonistic potencies against C5a are consistent with differences in receptors within these cell types. The nature of these differences is yet to be elucidated. PMID:10602324

  8. COL4A1 and COL4A2 mutations and disease: insights into pathogenic mechanisms and potential therapeutic targets

    PubMed Central

    Kuo, Debbie S.; Labelle-Dumais, Cassandre; Gould, Douglas B.

    2012-01-01

    Heterotrimers composed of collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) constitute one of the most abundant components of nearly all basement membranes. Accordingly, mutations in COL4A1 or COL4A2 are pleiotropic and contribute to a broad spectrum of disorders, including myopathy, glaucoma and hemorrhagic stroke. Here, we summarize the contributions of COL4A1 and COL4A2 mutations in human disease, integrate knowledge gained from model organisms and evaluate the implications for pathogenic mechanisms and therapeutic approaches. PMID:22914737

  9. 5. A PHOTO IMAGE FROM THE WEST SIDEWALK LOOKING EAST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. A PHOTO IMAGE FROM THE WEST SIDEWALK LOOKING EAST TOWARD THE ENTRY GATES AND PORTIONS OF RILEY PARK - Delphi Bridge on U.S. Route 421, Spanning Deer Creek at U.S. Route 421, Delphi, Carroll County, IN

  10. Synthesis, crystal structure, characterization and antifungal activity of pyrazolo[1,5-a]pyrimidines derivatives

    NASA Astrophysics Data System (ADS)

    Zhang, Jin; Peng, Ju-Fang; Wang, Tao; Wang, Ping; Zhang, Zun-Ting

    2016-09-01

    Under microwave radiation, isomers 2-(pyrazolo[1,5-a]pyrimidin-5-yl)phenols (3) and 2-(pyrazolo[1,5-a]pyrimidin-7-yl)phenols (4) were simultaneously obtained by the condensation of chromones and 3-aminopyrazoles. These two isomers were fully characterized by IR, 1H NMR, 13C NMR and HRMS. In addition, a representative product 5-chloro-2-(2-methyl-pyrazolo[1,5-a] pyrimidin-5-yl)phenol (3e) was further conformed by the single crystal X-ray diffraction. The antifungal abilities of the obtained products 3 and 4 were evaluated against five phytopathogenic fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani and Fusarium solani). The results revealed that 2-(pyrazolo[1,5-a]pyrimidin-5-yl)phenol (3a) and 4-chloro-2-(2-methylpyrazolo[1,5-a]pyrimidin-7-yl)phenol (4e) exhibited good antifungal abilities against Colletotrichum gloeosporioides with the IC50 values of 24.90 and 28.28 μg/mL, respectively.

  11. Wnt5a Suppresses β-catenin Signaling during Hair Follicle Regeneration

    PubMed Central

    Xing, Yizhan; Ma, Xiaogen; Guo, Haiying; Deng, Fang; Yang, Jin; Li, Yuhong

    2016-01-01

    Hair follicles display periodic growth. Wnt signaling is a critical regulator for hair follicle regeneration. Previously, we reported that Wnt5a inhibits the telogen-to-anagen transition of hair follicles, but the mechanism by which this process occurs has not yet been reported. Here, we determined the expression patterns of Wnt signaling pathway molecules by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry and found that β-catenin signaling was suppressed by Wnt5a. We then compared the phenotypes and expression patterns following β-catenin knockdown and Wnt5a overexpression during hair follicle regeneration induced by hair depilation and observed similar patterns. In addition, we performed a rescue experiment in the JB6 cell line and found that the inhibitory effect of Wnt5a on cell proliferation could be rescued by the addition of Wnt3a. Our data reveal that Wnt5a suppresses the activation of β-catenin signaling during hair follicle regeneration. PMID:27499692

  12. Apolipoprotein A1/C3/A5 haplotypes and serum lipid levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The association of single nucleotide polymorphisms (SNPs) in the apolipoprotein (Apo) A1/C3/A4/A5 gene cluster and serum lipid profiles is inconsistent. The present study was undertaken to detect the association between the ApoA1/C3/A5 gene polymorphisms and their haplotypes with serum lipid levels ...

  13. Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach

    PubMed Central

    Liu, Dandan; Ji, Juan; Ndongwe, Tanya P.; Michailidis, Eleftherios; Rice, Charles M.; Ralston, Robert

    2015-01-01

    While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations. PMID:25845863

  14. Conversion of abiraterone to D4A drives antitumor activity in prostate cancer

    PubMed Central

    Li, Zhenfei; Bishop, Andrew; Alyamani, Mohammad; Garcia, Jorge A.; Dreicer, Robert; Bunch, Dustin; Liu, Jiayan; Upadhyay, Sunil K.; Auchus, Richard J.; Sharifi, Nima

    2015-01-01

    Summary Prostate cancer resistance to castration occurs because tumors acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signaling by the androgen receptor (AR) and the development of castration-resistant prostate cancer (CRPC)1–3. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly require enzymatic reactions by 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A) and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes4,5. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival6,7. We hypothesized that abiraterone is converted by an enzyme to the more active Δ4-abiraterone (D4A) that blocks multiple steroidogenic enzymes and antagonizes the androgen receptor (AR), providing an additional explanation for abiraterone’s clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3βHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive AR antagonism by D4A is comparable to the potent antagonist, enzalutamide. D4A also has more potent antitumor activity against xenograft tumors than abiraterone. Our findings suggest an additional explanation – conversion to a more active agent – for abiraterone’s survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment. PMID:26030522

  15. Determinants of Dengue Virus NS4A Protein Oligomerization

    PubMed Central

    Lee, Chia Min; Xie, Xuping; Zou, Jing; Li, Shi-Hua; Lee, Michelle Yue Qi; Dong, Hongping; Qin, Cheng-Feng; Kang, Congbao

    2015-01-01

    ABSTRACT Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication. IMPORTANCE We report that DENV NS4A forms oligomers. Such NS4A oligomerization is mediated mainly through amino acids 50 to 76 (spanning the first

  16. Streptococcal C5a peptidase is a highly specific endopeptidase.

    PubMed Central

    Cleary, P P; Prahbu, U; Dale, J B; Wexler, D E; Handley, J

    1992-01-01

    Compositional analysis of streptococcal C5a peptidase (SCPA) cleavage products from a synthetic peptide corresponding to the 20 C-terminal residues of C5a demonstrated that the target cleavage site is His-Lys rather than Lys-Asp, as previously suggested. A C5a peptide analog with Lys replaced by Gln was also subject to cleavage by SCPA. This confirmed that His-Lys rather than Lys-Asp is the scissile bond. Cleavage at histidine is unusual but is the same as that suggested for a peptidase produced by group B streptococci. Native C5 protein was also resistant to SCPA, suggesting that the His-Lys bond is inaccessible prior to proteolytic cleavage by C5 convertase. These experiments showed that the streptococcal C5a peptidase is highly specific for C5a and suggest that its function is not merely to process protein for metabolic consumption but to act primarily to eliminate this chemotactic signal from inflammatory foci. Images PMID:1452354

  17. Wnt5a is essential for intestinal elongation in mice

    PubMed Central

    Cervantes, Sara; Yamaguchi, Terry P.; Hebrok, Matthias

    2009-01-01

    Summary Morphogenesis of the mammalian small intestine entails extensive elongation and folding of the primitive gut into a tightly coiled digestive tube. Surprisingly, little is known about the cellular and molecular mechanisms that mediate the morphological aspects of small intestine formation. Here, we demonstrate that Wnt5a, a member of the Wnt family of secreted proteins, is essential for the development and elongation of the small intestine from the midgut region. We found that the small intestine in mice lacking Wnt5a was dramatically shortened and duplicated, forming a bifurcated lumen instead of a single tube. In addition, cell proliferation was reduced and re-intercalation of post-mitotic cells into the elongating gut tube epithelium was disrupted. Thus, our study demonstrates that Wnt5a functions as a critical regulator of midgut formation and morphogenesis in mammals. PMID:19100728

  18. A structural study of Hypocrea jecorina Cel5A

    PubMed Central

    Lee, Toni M; Farrow, Mary F; Arnold, Frances H; Mayo, Stephen L

    2011-01-01

    Interest in generating lignocellulosic biofuels through enzymatic hydrolysis continues to rise as nonrenewable fossil fuels are depleted. The high cost of producing cellulases, hydrolytic enzymes that cleave cellulose into fermentable sugars, currently hinders economically viable biofuel production. Here, we report the crystal structure of a prevalent endoglucanase in the biofuels industry, Cel5A from the filamentous fungus Hypocrea jecorina. The structure reveals a general fold resembling that of the closest homolog with a high-resolution structure, Cel5A from Thermoascus aurantiacus. Consistent with previously described endoglucanase structures, the H. jecorina Cel5A active site contains a primarily hydrophobic substrate binding groove and a series of hydrogen bond networks surrounding two catalytic glutamates. The reported structure, however, demonstrates stark differences between side-chain identity, loop regions, and the number of disulfides. Such structural information may aid efforts to improve the stability of this protein for industrial use while maintaining enzymatic activity through revealing nonessential and immutable regions. PMID:21898652

  19. A structural study of Hypocrea jecorina Cel5A.

    PubMed

    Lee, Toni M; Farrow, Mary F; Arnold, Frances H; Mayo, Stephen L

    2011-11-01

    Interest in generating lignocellulosic biofuels through enzymatic hydrolysis continues to rise as nonrenewable fossil fuels are depleted. The high cost of producing cellulases, hydrolytic enzymes that cleave cellulose into fermentable sugars, currently hinders economically viable biofuel production. Here, we report the crystal structure of a prevalent endoglucanase in the biofuels industry, Cel5A from the filamentous fungus Hypocrea jecorina. The structure reveals a general fold resembling that of the closest homolog with a high-resolution structure, Cel5A from Thermoascus aurantiacus. Consistent with previously described endoglucanase structures, the H. jecorina Cel5A active site contains a primarily hydrophobic substrate binding groove and a series of hydrogen bond networks surrounding two catalytic glutamates. The reported structure, however, demonstrates stark differences between side-chain identity, loop regions, and the number of disulfides. Such structural information may aid efforts to improve the stability of this protein for industrial use while maintaining enzymatic activity through revealing nonessential and immutable regions. PMID:21898652

  20. Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

    PubMed Central

    Sebastian, Katrin; Detro-Dassen, Silvia; Rinis, Natalie; Fahrenkamp, Dirk; Müller-Newen, Gerhard; Merk, Hans F.; Schmalzing, Günther

    2013-01-01

    Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration. PMID:24376674

  1. Dual functions for WNT5A during cartilage development and in disease.

    PubMed

    Hosseini-Farahabadi, Sara; Geetha-Loganathan, Poongodi; Fu, Katherine; Nimmagadda, Suresh; Yang, Hoe Joong; Richman, Joy M

    2013-06-24

    Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the

  2. 5 A's Dean's Grant Second Year Monograph. 1980-82.

    ERIC Educational Resources Information Center

    Melnick, Curtis C., Ed.

    The monograph presents 12 papers on aspects of retraining College of Education faculty regarding mainstreaming of handicapped children. Papers grew out of the 5 A's Dean's Grant (Awareness, Access, Appropriateness, Assessment, and Accountability) at Roosevelt University. The following titles and authors are represented: "On Loving the Unlovable"…

  3. Historical review of C-5A lift distribution control systems

    NASA Technical Reports Server (NTRS)

    Disney, T. E.; Eckholdt, D. C.

    1976-01-01

    Analytical and experimental development work on various load alleviation systems for the C-5A is reviewed to trace the development of the technical and hardware concepts to the present time. Variations in system objectives, means of implementation and effects on loads and airplane performance, stability and control are discussed.

  4. Transmembrane water-flux through SLC4A11: a route defective in genetic corneal diseases

    PubMed Central

    Vilas, Gonzalo L.; Loganathan, Sampath K.; Liu, Jun; Riau, Andri K.; Young, James D.; Mehta, Jodhbir S.; Vithana, Eranga N.; Casey, Joseph R.

    2013-01-01

    Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11−/− mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues. PMID:23813972

  5. Mouse Models of SCN5A-Related Cardiac Arrhythmias

    PubMed Central

    Derangeon, Mickael; Montnach, Jérôme; Baró, Isabelle; Charpentier, Flavien

    2012-01-01

    Mutations of SCN5A gene, which encodes the α-subunit of the voltage-gated Na+ channel NaV1.5, underlie hereditary cardiac arrhythmic syndromes such as the type 3 long QT syndrome, cardiac conduction diseases, the Brugada syndrome, the sick sinus syndrome, a trial standstill, and numerous overlap syndromes. Patch-clamp studies in heterologous expression systems have provided important information to understand the genotype-phenotype relationships of these diseases. However, they could not clarify how SCN5A mutations can be responsible for such a large spectrum of diseases, for the late age of onset or the progressiveness of some of these diseases and for the overlapping syndromes. Genetically modified mice rapidly appeared as promising tools for understanding the pathophysiological mechanisms of cardiac SCN5A-related arrhythmic syndromes and several mouse models have been established. This review presents the results obtained on these models that, for most of them, recapitulate the clinical phenotypes of the patients. This includes two models knocked out for Nav1.5 β1 and β3 auxiliary subunits that are also discussed. Despite their own limitations that we point out, the mouse models still appear as powerful tools to elucidate the pathophysiological mechanisms of SCN5A-related diseases and offer the opportunity to investigate the secondary cellular consequences of SCN5A mutations such as the expression remodeling of other genes. This points out the potential role of these genes in the overall human phenotype. Finally, they constitute useful tools for addressing the role of genetic and environmental modifiers on cardiac electrical activity. PMID:22737129

  6. CYP3A in horse intestines.

    PubMed

    Tydén, Eva; Olsén, Lena; Tallkvist, Jonas; Larsson, Pia; Tjälve, Hans

    2004-12-01

    The intestinal enterocytes provide the initial site for cytochrome P450 (CYP)-mediated metabolism of orally absorbed xenobiotics. In man and some animal species, the CYP3A subfamily is highly expressed in the intestines and considered to be important in the first-pass metabolism of drugs and other xenobiotics. The aim of the present study was to investigate the mRNA expression, immunohistochemical localization and catalytic activity of CYP3A in the intestines of horse. Real-time RT-PCR analyses showed that the highest CYP3A mRNA expression was present in the duodenum with a decreasing level towards jejunum, ileum, cecum, and colon. The CYP3A mRNA expression in the liver was similar as in the anterior part of the jejunum, but about 4.5 times lower than in the anterior part of the duodenum. Immunohistochemistry showed CYP3A immunoreactivity in the cytoplasm of the enterocytes, which decreased distally along the intestinal tract. CYP3A-dependent metabolic activity rose slightly from the anterior to the distal part of the duodenum and the anterior part of the jejunum and then declined to the middle and distal parts of the jejunum and the ileum, cecum, and colon. Our results suggest that CYP3A in the small intestine plays a major role in first-pass metabolism and may affect bioavailability and therapeutic efficiency of some orally administrated drugs in horse. PMID:15541751

  7. Blockade of C5a and C5b-9 generation inhibits leukocyte and platelet activation during extracorporeal circulation.

    PubMed Central

    Rinder, C S; Rinder, H M; Smith, B R; Fitch, J C; Smith, M J; Tracey, J B; Matis, L A; Squinto, S P; Rollins, S A

    1995-01-01

    Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression). to specifically target complement activation in this model, we added a blocking mAb directed at the human C5 complement component and assessed its effect on complement and cellular activation. Compared with a control mAB, the anti-human C5 mAb profoundly inhibited C5a and soluble C5b-9 generation and serum complement hemolytic activity but had no effect on C3a generation. Additionally, the anti-human C5 mAb significantly inhibited neutrophil CD11b upregulation and abolished the increase in P-selectin-positive platelets and leukocyte-platelet conjugate formation compared to experiments performed with the control mAb. This suggests that the terminal components C5a and C5b-9, but not C3a, directly contribute to platelet and neutrophil activation during extracorporeal circulation. Furthermore, these data identify the C5 component as a site for therapeutic intervention in cardiopulmonary bypass. PMID:7657827

  8. 49 CFR 173.4a - Excepted quantities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 2 2014-10-01 2014-10-01 false Excepted quantities. 173.4a Section 173.4a Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SHIPPERS-GENERAL REQUIREMENTS FOR SHIPMENTS AND PACKAGINGS General §...

  9. 15 CFR 4a.5 - Duration of classification.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Duration of classification. 4a.5 Section 4a.5 Commerce and Foreign Trade Office of the Secretary of Commerce CLASSIFICATION..., except as provided in § 1.6(d) of E.O. 12958. Under E.O. 12958, information may be exempted...

  10. 15 CFR 4a.2 - Deputy Assistant Secretary for Security.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Deputy Assistant Secretary for Security. 4a.2 Section 4a.2 Commerce and Foreign Trade Office of the Secretary of Commerce CLASSIFICATION... Security. The Deputy Assistant Secretary for Security (DAS) is responsible for implementing E.O. 12958...

  11. 49 CFR 173.4a - Excepted quantities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false Excepted quantities. 173.4a Section 173.4a Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SHIPPERS-GENERAL REQUIREMENTS FOR SHIPMENTS AND PACKAGINGS General §...

  12. YO-3A parked on ramp

    NASA Technical Reports Server (NTRS)

    1997-01-01

    NASA's YO-3A parked on the Dryden ramp. The YO-3A aircraft was originally a Schweizer SGS-2-23 sailplane. During the late 1960s Lockheed modified over a dozen of these sailplanes to create ultra-quiet observation aircraft for use over South Vietnam during the conflict there. This particular YO-3A flew combat missions and was later sold to an airframe and powerplant mechanics school. NASA's Ames Research Center at Mountain Veiw, California, acquired the aircraft from the school in 1978. It restored the YO-3A to flight status and fitted it with wing- and tail-mounted microphones as an accoustic research aircraft. Ames operated it at Edwards Air Force Base for noise measurements of helicopters and tilt rotor aircraft. One set of tests in December 1995 obtained free-flight noise data on the XV-15 tilt rotor. NASA also used the YO-3A for sonic boom measurements of a NASA SR-71 assigned to the Dryden Flight Research Center. NASA transferred the YO-3A to Dryden in December 1997, and as of April 2001 it was in flyable storage there. The designation YO-3A indicates that this aircraft was a pre-production (Y) observation (O) aircraft. Even though the YO-3A saw operational use, the Y designation was never removed. Its 210-horsepower Continental V-6 was modified to reduce noise. The engine was connected to a propeller through a belt-driven reduction system. This reduced the propeller's rotation speed. The propeller blades themselves were made of birch plywood and were wider than standard propellers. The result of these modifications was an aircraft so quiet that its noise was drowned out by the background sounds.

  13. Absolute Radiometric Calibration of KOMPSAT-3A

    NASA Astrophysics Data System (ADS)

    Ahn, H. Y.; Shin, D. Y.; Kim, J. S.; Seo, D. C.; Choi, C. U.

    2016-06-01

    This paper presents a vicarious radiometric calibration of the Korea Multi-Purpose Satellite-3A (KOMPSAT-3A) performed by the Korea Aerospace Research Institute (KARI) and the Pukyong National University Remote Sensing Group (PKNU RSG) in 2015.The primary stages of this study are summarized as follows: (1) A field campaign to determine radiometric calibrated target fields was undertaken in Mongolia and South Korea. Surface reflectance data obtained in the campaign were input to a radiative transfer code that predicted at-sensor radiance. Through this process, equations and parameters were derived for the KOMPSAT-3A sensor to enable the conversion of calibrated DN to physical units, such as at-sensor radiance or TOA reflectance. (2) To validate the absolute calibration coefficients for the KOMPSAT-3A sensor, we performed a radiometric validation with a comparison of KOMPSAT-3A and Landsat-8 TOA reflectance using one of the six PICS (Libya 4). Correlations between top-of-atmosphere (TOA) radiances and the spectral band responses of the KOMPSAT-3A sensors at the Zuunmod, Mongolia and Goheung, South Korea sites were significant for multispectral bands. The average difference in TOA reflectance between KOMPSAT-3A and Landsat-8 image over the Libya 4, Libya site in the red-green-blue (RGB) region was under 3%, whereas in the NIR band, the TOA reflectance of KOMPSAT-3A was lower than the that of Landsat-8 due to the difference in the band passes of two sensors. The KOMPSAT-3Aensor includes a band pass near 940 nm that can be strongly absorbed by water vapor and therefore displayed low reflectance. Toovercome this, we need to undertake a detailed analysis using rescale methods, such as the spectral bandwidth adjustment factor.

  14. Calcium and cargoes as regulators of myosin 5a activity

    SciTech Connect

    Sellers, James R. Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-04-25

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

  15. Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons*

    PubMed Central

    Godoy, Juan A.; Arrázola, Macarena S.; Ordenes, Daniela; Silva-Alvarez, Carmen; Braidy, Nady; Inestrosa, Nibaldo C.

    2014-01-01

    The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca2+), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca2+ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders. PMID:25336659

  16. A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair.

    PubMed

    Encarnação, Marisa; Espada, Lília; Escrevente, Cristina; Mateus, Denisa; Ramalho, José; Michelet, Xavier; Santarino, Inês; Hsu, Victor W; Brenner, Michael B; Barral, Duarte; Vieira, Otília V

    2016-06-20

    Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis. PMID:27325790

  17. Rare hereditary COL4A3/COL4A4 variants may be mistaken for familial focal segmental glomerulosclerosis

    PubMed Central

    Malone, Andrew F; Phelan, Paul J; Hall, Gentzon; Cetincelik, Umran; Homstad, Alison; Alonso, Andrea; Jiang, Ruiji; Lindsey, Thomas; Wu, Guanghong; Sparks, Matthew A; Smith, Stephen R; Webb, Nicholas J A; Kalra, Philip; Adeyemo, Adebowale; Shaw, Andrey S; Conlon, Peter J; Jennette, J Charles; Howell, David N; Winn, Michelle P; Gbadegesin, Rasheed A

    2014-01-01

    Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes including inherited genetic defects with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome, thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane findings. Secondary FSGS is known to develop in classic Alport Syndrome at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical findings at diagnosis were proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin glomerular basement membrane, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes. PMID:25229338

  18. The NPG 7120.5A Electronic Review Process

    NASA Technical Reports Server (NTRS)

    McBrayer, Robert; Ives, Mark

    1998-01-01

    The use of electronics to review a document is well within the technical realm of today's state-of-the-art workplace. File servers and web site interaction are common tools for many NASA employees. The electronic comment processing described here was developed for the NPG 7120.5A review to augment the existing NASA Online Directives Information System (NODIS). The NODIS system is NASA's official system for formal review, approval and storage of NASA Directives. The electronic review process worked so well that NASA and other agencies may want to consider it as one of our "best practices." It was participatory decision making at its very best, a process that attracted dozens of very good ideas to improve the document as well as the way we can be managing projects far more effectively. The revision of NPG 7120.5A has significant implications for the way all elements of the Agency accomplish program and project management. Therefore, the review of NPG 7120.5A was an Agencywide effort with high visibility, heavy participation and a short schedule. The level of involvement created interest in supplementing the formal NODIS system with a system to collect comments efficiently and to allow the Centers and Codes to review and consolidate their comments into the official system in a short period of time. In addition, the Program Management Council Working Group (PMCWG), responsible for the revision of the document and the disposition of official comments, needed an electronic system to manage the disposition of comments, obtain PMCWG consensus on each disposition, and coordinate the disposition with the appropriate Headquarters Code that had submitted the official comment. The combined NASA and contractor talents and resources provided a system that supplemented the NODIS system and its operating personnel to produce a thorough review and approval of NPG 7120.5A on April 3, 1998, 7.5 months from the start of the process. The original six-month schedule is indicated. All

  19. 10. View looking south at Trestle 4, a typical low ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. View looking south at Trestle 4, a typical low trestle. The Del Dios Highway is visible in the background. - Lake Hodges Flume, Along San Dieguito River between Lake Hodges & San Dieguito Reservoir, Rancho Santa Fe, San Diego County, CA

  20. 4. A VIEW LOOKING TOWARDS THE TOWN OF WABASH, TO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. A VIEW LOOKING TOWARDS THE TOWN OF WABASH, TO THE NORTH, THROUGH THE BRIDGE, SHOWING ITS PORTAL, DECK, GUARD RAILS AND SUPERSTRUCTURE. - Wabash County Bridge No. 509, Spanning Wabash River at Carroll & Smith Streets, Wabash, Wabash County, IN

  1. 4. A VIEW LOOKING NORTH FROM THE EAST BANK OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. A VIEW LOOKING NORTH FROM THE EAST BANK OF LITTLE WALNUT CREEK. THE NARROW ARCH OF THE SPAN IS CLEARLY SHOWN - Putnam County Bridge No. 111, Spanning Little Walnut Creek on County Road 50, Greencastle, Putnam County, IN

  2. Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib.

    PubMed

    Towles, Joanna K; Clark, Rebecca N; Wahlin, Michelle D; Uttamsingh, Vinita; Rettie, Allan E; Jackson, Klarissa D

    2016-10-01

    Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinib-induced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (Km and kcat) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 kcat/Km = 6.8 μM(-1) min(-1) versus CYP3A5 kcat/Km = 1.3 μM(-1) min(-1)). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by kcat (maximum relative GSH adduct levels)/Km (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A- and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation. PMID:27450182

  3. Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin

    PubMed Central

    Russkamp, Norman F.; Ruemmler, Robert; Roewe, Julian; Moore, Bethany B.; Ward, Peter A.; Bosmann, Markus

    2015-01-01

    Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement-derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6-G+CD11b+ leukocytes to the alveolar spaces and severe alveolar-capillary barrier dysfunction (C5a-ALI; EC50[C5a] = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5adesArg) did not induce alveolar inflammation. The severity of C5a-ALI was aggravated in C5-deficient mice. Depletion of Ly6-G+ cells and use of C5aR1−/− bone marrow chimeras suggested an essential role of C5aR1+ hematopoietic cells in C5a-ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC-chemokines. Mice with genetic deficiency of CC-chemokine receptor (CCR) type 5, the common receptor of chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a-ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.—Russkamp, N. F., Ruemmler, R., Roewe, J., Moore, B. B., Ward, P. A., Bosmann, M. Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin. PMID:25999468

  4. MB3a Infrasound Sensor Evaluation.

    SciTech Connect

    Merchant, Bion J.; McDowell, Kyle D.

    2014-11-01

    Sandia National Laboratories has tested and evaluated a new infrasound sensor, the MB3a, manufactured by Seismo Wave. These infrasound sensors measure pressure output by a methodology developed by researchers at the French Alternative Energies and Atomic Energy Commission (CEA) and the technology was recently licensed to Seismo Wave for production and sales. The purpose of the infrasound sensor evaluation was to determine a measured sensitivity, transfer function, power, self-noise, dynamic range, seismic sensitivity, and self- calibration ability. The MB3a infrasound sensors are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

  5. COL4A1 Mutation in Preterm Intraventricular Hemorrhage

    PubMed Central

    Bilguvar, Kaya; DiLuna, Michael L.; Bizzarro, Matthew J.; Bayri, Yasar; Schneider, Karen C.; Lifton, Richard P.; Gunel, Murat; Ment, Laura R.

    2010-01-01

    Intraventricular hemorrhage is a common complication of preterm infants. Mutations in the type IV procollagen gene, COL4A1, are associated with cerebral small vessel disease with hemorrhage in adults and fetuses. We report a rare variant in COL4A1 associated with intraventricular hemorrhage in dizygotic preterm twins. These results expand the spectrum of diseases attributable to mutations in type IV procollagens. PMID:19840616

  6. Mark 4-A DSCC (Magellan-era) telemetry system description

    NASA Technical Reports Server (NTRS)

    Ross, D. L.

    1990-01-01

    An update to the description of the Deep Space Communications Complex portion of the Mark 4-A Telemetry system is given. This system is currently implemented at all signal processing centers. The upgrade of the telemetry system was undertaken primarily in support of the Voyager-Neptune Encounter and the Magellan mission. The Mark 3 Telemetry System is the predecessor of the Mark 4-A system.

  7. Establishment of totipotency does not depend on Oct4A

    PubMed Central

    Wu, Guangming; Han, Dong; Gong, Yu; Sebastiano, Vittorio; Gentile, Luca; Singhal, Nishant; Adachi, Kenjiro; Fischedick, Gerrit; Ortmeier, Claudia; Sinn, Martina; Radstaak, Martina; Tomilin, Alexey; Schöler, Hans R.

    2013-01-01

    SUMMARY Oct4A is a core component of the regulatory network of pluripotent cells, and by itself can reprogram neural stem cells into pluripotent cells in mouse and humans. However, its role in defining totipotency and inducing pluripotency during embryonic development is still unclear. We genetically eliminated maternal Oct4A using a Cre-lox approach in mouse and found that the establishment of totipotency was not affected, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4A expression, the embryos still formed Oct4-GFP– and Nanog–expressing inner cell masses, albeit non-pluripotent, indicating that Oct4A is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4A-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results clearly demonstrate that, in contrast to its role in the maintenance of pluripotency, maternal Oct4A is crucial for neither the establishment of totipotency in embryos, nor the induction of pluripotency in somatic cells using oocytes. PMID:23934214

  8. C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients

    PubMed Central

    Morris, Andrew Conway; Kefala, Kallirroi; Wilkinson, Thomas S.; Dhaliwal, Kevin; Farrell, Lesley; Walsh, Tim; Mackenzie, Simon J.; Reid, Hamish; Davidson, Donald J.; Haslett, Chris; Rossi, Adriano G.; Sallenave, Jean-Michel; Simpson, A. John

    2010-01-01

    Rationale Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood. Objectives To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment. Methods Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions. Measurements and Main Results Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody. Conclusions Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs

  9. Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3.

    PubMed

    Akbas, F; Aydin, Z

    2012-01-01

    Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels. PMID:22576912

  10. Yeast RNA polymerase II at 5 A resolution.

    PubMed

    Fu, J; Gnatt, A L; Bushnell, D A; Jensen, G J; Thompson, N E; Burgess, R R; David, P R; Kornberg, R D

    1999-09-17

    Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution. The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography. Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel. PMID:10499797

  11. Neuroimmune semaphorin 4A downregulates the severity of allergic response.

    PubMed

    Nkyimbeng-Takwi, E H; Shanks, K; Smith, E; Iyer, A; Lipsky, M M; Detolla, L J; Kikutani, H; Keegan, A D; Chapoval, S P

    2012-07-01

    To define the role of semaphorin 4A (Sema4A) in allergic response, we employed Sema4A⁻/⁻ and wild-type (WT) mice in the experimental model of ovalbumin (OVA)-induced allergic airway inflammation. We observed a selective increase in eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A⁻/⁻ mice relative to WT mice. This enhanced inflammatory response was associated with a selective increase in bronchoalveolar lavage (BAL) interleukin 13 (IL-13) content, augmented airway hyperreactivity, and lower regulatory T cell (Treg) numbers. In vivo allergen-primed Sema4A⁻/⁻ CD4+ T cells were more effective in transferring T helper type 2 (Th2) response to naive mice as compared with WT CD4+ T cells. T-cell proliferation and IL-13 productions in OVA₃₂₃₋₃₃₉-restimulated Sema4A⁻/⁻ cell cultures were upregulated. Generated bone marrow chimeras showed an equal importance of both lung-resident cell and inflammatory cell Sema4A expression in optimal disease regulation. These data provide a new insight into Sema4A biology and define Sema4A as an important regulator of Th2-driven lung pathophysiology. PMID:22472774

  12. Performance of MCNP4A on seven computing platforms

    SciTech Connect

    Hendricks, J.S.; Brockhoff, R.C.

    1994-12-31

    The performance of seven computer platforms has been evaluated with the MCNP4A Monte Carlo radiation transport code. For the first time we report timing results using MCNP4A and its new test set and libraries. Comparisons are made on platforms not available to us in previous MCNP timing studies. By using MCNP4A and its 325-problem test set, a widely-used and readily-available physics production code is used; the timing comparison is not limited to a single ``typical`` problem, demonstrating the problem dependence of timing results; the results are reproducible at the more than 100 installations around the world using MCNP; comparison of performance of other computer platforms to the ones tested in this study is possible because we present raw data rather than normalized results; and a measure of the increase in performance of computer hardware and software over the past two years is possible. The computer platforms reported are the Cray-YMP 8/64, IBM RS/6000-560, Sun Sparc10, Sun Sparc2, HP/9000-735, 4 processor 100 MHz Silicon Graphics ONYX, and Gateway 2000 model 4DX2-66V PC. In 1991 a timing study of MCNP4, the predecessor to MCNP4A, was conducted using ENDF/B-V cross-section libraries, which are export protected. The new study is based upon the new MCNP 25-problem test set which utilizes internationally available data. MCNP4A, its test problems and the test data library are available from the Radiation Shielding and Information Center in Oak Ridge, Tennessee, or from the NEA Data Bank in Saclay, France. Anyone with the same workstation and compiler can get the same test problem sets, the same library files, and the same MCNP4A code from RSIC or NEA and replicate our results. And, because we report raw data, comparison of the performance of other compute platforms and compilers can be made.

  13. Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction

    PubMed Central

    Pu, Xiangyuan; Ren, Meixia; An, Weiwei; Zhang, Ruoxin; Yan, Shunying; Situ, Haiteng; He, Xinjie; Chen, Yequn; Tan, Xuerui; Xiao, Qingzhong; Tucker, Arthur T.; Caulfield, Mark J.; Ye, Shu

    2016-01-01

    Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD

  14. Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction.

    PubMed

    Yang, Wei; Ng, Fu Liang; Chan, Kenneth; Pu, Xiangyuan; Poston, Robin N; Ren, Meixia; An, Weiwei; Zhang, Ruoxin; Wu, Jingchun; Yan, Shunying; Situ, Haiteng; He, Xinjie; Chen, Yequn; Tan, Xuerui; Xiao, Qingzhong; Tucker, Arthur T; Caulfield, Mark J; Ye, Shu

    2016-07-01

    Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD

  15. SOY PROTEIN ISOLATE INDUCES CYP3A1 AND CYP3A2 IN PREPUBERTAL RATS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feeding soy diets has been shown to induce cytochrome P450s in gene family CYP3A in Sprague-Dawley rat liver. We compared expression of CYP3A enzymes on PND33 rats fed casein or soy protein isolate (SPI+)-based AIN-93G diets continuously from gestational day 4 through PND 33 or the diets were switc...

  16. 4. A member of a buoy tender's deck force prepares ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. A member of a buoy tender's deck force prepares to trip the chain stopper. This will release the buoy's anchor for the plunge to the sea-floor. - U.S. Coast Guard Buoy Tenders, 180' Class, U.S. Coast Guard Headquarters, 2100 Second Street Southwest, Washington, District of Columbia, DC

  17. The Deep Space Network Tracking System, Mark 4-A, 1986

    NASA Technical Reports Server (NTRS)

    Wackley, J. A.

    1986-01-01

    The Deep Space Network (DSN) Tracking System provides highly precise measurements of spacecraft Doppler and range. It was recently extensively modified as part of the Mark 4-A implementation. The DSN Tracking System as currently implemented, its performance in support of Voyager 2, and plans for new implementation are described.

  18. Mark 4A DSN receiver-exciter and transmitter subsystems

    NASA Astrophysics Data System (ADS)

    Wick, M. R.

    1986-05-01

    The present configuration of the Mark 4A DSN Receiver-Exciter and Transmitter Subsystems is described. Functional requirements and key characteristics are given to show the differences in the capabilities required by the Networks Consolidation task for combined High Earth Orbiter and Deep Space Network tracking support.

  19. Mark 4A DSN receiver-exciter and transmitter subsystems

    NASA Technical Reports Server (NTRS)

    Wick, M. R.

    1986-01-01

    The present configuration of the Mark 4A DSN Receiver-Exciter and Transmitter Subsystems is described. Functional requirements and key characteristics are given to show the differences in the capabilities required by the Networks Consolidation task for combined High Earth Orbiter and Deep Space Network tracking support.

  20. Hepatitis C Virus NS3/4A Protease Inhibitors.

    PubMed

    López-Labrador, Francesc-Xavier

    2008-11-01

    Chronic hepatitis C virus infection is a global problem worldwide due to the lack of an effective therapy (the current standard of care treatment is effective in about 40-50% of the cases), and the difficulties in developing a protective vaccine. Chronic infection progresses to end-stage liver disease and liver failure in a considerable number of infected individuals. Once liver function is compromised, the only reliable therapeutic intervention is liver transplantation. Unfortunately, re-infection of the graft is unavoidable, and a new chronic hepatitis is early established in transplant recipients, that can result in graft loss. Thus, there is an urgent need for new, specifically targeted therapies for the treatment of HCV chronic infection. Among the viral proteins, the NS3/4A protease and the NS5b RNA-dependent RNA-polymerase, essential for the virus life cycle, have concentrated the efforts in the development of new antivirals, and some promising ones have already entered clinical trials. In particular, inhibitors of the HCV NS3/4A protease are the most advanced in clinical development. This review summarizes the available data for the most important HCV NS3/4A protease inhibitors in development, the most recent patents of these type of compounds, the envisioned options for future HCV therapies, and the eventual impact of HCV genetic variability on resistance to new NS3/4A protease inhibitors. PMID:18991798

  1. 4. A PHOTOGRAPH TAKEN FROM THE NORTH PORTAL, LOOKING SOUTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. A PHOTOGRAPH TAKEN FROM THE NORTH PORTAL, LOOKING SOUTH ON US 421. THE BANNISTER AND SIDEWALKS ARE THE ONLY MANIFESTATIONS OF THE BRIDGE. - Delphi Bridge on U.S. Route 421, Spanning Deer Creek at U.S. Route 421, Delphi, Carroll County, IN

  2. Mosaic hoxb4a Neuronal Pleiotropism in Zebrafish Caudal Hindbrain

    PubMed Central

    Ma, Leung-Hang; Punnamoottil, Beena; Rinkwitz, Silke; Baker, Robert

    2009-01-01

    To better understand how individual genes and experience influence behavior, the role of a single homeotic unit, hoxb4a, was comprehensively analyzed in vivo by clonal and retrograde fluorescent labeling of caudal hindbrain neurons in a zebrafish enhancer-trap YFP line. A quantitative spatiotemporal neuronal atlas showed hoxb4a activity to be highly variable and mosaic in rhombomere 7–8 reticular, motoneuronal and precerebellar nuclei with expression decreasing differentially in all subgroups through juvenile stages. The extensive Hox mosaicism and widespread pleiotropism demonstrate that the same transcriptional protein plays a role in the development of circuits that drive behaviors from autonomic through motor function including cerebellar regulation. We propose that the continuous presence of hoxb4a positive neurons may provide a developmental plasticity for behavior-specific circuits to accommodate experience- and growth-related changes. Hence, the ubiquitous hoxb4a pleitropism and modularity likely offer an adaptable transcriptional element for circuit modification during both growth and evolution. PMID:19536294

  3. Sulfated Tyrosines Contribute to the Formation of the C5a Docking Site of the Human C5a Anaphylatoxin Receptor

    PubMed Central

    Farzan, Michael; Schnitzler, Christine E.; Vasilieva, Natalya; Leung, Doris; Kuhn, Jens; Gerard, Craig; Gerard, Norma P.; Choe, Hyeryun

    2001-01-01

    The complement anaphylatoxin C5a and its seven-transmembrane segment (7TMS) receptor play an important role in host defense and in a number of inflammation-associated pathologies. The NH2-terminal domain of the C5a receptor (C5aR/CD88) contributes substantially to its ability to bind C5a. Here we show that the tyrosines at positions 11 and 14 of the C5aR are posttranslationally modified by the addition of sulfate groups. The sulfate moieties of each of these tyrosines are critical to the ability of the C5aR to bind C5a and to mobilize calcium. A C5aR variant lacking these sulfate moieties efficiently mobilized calcium in response to a small peptide agonist, but not to C5a, consistent with a two-site model of ligand association in which the tyrosine-sulfated region of the C5aR mediates the initial docking interaction. A peptide based on the NH2 terminus of the C5aR and sulfated at these two tyrosines, but not its unsulfated analogue or a doubly sulfated control peptide, partially inhibited C5a association with its receptor. These observations clarify structural and mutagenic studies of the C5a/C5aR association and suggest that related 7TMS receptors are also modified by functionally important sulfate groups on their NH2-terminal tyrosines. PMID:11342590

  4. Crystal structure of recombinant human T-cell cyclophilin A at 2.5 A resolution.

    PubMed

    Ke, H M; Zydowsky, L D; Liu, J; Walsh, C T

    1991-11-01

    The structure of the unligated human T-cell recombinant cyclophilin has been determined at 3 A resolution by multipole isomorphous replacement methods and refined at 2.5 A resolution to an R factor of 0.209. The root-mean-square errors of the bond lengths and bond angles are 0.013 A and 2.8 degrees from ideal geometry, respectively. The overall structure is a beta-barrel, consisting of eight antiparallel beta-strands wrapping around the barrel surface and two alpha-helices sitting on the top and the bottom closing the barrel. Inside the barrel, seven aromatic and other hydrophobic residues form a compact hydrophobic core. A loop of Lys-118 to His-126 and four beta-strands (B3-B6) constitute a pocket we speculate to be the binding site of cyclosporin A. PMID:1946361

  5. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro. PMID:15155547

  6. Familial Ehlers-Danlos syndrome with lethal arterial events caused by a mutation in COL5A1.

    PubMed

    Monroe, Glen R; Harakalova, Magdalena; van der Crabben, Saskia N; Majoor-Krakauer, Danielle; Bertoli-Avella, Aida M; Moll, Frans L; Oranen, Björn I; Dooijes, Dennis; Vink, Aryan; Knoers, Nine V; Maugeri, Alessandra; Pals, Gerard; Nijman, Isaac J; van Haaften, Gijs; Baas, Annette F

    2015-06-01

    Different forms of Ehlers-Danlos syndrome (EDS) exist, with specific phenotypes and associated genes. Vascular EDS, caused by heterozygous mutations in the COL3A1 gene, is characterized by fragile vasculature with a high risk of catastrophic vascular events at a young age. Classic EDS, caused by heterozygous mutations in the COL5A1 or COL5A2 genes, is characterized by fragile, hyperextensible skin and joint laxity. To date, vessel rupture in four unrelated classic EDS patients with a confirmed COL5A1 mutation has been reported. We describe familial occurrence of a phenotype resembling vascular EDS in a mother and her two sons, who all died at an early age from arterial ruptures. Diagnostic Sanger sequencing in the proband failed to detect aberrations in COL3A1, COL1A1, COL1A2, TGFBR1, TGFBR2, SMAD3, and ACTA2. Next, the proband's DNA was analyzed using a next-generation sequencing approach targeting 554 genes linked to vascular disease (VASCULOME project). A novel heterozygous mutation in COL5A1 was detected, resulting in an essential glycine substitution at the C-terminal end of the triple helix domain (NM_000093.4:c.4610G>T; p.Gly1537Val). This mutation was also present in DNA isolated from autopsy material of the index's brother. No material was available from the mother, but the mutation was excluded in her parents, siblings and in the father of her sons, suggesting that the COL5A1 mutation occurred in the mother's genome de novo. In conclusion, we report familial occurrence of lethal arterial events caused by a COL5A1 mutation. PMID:25845371

  7. Loxoprofen sodium induces the production of complement C5a in human serum.

    PubMed

    Kumagai, Tomoaki; Yamaguchi, Nozomi; Hirai, Hiroyuki; Kojima, Shigeyuki; Kodani, Yoshiko; Hashiguchi, Akihiko; Haida, Michiko; Nakamura, Masataka

    2016-04-01

    Basophil activation test (BAT) is an in vitro allergy test that is useful to identify allergens that cause IgE-dependent allergies. The test has been used to detect not only food allergies and allergies caused by environmental factors but also to detect drug hypersensitivity, which has been known to include IgE-independent reactions. In our preliminary studies in which BAT was applied to detect hypersensitivity of loxoprofen, a non-steroidal anti-inflammatory drug (NSAID), conventional BAT with incubation for 30min did not show basophil activation by means of increased CD203c expression. In this study, we extended the incubation time to 24h on the basis of the hypothesis that loxoprofen indirectly activates basophils. Basophils from healthy control donors as well as allergic patients showed up-regulation of CD203c after incubation with loxoprofen for 24h. Activation was induced using loxoprofen-treated serum. Proteomic and pharmacologic analyses revealed that serum incubation with loxoprofen generated an active complement component C5a, which induced CD203c expression via binding to the C5a receptor on basophils. Because C3a production was also detected after incubation for 24h, loxoprofen is likely to stimulate the complement classical pathway. Our findings suggest that the complement activation is involved in drug hypersensitivity and the suppression of this activation may contribute to the elimination of false positive of BAT for drug allergies. PMID:26854577

  8. 26 CFR 1.668(b)-3A - Computation of the beneficiary's income and tax for a prior taxable year.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... for a prior taxable year. 1.668(b)-3A Section 1.668(b)-3A Internal Revenue INTERNAL REVENUE SERVICE... Distributions of Trusts Applicable to Taxable Years Beginning Before January 1, 1969 § 1.668(b)-3A Computation... required to be furnished by him under § 1.668(b)- 4A(a). The gross income, related deductions, and...

  9. Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

    PubMed Central

    Papazachariou, Louiza; Demosthenous, Panayiota; Pieri, Myrtani; Papagregoriou, Gregory; Savva, Isavella; Stavrou, Christoforos; Zavros, Michael; Athanasiou, Yiannis; Ioannou, Kyriakos; Patsias, Charalambos; Panagides, Alexia; Potamitis, Costas; Demetriou, Kyproula; Prikis, Marios; Hadjigavriel, Michael; Kkolou, Maria; Loukaidou, Panayiota; Pastelli, Androulla; Michael, Aristos; Lazarou, Akis; Arsali, Maria; Damianou, Loukas; Goutziamani, Ioanna; Soloukides, Andreas; Yioukas, Lakis; Elia, Avraam; Zouvani, Ioanna; Polycarpou, Polycarpos; Pierides, Alkis; Voskarides, Konstantinos; Deltas, Constantinos

    2014-01-01

    Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies. PMID:25514610

  10. Frequency of COL4A3/COL4A4 mutations amongst families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response. Focal and segmental glomerulosclerosis is a frequent development during ageing.

    PubMed

    Papazachariou, Louiza; Demosthenous, Panayiota; Pieri, Myrtani; Papagregoriou, Gregory; Savva, Isavella; Stavrou, Christoforos; Zavros, Michael; Athanasiou, Yiannis; Ioannou, Kyriakos; Patsias, Charalambos; Panagides, Alexia; Potamitis, Costas; Demetriou, Kyproula; Prikis, Marios; Hadjigavriel, Michael; Kkolou, Maria; Loukaidou, Panayiota; Pastelli, Androulla; Michael, Aristos; Lazarou, Akis; Arsali, Maria; Damianou, Loukas; Goutziamani, Ioanna; Soloukides, Andreas; Yioukas, Lakis; Elia, Avraam; Zouvani, Ioanna; Polycarpou, Polycarpos; Pierides, Alkis; Voskarides, Konstantinos; Deltas, Constantinos

    2014-01-01

    Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies. PMID:25514610

  11. 4. A photo, looking north, northwest, from the stream bed ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. A photo, looking north, northwest, from the stream bed east of the bridge. In this view of the north entrados of the concrete arch, cracks in the concrete castings are evident, as are some rebars in the faults. Graffiti is evident on the underside of the arch - Vigo County Bridge No. 139, Spanning Sugar Creek at Seventy-fourth Place, Terre Haute, Vigo County, IN

  12. Syntax Editing for Mark 4-A System Performance Test Software

    NASA Technical Reports Server (NTRS)

    Jacobson, G. N.

    1983-01-01

    This article describes the syntax editing concepts used by the Operations Sustaining Engineering Section in implementing System Performance Test software for the Mark 4-A era. The processing functions are discussed, as well as the necessary data structures and table generation macros used in implementing those functions. In addition, the procedural and software interfaces which have been developed for users of the syntax editor are described, including the forms required for establishing directive and parameter characteristics.

  13. Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a

    PubMed Central

    Yao, Lin-Lin; Cao, Qing-Juan; Zhang, Hai-Man; Zhang, Jie; Cao, Yang; Li, Xiang-dong

    2015-01-01

    The tail-inhibition model is generally accepted for the regulation of myosin-5a motor function. Inhibited myosin-5a is in a folded conformation in which its globular tail domain (GTD) interacts with its head and inhibits its motor function, and high Ca2+ or cargo binding may reduce the interaction between the GTD and the head of myosin-5a, thus activating motor activity. Although it is well established that myosin-5a motor function is regulated by Ca2+, little is known about the effects of cargo binding. We previously reported that melanophilin (Mlph), a myosin-5a cargo-binding protein, is capable of activating myosin-5a motor function. Here, we report that Mlph-GTBDP, a 26 amino-acid-long peptide of Mlph, is sufficient for activating myosin-5a motor function. We demonstrate that Mlph-GTBDP abolishes the interaction between the head and GTD of myosin-5a, thereby inducing a folded-to-extended conformation transition for myosin-5a and activating its motor function. Mutagenesis of the GTD shows that the GTD uses two distinct, non-overlapping regions to interact with Mlph-GTBDP and the head of myosin-5a. We propose that the GTD is an allosteric protein and that Mlph allosterically inhibits the interaction between the GTD and head of myosin-5a, thereby activating myosin-5a motor function. PMID:26039755

  14. Differential Roles for "Nr4a1" and "Nr4a2" in Object Location vs. Object Recognition Long-Term Memory

    ERIC Educational Resources Information Center

    McNulty, Susan E.; Barrett, Ruth M.; Vogel-Ciernia, Annie; Malvaez, Melissa; Hernandez, Nicole; Davatolhagh, M. Felicia; Matheos, Dina P.; Schiffman, Aaron; Wood, Marcelo A.

    2012-01-01

    "Nr4a1" and "Nr4a2" are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either "Nr4a1" or "Nr4a2", we found that "Nr4a2" is necessary for both long-term…

  15. MADANALYSIS 5, a user-friendly framework for collider phenomenology

    NASA Astrophysics Data System (ADS)

    Conte, Eric; Fuks, Benjamin; Serret, Guillaume

    2013-01-01

    We present MADANALYSIS 5, a new framework for phenomenological investigations at particle colliders. Based on a C++ kernel, this program allows us to efficiently perform, in a straightforward and user-friendly fashion, sophisticated physics analyses of event files such as those generated by a large class of Monte Carlo event generators. MADANALYSIS 5 comes with two modes of running. The first one, easier to handle, uses the strengths of a powerful PYTHON interface in order to implement physics analyses by means of a set of intuitive commands. The second one requires one to implement the analyses in the C++ programming language, directly within the core of the analysis framework. This opens unlimited possibilities concerning the level of complexity which can be reached, being only limited by the programming skills and the originality of the user. Program summaryProgram title: MadAnalysis 5 Catalogue identifier: AENO_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENO_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Permission to use, copy, modify and distribute this program is granted under the terms of the GNU General Public License. No. of lines in distributed program, including test data, etc.: 31087 No. of bytes in distributed program, including test data, etc.: 399105 Distribution format: tar.gz Programming language: PYTHON, C++. Computer: All platforms on which Python version 2.7, Root version 5.27 and the g++ compiler are available. Compatibility with newer versions of these programs is also ensured. However, the Python version must be below version 3.0. Operating system: Unix, Linux and Mac OS operating systems on which the above-mentioned versions of Python and Root, as well as g++, are available. Classification: 11.1. External routines: ROOT (http://root.cern.ch/drupal/) Nature of problem: Implementing sophisticated phenomenological analyses in high-energy physics through a

  16. Methyl 5-ferrocenyl-5a-hydr­oxy-1-methyl-10-oxo-2,3,3a,4,5a,10-hexa­hydro-1H-indeno[1,2:2′,3′]furo[3′,4′-b]pyrrole-3a-carboxyl­ate

    PubMed Central

    Kamala, E. Theboral Sugi; Nirmala, S.; Sudha, L.; Kathiravan, S.; Raghunathan, R.

    2009-01-01

    In the title compound, [Fe(C5H5)(C21H20NO5)], the pyrrolidine and cyclo­penta­none rings exhibit a twist conformation. The pyrrolidine ring is almost perpendicular to the cyclo­penta­none ring, making a dihedral angle of 81.91 (6)°. The mol­ecular conformation is stabilized by an intra­molecular O—H⋯N hydrogen bond and C—H⋯O inter­actions. The crystal structure is stabilized by inter­molecular C—H⋯O inter­actions. PMID:21583045

  17. Crystal structure of rac-(3aR,4S,5aR,6S,9R,10aS,10bR)-3a,5a,9-tri­methyl­tetra­deca­hydro-6,9-ep­oxy­cyclo­hepta­[e]inden-4-ol monohydrate

    PubMed Central

    Schäfer, Andreas; Golz, Christopher; Preut, Hans; Strohmann, Carsten; Hiersemann, Martin

    2015-01-01

    The title hydrate, C17H28O2·H2O, was synthesized in order to determine the relative configuration of the tetra­cyclic framework. The fused 5,6,7-tricarbocyclic core exhibits an entire cis-annulation, featuring a 1,4-cis-relation of the angular methyl groups in the six-membered ring. The oxa bridge of the ep­oxy­cyclo­heptane moiety is oriented towards the concave face of the boat-shaped mol­ecule, whereas the angular methyl groups are directed towards the convex face. The asymmetric unit of the crystal contains two nearly identical formula units, which are related via a pseudo-centre of symmetry. The structure could be solved in the space groups I-4 and I41/a. The refinement in the acentric space group, however, gave significantly better results and these are used in this paper. O—H⋯O hydrogen bonds are observed between the organic mol­ecules, between the organic mol­ecules and the water mol­ecules, and between the water mol­ecules, forming a chain along the c-axis direction. PMID:26396907

  18. Nuclear AP/sub 4/A-binding activity of sea urchin embryos changes in relation to the initiation of S phase

    SciTech Connect

    Morioka, M.; Shimada, H.

    1986-01-01

    The AP/sub 4/A-binding activity of sea urchin embryos was studied using radioactively labelled diadenosine 5', 5'''-P/sup 1/,P/sup 4/-tetraphosphate (Ap/sub 4/A). Among various subcellular components that can bind (/sup 3/H)AP/sub 4/A, nuclei alone showed the highly specific Ap/sub 4/A-binding activity which was not influenced by the presence of AP/sub 4/A, AP/sub 5/A and GP/sub 4/G. The addition of an excess amount of ATP only slightly reduced the binding of (/sup 3/H)AP/sub 4/A to the nuclei. It was found that AP/sub 4/A binds to the residual proteinaceous structure of nuclei which was resistant to the extraction with 2 M NaCl. The nuclear AP/sub 4/A-binding activity fluctuated cyclically during each cell cycle, with at transient increase at the beginning of S phase followed by an abrupt-decrease within 10 min. When the initiation of S phase was blocked, the increase in the AP/sub 4/A-binding activity was also prevented. It seems that the binding of AP/sub 4/A to the nuclear structural protein is involved in the initiation of S phase.

  19. Current role of the minimally invasive direct aortic surgery for 3-A repair (MIDAS-3A).

    PubMed

    de Donato, Gaetano; Sarradon, Pierre; Weber, George; de Donato, Gianmarco

    2003-01-01

    Open aneurysmectomy and aortic graft is still associated with a relatively high morbidity and mortality. To decrease this surgical stress, less invasive procedure, MIDAS-3A technique (Minimally Invasive Direct Aortic Surgery for AAA) was developed, utilizing a 5 cm abdominal incision and a video-laparoscopic assistance (gas-less) to reach the AAA retroperitoneally. From Nov. 1999 to Dec. 2002, 80 patients underwent surgery. This technique provides all the benefits of an open surgical approach, to be combined with the advantages derived from minimized tissue trauma. A comparison between MIDAS-3A and CL (Conventional Laparotomy) was performed, monitorizing-nasogastric drainage;--initial feeding;--pulmonary functions (Vital Capacity, and Forced Expiration Volume);--Intensive Care Unit recovery (long stay);--length of hospital stay;--operative time;--blood loss. The perioperative (30 days) mortality (2.5%), and the morbidity (7.5%) was equal in both groups. No conversion to conventional laparotomy occurred. MIDAS-3A has significantly reduced length of hospital stay (3.5 days), and pulmonary dysfunctions. This technique provides all the benefits of open surgical approach, to be combined with the advantages derived from minimized tissue trauma. MIDAS-3A reduced trauma and pain, which resulted in a shorter hospital stay, and so lower expense and better financial consequences. PMID:14587105

  20. Inhibition of AKT promotes FOXO3a-dependent apoptosis in prostate cancer

    PubMed Central

    Das, T P; Suman, S; Alatassi, H; Ankem, M K; Damodaran, C

    2016-01-01

    Growth factor-induced activation of protein kinase-B (PKB), also known as AKT, induces pro-survival signaling and inhibits activation of pro-apoptotic signaling molecules including the Forkhead box O-3a (FOXO3a) transcription factor and caspase in transformed prostate cells in vitro. Earlier we reported that Withaferin-A (WA), a small herbal molecule, induces pro-apoptotic response-4 (Par-4) mediated apoptosis in castration-resistant prostate cancer (CRPC) cells. In the present study, we demonstrate that inhibition of AKT facilitates nuclear shuttling of FOXO3a where it regulates Par-4 transcription in CRPC cells. FOXO3a is upstream of Par-4 signaling, which is required for induction of apoptosis in CRPC cells. Promoter bashing studies and Ch-IP analysis confirm a direct interaction of FOXO3a and Par-4; a sequential deletion of FOXO3a-binding sites in the Par-4 promoter fails to induce Par-4 activation. To confirm these observations, we either overexpressed AKT or silenced FOXO3a activation in CRPC cells. Both methods inhibit Par-4 function and apoptosis is significantly compromised. In xenograft tumors derived from AKT-overexpressed CRPC cells, FOXO3a and Par-4 expression is downregulated, leading to aggressive tumor growth. Oral administration of WA to mice with xenograft tumors restores FOXO3a-mediated Par-4 functions and results in inhibited tumor growth. Finally, an inverse correlation of nuclear localization of AKT expression corresponds to cytoplasmic Par-4 localization in human prostate tissue array. Our studies suggest that Par-4 is one of the key transcriptional targets of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a appears to be an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC. PMID:26913603

  1. Inhibition of AKT promotes FOXO3a-dependent apoptosis in prostate cancer.

    PubMed

    Das, T P; Suman, S; Alatassi, H; Ankem, M K; Damodaran, C

    2016-01-01

    Growth factor-induced activation of protein kinase-B (PKB), also known as AKT, induces pro-survival signaling and inhibits activation of pro-apoptotic signaling molecules including the Forkhead box O-3a (FOXO3a) transcription factor and caspase in transformed prostate cells in vitro. Earlier we reported that Withaferin-A (WA), a small herbal molecule, induces pro-apoptotic response-4 (Par-4) mediated apoptosis in castration-resistant prostate cancer (CRPC) cells. In the present study, we demonstrate that inhibition of AKT facilitates nuclear shuttling of FOXO3a where it regulates Par-4 transcription in CRPC cells. FOXO3a is upstream of Par-4 signaling, which is required for induction of apoptosis in CRPC cells. Promoter bashing studies and Ch-IP analysis confirm a direct interaction of FOXO3a and Par-4; a sequential deletion of FOXO3a-binding sites in the Par-4 promoter fails to induce Par-4 activation. To confirm these observations, we either overexpressed AKT or silenced FOXO3a activation in CRPC cells. Both methods inhibit Par-4 function and apoptosis is significantly compromised. In xenograft tumors derived from AKT-overexpressed CRPC cells, FOXO3a and Par-4 expression is downregulated, leading to aggressive tumor growth. Oral administration of WA to mice with xenograft tumors restores FOXO3a-mediated Par-4 functions and results in inhibited tumor growth. Finally, an inverse correlation of nuclear localization of AKT expression corresponds to cytoplasmic Par-4 localization in human prostate tissue array. Our studies suggest that Par-4 is one of the key transcriptional targets of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a appears to be an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC. PMID:26913603

  2. Cytochrome P450 3A Conjugation to Ubiquitin in a Process Distinct from Classical Ubiquitination Pathway

    SciTech Connect

    Zangar, Richard C. ); Kimzey, Amy L.; Okita, Janice R.; Wunschel, David S. ); Edwards, Robert J.; Kim, Hyesook; Okita, Richard T.

    2001-12-01

    We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+), or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.

  3. Food-drug interactions via human cytochrome P450 3A (CYP3A).

    PubMed

    Fujita, Ken-ichi

    2004-01-01

    Food-drug interactions have been reported to occur in various systems in the body. The causes of these interactions are mainly divided into pharmacodynamic and pharmacokinetic processes. Among these processes, drug metabolism plays a crucial role in drug interactions. Metabolic food-drug interactions occur when a certain food alters the activity of a drug-metabolizing enzyme, leading to a modulation of the pharmacokinetics of drugs metabolized by the enzyme. A variety of interactions have been documented so far. Foods consisting of complex chemical mixtures, such as fruits, alcoholic beverages, teas, and herbs, possess the ability to inhibit or induce the activity of drug-metabolizing enzymes. According to results obtained thus far, cytochrome P450 3A4 (CYP3A4) appears to be a key enzyme in food-drug interactions. For example, interactions of grapefruit juice with felodipine and cyclosporine, red wine with cyclosporine, and St John's wort with various medicines including cyclosporine, have been demonstrated. The results indicate the requirement of dosage adjustment to maintain drug concentrations within their therapeutic windows. The CYP3A4-related interaction by food components may be related to the high level of expression of CYP3A4 in the small intestine, as well as its broad substrate specificity, as CYP3A4 is responsible for the metabolism of more than 50% of clinical pharmaceuticals. This review article summarizes the findings obtained to date concerning food-drug interactions and their clinical implications. It seems likely that more information regarding such interactions will accumulate in the future, and awareness is necessary for achieving optimal drug therapy. PMID:15663291

  4. WINCO maintenance before and after DOE 4330.4A

    SciTech Connect

    Remsburg, R.E.

    1993-04-27

    This report discusses numerous elements of the maintenance program at the Idaho Chemical Processing Plant (ICPP) which have undergone changes during the last 5 years. Looking back, many of the maintenance program changes prior to 1991 were fragmented. Maintenance program elements were being ratcheted to address specific productivity and safety concerns. The improvements were warranted, but often improvements were implemented without a systematic review of how they impacted other maintenance program elements. After 1990 when WINCO began using the Chapter II guidelines of DOE Order 4330.4A as a maintenance program model, there was a marked improvement in the quality of the maintenance program changes. The post 1990 changes have been part of a systematic plan that has been communicated to all of the maintenance players. The Chapter II guidelines and the maintenance implementation plan (MIP) personnel with a focus for improvement that was missing prior to 1991.

  5. MBE-4, a heavy ion multiple-beam experiment

    SciTech Connect

    Avery, R.T.; Chavis, C.S.; Fessenden, T.J.; Gough, D.E.; Henderson, T.F.; Keefe, D.; Meneghetti, J.R.; Pike, C.D.; Vanecek, D.L.; Warwick, A.I.

    1985-05-01

    MBE-4, a heavy-ion multiple beam induction linac being built at LBL in FY85/86, will model many features of a much longer device. It will accelerate four space-charge-dominated cesium ion beams from, for example, 0.2 MeV, 5 mA/beam, 3.0 ..mu..sec, 1.6 m length at injection to approx.0.8 MeV, 15 mA/beam, 1.0 ..mu..sec, 1.1 m length at the exit. It will permit study of simultaneous focussing, acceleration, current amplification and emittance growth of multiple space-charge-dominated ion beams. Some features of this accelerator are described. 11 refs., 5 figs.

  6. 75 FR 28188 - Airworthiness Directives; General Electric Company CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-20

    ... 7, 2010 (75 FR 910), we published a final rule AD, FR Doc, E9-30471, in the Federal Register. That... (GE) CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1 turbofan engines. The GE alert service bulletin...

  7. Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo

    PubMed Central

    Andersson, Emma R.; Prakash, Nilima; Bryja, Vitezslav; Bryjova, Lenka; Yamaguchi, Terry P.; Hall, Anita C.

    2008-01-01

    Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9–10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5–E13.5. Analysis of Wnt5a−/− mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a−/− mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors. PMID:18953410

  8. Correlation between NS5A Dimerization and Hepatitis C Virus Replication*

    PubMed Central

    Lim, Precious J.; Chatterji, Udayan; Cordek, Daniel; Sharma, Suresh D.; Garcia-Rivera, Jose A.; Cameron, Craig E.; Lin, Kai; Targett-Adams, Paul; Gallay, Philippe A.

    2012-01-01

    Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1–240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn2+ but not by Mg2+ or Mn2+. Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies. PMID:22801423

  9. β-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.

    PubMed

    Reynolds, Merrick S; Hancock, Chad R; Ray, Jason D; Kener, Kyle B; Draney, Carrie; Garland, Kevin; Hardman, Jeremy; Bikman, Benjamin T; Tessem, Jeffery S

    2016-07-01

    β-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study, we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here, we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion. PMID:27221116

  10. The Major Prognostic Features of Nuclear Receptor NR5A2 in Infiltrating Ductal Breast Carcinomas

    PubMed Central

    Chang, Li-Yun; Liu, Li-Yu D.; Roth, Don A.; Kuo, Wen-Hung; Hwa, Hsiao-Lin; Chang, King-Jen; Hsieh, Fon-Jou

    2015-01-01

    Background. Gene expression profiles of 181 breast cancer samples were analyzed to identify prognostic features of nuclear receptors NR5A1 and NR5A2 based upon their associated transcriptional networks. Methods. A supervised network analysis approach was used to build the NR5A-mediated transcriptional regulatory network. Other bioinformatic tools and statistical methods were utilized to confirm and extend results from the network analysis methodology. Results. NR5A2 expression is a negative factor in breast cancer prognosis in both ER(−) and ER(−)/ER(+) mixed cohorts. The clinical and cohort significance of NR5A2-mediated transcriptional activities indicates that it may have a significant role in attenuating grade development and cancer related signal transduction pathways. NR5A2 signature that conditions poor prognosis was identified based upon results from 15 distinct probes. Alternatively, the expression of NR5A1 predicts favorable prognosis when concurrent NR5A2 expression is low. A favorable signature of eight transcription factors mediated by NR5A1 was also identified. Conclusions. Correlation of poor prognosis and NR5A2 activity is identified by NR5A2-mediated 15-gene signature. NR5A2 may be a potential drug target for treating a subset of breast cancer tumors across breast cancer subtypes, especially ER(−) breast tumors. The favorable prognostic feature of NR5A1 is predicted by NR5A1-mediated 8-gene signature. PMID:26366408

  11. Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

    SciTech Connect

    Costa-Neto, Claudio M. . E-mail: claudio@fmrp.usp.br; Parreiras-e-Silva, Lucas T.; Ruller, Roberto; Oliveira, Eduardo B.; Miranda, Antonio; Oliveira, Laerte; Ward, Richard J.

    2006-09-01

    The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.

  12. Structure-Based Drug Design of Novel Potent and Selective Tetrahydropyrazolo[1,5-a]pyrazines as ATR Inhibitors

    PubMed Central

    2014-01-01

    A saturation strategy focused on improving the selectivity and physicochemical properties of ATR inhibitor HTS hit 1 led to a novel series of highly potent and selective tetrahydropyrazolo[1,5-a]pyrazines. Use of PI3Kα mutants as ATR crystal structure surrogates was instrumental in providing cocrystal structures to guide the medicinal chemistry designs. Detailed DMPK studies involving cyanide and GSH as trapping agents during microsomal incubations, in addition to deuterium-labeled compounds as mechanistic probes uncovered the molecular basis for the observed CYP3A4 TDI in the series. PMID:25589927

  13. S4: A Spatial-spectral model for Speckle Suppression

    NASA Astrophysics Data System (ADS)

    Fergus, Rob; Hogg, David W.; Oppenheimer, Rebecca; Brenner, Douglas; Pueyo, Laurent

    2014-10-01

    High dynamic range imagers aim to block or eliminate light from a very bright primary star in order to make it possible to detect and measure far fainter companions; in real systems, a small fraction of the primary light is scattered, diffracted, and unocculted. We introduce S4, a flexible data-driven model for the unocculted (and highly speckled) light in the P1640 spectroscopic coronagraph. The model uses principal components analysis (PCA) to capture the spatial structure and wavelength dependence of the speckles, but not the signal produced by any companion. Consequently, the residual typically includes the companion signal. The companion can thus be found by filtering this error signal with a fixed companion model. The approach is sensitive to companions that are of the order of a percent of the brightness of the speckles, or up to 10-7 times the brightness of the primary star. This outperforms existing methods by a factor of two to three and is close to the shot-noise physical limit.

  14. S4: A spatial-spectral model for speckle suppression

    SciTech Connect

    Fergus, Rob; Hogg, David W.; Oppenheimer, Rebecca; Brenner, Douglas; Pueyo, Laurent

    2014-10-20

    High dynamic range imagers aim to block or eliminate light from a very bright primary star in order to make it possible to detect and measure far fainter companions; in real systems, a small fraction of the primary light is scattered, diffracted, and unocculted. We introduce S4, a flexible data-driven model for the unocculted (and highly speckled) light in the P1640 spectroscopic coronagraph. The model uses principal components analysis (PCA) to capture the spatial structure and wavelength dependence of the speckles, but not the signal produced by any companion. Consequently, the residual typically includes the companion signal. The companion can thus be found by filtering this error signal with a fixed companion model. The approach is sensitive to companions that are of the order of a percent of the brightness of the speckles, or up to 10{sup –7} times the brightness of the primary star. This outperforms existing methods by a factor of two to three and is close to the shot-noise physical limit.

  15. Real-Time Imaging of Interactions of Neutrophils with Cryptococcus neoformans Demonstrates a Crucial Role of Complement C5a-C5aR Signaling

    PubMed Central

    Sun, Donglei; Zhang, Mingshun; Liu, Gongguan; Wu, Hui; Zhu, Xiaoping; Zhou, Hong

    2015-01-01

    Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live-cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real time. Neutrophils were directly seen to chase C. neoformans cells and then rapidly internalize them. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in optimal phagocytosis and subsequent killing of the organisms. The addition of recombinant complement C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that the C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 mitogen-activated protein (MAP) kinases (MAPKs) in neutrophils. Inhibition of the p38 MAPK pathway, but not the Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils toward C. neoformans but almost completely abolished phagocytosis and killing of the organisms by neutrophils. C5a-C5aR signaling induced enhanced surface expression of CD11b. Interestingly, the original surface expression of CD11b was essential and sufficient for neutrophils to attach to C. neoformans but was unable to mediate phagocytosis. In contrast, the enhanced surface expression of CD11b induced by C5a-C5aR signaling was essential for neutrophil phagocytosis and subsequent killing of yeast cells. Collectively, this is the first report of the dynamic interactions of neutrophils with C. neoformans, demonstrating a crucial role of C5a-C5aR signaling in neutrophil killing of C. neoformans in real time. PMID:26502909

  16. Functional Roles for C5a and C5aR but Not C5L2 in the Pathogenesis of Human and Experimental Cerebral Malaria

    PubMed Central

    Kim, Hani; Erdman, Laura K.; Lu, Ziyue; Serghides, Lena; Zhong, Kathleen; Dhabangi, Aggrey; Musoke, Charles; Gerard, Craig; Cserti-Gazdewich, Christine; Liles, W. Conrad

    2014-01-01

    The host immune response plays an important role in the onset and progression of cerebral malaria (CM). The complement system is an essential component of the innate immune response to malaria, and its activation generates the anaphylatoxin C5a. To test the hypothesis that C5a signaling contributes to the pathogenesis of CM, we investigated a causal role for the C5a receptors C5aR and C5L2 in a mouse model of experimental CM (ECM) induced by Plasmodium berghei ANKA infection, and using a case-control design, we examined levels of C5a in plasma samples from Ugandan children presenting with CM or uncomplicated malaria (UM). In the ECM model, C5aR−/− mice displayed significantly improved survival compared to their wild-type (WT) counterparts (P = 0.004), whereas C5L2−/− mice showed no difference in survival from WT mice. Improved survival in C5aR−/− mice was associated with reduced levels of the proinflammatory cytokines tumor necrosis factor (TNF) and gamma interferon (IFN-γ) and the chemokine, monocyte chemoattractant protein 1 (MCP-1) (CCL2). Furthermore, endothelial integrity was enhanced, as demonstrated by increased levels of angiopoietin-1, decreased levels of angiopoietin-2 and soluble ICAM-1, and decreased Evans blue extravasation into brain parenchyma. In the case-control study, the median levels of C5a at presentation were significantly higher in children with CM versus those in children with UM (43.7 versus 22.4 ng/ml; P < 0.001). These findings demonstrate that C5a is dysregulated in human CM and contributes to the pathogenesis of ECM via C5aR-dependent inflammation and endothelial dysfunction. PMID:24191300

  17. Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins*

    PubMed Central

    Nguyen, Suong; Leija, Chrisopher; Kinch, Lisa; Regmi, Sandesh; Li, Qiong; Grishin, Nick V.; Phillips, Margaret A.

    2015-01-01

    The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens. PMID:26082486

  18. Broadcasting Satellite-3A and -3B (BS-3A and 3B)

    NASA Technical Reports Server (NTRS)

    Horii, M.; Funakawa, K.

    1991-01-01

    The BS-3A and -3B will provide direct color TV broadcasting to the Japanese mainland and remote islands. The satellites will be launched from Tanegashima Space Center by a type H-1 launch vehicle. The coverage will consist of the 26-m antenna and the 34-m antenna as a backup support for the transfer and drift orbits. Maximum support will consist of one 8-hour track per station for a seven day period, plus 23 days of contingency support from all complexes. Information is given in tabular form for Deep Space Network support, frequency assignments, telemetry, command, and tracking support responsibility.

  19. Galectin-4, a novel neuronal regulator of myelination.

    PubMed

    Stancic, Mirjana; Slijepcevic, Davor; Nomden, Anita; Vos, Michel J; de Jonge, Jenny C; Sikkema, Arend H; Gabius, Hans-J; Hoekstra, Dick; Baron, Wia

    2012-05-01

    Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin-specific proteins and lipids. Galectin-4, a tandem-repeat-type lectin with affinity to sulfatide and nonsialylated termini of N-glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin-4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin-4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin-4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin-4 or anti-galectin-4 antibodies were added to co-cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin-4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin-4. Interestingly, in co-cultures, a reduced release of endogenous galectin-4 correlated with the onset of myelination. Moreover, galectin-4-reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin-4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination. © 2012 Wiley Periodicals, Inc. PMID:22431161

  20. Human colonic epithelial cells detect and respond to C5a via apically expressed C5aR through the ERK pathway.

    PubMed

    Cao, Qi; McIsaac, Shayla M; Stadnyk, Andrew W

    2012-06-15

    Intestinal epithelial cells (IECs) exhibit numerous adaptations to maintain barrier function as well as play sentinel roles by expressing receptors for microbial products and antimicrobial peptides. The complement system is another important innate sensing and defense mechanism of the host against bacteria and increasing evidence shows that complement plays a role in colitis. The split component C5a is a potent proinflammatory molecule, and the C5a receptor (C5aR) CD88 has been reported on multiple cell types. Here, we examined the question of whether human colonic cell lines can detect activated complement via C5aR and what signaling pathway is critical in the subsequent responses. T84, HT29, and Caco2 cell lines all possessed mRNA and protein for C5aR and the decoy receptor C5L2. Polarized cells expressed the proteins on the apical cell membrane. C5a binding to the C5aR on human IECs activates the ERK pathway, which proved critical for a subsequent upregulation of IL-8 mRNA, increased permeability of monolayers, and enhanced proliferation of the cells. The fact that human IECs are capable of detecting complement activation in the lumen via this anaphylatoxin receptor highlights the potential for IECs to detect pathogens indirectly through complement activation and be primed to amplify the host response through heightened inflammatory mediator expression to further recruit immune cells. PMID:22496247

  1. Calystegine B4, a novel trehalase inhibitor from Scopolia japonica.

    PubMed

    Asano, N; Kato, A; Kizu, H; Matsui, K; Watson, A A; Nash, R J

    1996-10-31

    GLC-MS analysis has been developed for screening plants of the family Solanaceae for new calystegines. GLC-MS analyses of the extract of Scopolia japonica showed the presence of a new tetrahydroxy-nor-tropane alkaloid in addition to the known calystegines A3, A5, B1, B2, B3, and C1. We gave this new alkaloid the trivial name calystegine B4. The structure of calystegine B4 was determined as 1 alpha, 2 beta, 3 alpha, 4 alpha-tetrahydroxy-nor-tropane from a variety of NMR spectral data. Calystegines B1, B2, and C1 are potent competitive inhibitors with Ki values ranging from 10(-6) to 10(-7) M for almond beta-glucosidase, while calystegine B4 inhibited this enzyme in a competitive manner, with a Ki value of 7.3 microM. Calystegine B2 is also a potent inhibitor of green coffee bean alpha-galactosidase, whereas calystegine B4 exhibited no significant activity for this enzyme. Among rat intestinal glycosidases, only trehalase was potently inhibited by calystegine B4, with an IC50 value of 9.8 microM. Furthermore, calystegine B4 potently inhibited pig kidney trehalase in a competitive manner, with a Ki value of 1.2 microM, but it was almost inactive against yeast and fungal trehalases. PMID:8938376

  2. WNT5A-NFAT Signaling Mediates Resistance to Apoptosis in Pancreatic Cancer1 2

    PubMed Central

    Griesmann, Heidi; Ripka, Stefanie; Pralle, Moritz; Ellenrieder, Volker; Baumgart, Sandra; Buchholz, Malte; Pilarsky, Christian; Aust, Daniela; Gress, Thomas M; Michl, Patrick

    2013-01-01

    Introduction WNT5A belongs to the Wnt family of secreted signaling molecules. Using transcriptional profiling, we previously identified WNT5A as target of the antiapoptotic transcription factor CUX1 and demonstrated high expression levels in pancreatic cancer. However, the impact of WNT5A on drug resistance and the signaling pathways employed by WNT5A remain to be elucidated. Objectives This project aims to decipher the impact of WNT5A on resistance to apoptosis and the signaling pathways employed by WNT5A in pancreatic cancer. Methods The impact of WNT5A and its downstream effectors on tumor growth and drug resistance was studied in vitro and in xenograft models in vivo. Tissue microarrays of pancreatic cancer specimens were employed for immunohistochemical studies. Results Knockdown of WNT5A results in a significant increase in drug-induced apoptosis. In contrast, overexpression of WNT5A or addition of recombinant WNT5A mediates resistance to apoptosis in vitro. In our attempt to identify downstream effectors of WNT5A, we identified the transcription factor nuclear factor of activated T cells c2 (NFATc2) as transcriptional target of WNT5A signaling. NFATc2 confers a strong antiapoptotic phenotype mediating at least in part the effects of WNT5A on drug resistance and tumor cell survival. In vivo, WNT5A expression leads to resistance to gemcitabine-induced apoptosis in a xenograft model, which is paralleled by up-regulation of NFATc2. Both WNT5A and NFATc2 proteins are highly expressed in human pancreatic cancer tissues and their expression levels correlated significantly. Conclusion We identified the WNT5A-NFATc2 axis as important mediator of drug resistance in pancreatic cancer. PMID:23359789

  3. Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

    PubMed

    Zhang, Jia-xiang; Zha, Wan-sheng; Ye, Liang-ping; Wang, Feng; Wang, Hui; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2016-02-01

    We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals. PMID:26095957

  4. Interplay between invertebrate C3a with vertebrate macrophages: functional characterization of immune activities of amphioxus C3a.

    PubMed

    Gao, Zhan; Li, Mengyang; Wu, Jie; Zhang, Shicui

    2013-10-01

    Our current knowledge of the structure and function of C3a comes from the study of vertebrate C3a anaphylatoxins, virtually nothing is known about the structure and function of C3a molecules in invertebrates. Here we demonstrated that C3a from the invertebrate chordate Branchiostoma japonicum, BjC3a, was similar to vertebrate C3a possessing potential antibacterial activity, as revealed by sequence analysis and computational modeling. The antibacterial activity of BjC3a was definitely confirmed by both antibacterial assay and TEM observation showing that recombinant BjC3a was directly bactericidal. Additionally, recombinant BjC3a, like vertebrate C3a, was capable of inducing sea bass macrophage migration and enhancing macrophage phagocytosis and respiratory burst response. Moreover, recombinant BjC3a-desArg (generated by removal of the C-terminal arginine), like mammalian C3a-desArg, retained the immunological activities of BjC3a such as antibacterial and respiratory burst-stimulating activities, indicating that the immunological functions of C3a-desArg were conserved throughout chordate evolution. Altogether, our findings show that invertebrate (amphioxus) BjC3a is able to interact with vertebrate (sea bass) macrophages and mediate immune activities, suggesting the emergence of the inflammatory pathway of the complement system similar to that of vertebrates in the basal chordate amphioxus. PMID:23954696

  5. Inhibition of hepatitis C virus infection by NS5A-specific aptamer.

    PubMed

    Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wang, Xiaohong; Yang, Darong; Qin, Yuwen; Yu, Rong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen

    2014-06-01

    To increase efficacy of hepatitis C treatment, future regiments will incorporate multiple direct-acting antiviral drugs. HCV NS5A protein was expressed and purified. Aptamers against NS5A were screened and obtained by the selective evolution of ligands by exponential enrichment approach and the antiviral actions of the aptamers were tested. The mechanisms through which the aptamers exert their antiviral activity were explored. The aptamers NS5A-4 and NS5A-5 inhibit HCV RNA replication and infectious virus production without causing cytotoxicity in human hepatocytes. The aptamers do not affect hepatitis B virus replication in HepG2.2.15 cells. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) are not induced by the aptamers in HCV-infected hepatocytes. Further study shows that domain I and domain III of NS5A protein are involved in the suppression of HCV RNA replication and infectious virus production by NS5A-4. Y2105H within NS5A is the major resistance mutation identified. NS5A aptamer disrupts the interaction of NS5A with core protein. The data suggest that the aptamers against NS5A protein may exert antiviral effects through inhibiting viral RNA replication, preventing the interaction of NS5A with core protein. Aptamers for NS5A may be used to understand the mechanisms of virus replication and assembly and served as potential therapeutic agents for hepatitis C. PMID:24713119

  6. Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator.

    PubMed Central

    Kato, N; Lan, K H; Ono-Nita, S K; Shiratori, Y; Omata, M

    1997-01-01

    The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells. Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis. PMID:9343247

  7. 26 CFR 1.148-5A - Yield and valuation of investments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Yield and valuation of investments. 1.148-5A Section 1.148-5A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME..., 1997 § 1.148-5A Yield and valuation of investments. (a) through (b)(2)(ii) . For guidance see §...

  8. S4 : A free electromagnetic solver for layered periodic structures

    NASA Astrophysics Data System (ADS)

    Liu, Victor; Fan, Shanhui

    2012-10-01

    We describe S4, a free implementation of the Fourier modal method (FMM), which has also been commonly referred to as rigorous coupled wave analysis (RCWA), for simulating electromagnetic propagation through 3D structures with 2D periodicity. We detail design aspects that allow S4 to be a flexible platform for these types of simulations. In particular, we highlight the ability to select different FMM formulations, user scripting, and extensibility of program capabilities for eigenmode computations. Program summary Program title: S4 Catalogue identifier: AEMO_v1_0. Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AEMO_v1_0..html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU General Public License, version 2 No. of lines in distributed program, including test data, etc.: 56910 No. of bytes in distributed program, including test data, etc.: 433883 Distribution format: Programming language: C, C++. Computer: Any computer with a Unix-like environment and a C++ compiler. Developed on 2.3 GHz AMD Phenom 9600. Operating system: Any Unix-like environment; developed under MinGW32 on Windows 7. Has the code been vectorized or parallelized?: Yes. Parallelized using MPI. RAM: Problem dependent (linearly proportional to number of layers and quadratic in number of Fourier components). A single layer calculation with approximately 100 Fourier components uses approximately 10 MB. Classification: 10. Electrostatics and Electromagnetics. External routines: Lua [1] and optionally exploits additional free software packages: FFTW [2], CHOLMOD [3], MPI message-passing interface [4], LAPACK and BLAS linear-algebra software [5], and Kiss FFT [6]. Nature of problem: Time-harmonic electromagnetism in layered bi-periodic structures. Solution method: The Fourier modal method (rigorous coupled wave analysis) and the scattering matrix method. Running time: Problem dependent and highly dependent on quality of the BLAS

  9. Try235Phe homozygous mutation of the steroid 5-a reductase type 2 (SRD5A2) gene in a Turkish patient.

    PubMed

    Parlak, Mesut; Durmaz, Erdem; Gursoy, Semin; Bircan, Iffet; Akcurin, Sema

    2014-01-01

    Steroid 5-a reductase type 2 isoenzyme (SRD5A2) deficiency is a male-limited autosomal recessive disorder that results in decreased conversion of testosterone to dihydrotestosterone with various de.gree of incomplete virilization in affected 46, XY infants. No clear genotype-phenotype relationship has been reported till date; moreover, the same mutation can result in considerable heterogeneity in clinical manifestations. Of 6 documented cases with Try235Phe homozygous mutation of the SRD5A2 gene, 3 patients had predominantly female external genitalia whereas the other 3 had predominantly male phenotype. We report Try235Phe homozygous mutation of the SRD5A2 gene in a Turkish patient who was initially assigned as a girl because of the predominantly female appearance of the external genitalia. PMID:25266188

  10. Collagen type IV-related nephropathies in Portugal: pathogenic COL4A3 and COL4A4 mutations and clinical characterization of 25 families.

    PubMed

    Nabais Sá, M J; Storey, H; Flinter, F; Nagel, M; Sampaio, S; Castro, R; Araújo, J A; Gaspar, M A; Soares, C; Oliveira, A; Henriques, A C; da Costa, A G; Abreu, C P; Ponce, P; Alves, R; Pinho, L; Silva, S E; de Moura, C P; Mendonça, L; Carvalho, F; Pestana, M; Alves, S; Carvalho, F; Oliveira, J P

    2015-11-01

    Pathogenic mutations in genes COL4A3/COL4A4 are responsible for autosomal Alport syndrome (AS) and thin basement membrane nephropathy (TBMN). We used Sanger sequencing to analyze all exons and splice site regions of COL4A3/COL4A4, in 40 unrelated Portuguese probands with clinical suspicion of AS/TBMN. To assess genotype-phenotype correlations, we compared clinically relevant phenotypes/outcomes between homozygous/compound heterozygous and apparently heterozygous patients. Seventeen novel and four reportedly pathogenic COL4A3/COL4A4 mutations were identified in 62.5% (25/40) of the probands. Regardless of the mutated gene, all patients with ARAS manifested chronic renal failure (CRF) and hearing loss, whereas a minority of the apparently heterozygous patients had CRF or extrarenal symptoms. CRF was diagnosed at a significantly younger age in patients with ARAS. In our families, the occurrence of COL4A3/COL4A4 mutations was higher, while the prevalence of XLAS was lower than expected. Overall, a pathogenic COL4A3/COL4A4/COL4A5 mutation was identified in >50% of patients with fewer than three of the standard diagnostic criteria of AS. With such a population background, simultaneous next-generation sequencing of all three genes may be recommended as the most expedite approach to diagnose collagen IV-related glomerular basement membrane nephropathies. PMID:25307543

  11. Eukaryotic Translation Initiation Factor 5A (EIF5A) Regulates Pancreatic Cancer Metastasis by Modulating RhoA and Rho-associated Kinase (ROCK) Protein Expression Levels.

    PubMed

    Fujimura, Ken; Choi, Sunkyu; Wyse, Meghan; Strnadel, Jan; Wright, Tracy; Klemke, Richard

    2015-12-11

    Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with an overall survival rate of less than 5%. The poor patient outcome in PDAC is largely due to the high prevalence of systemic metastasis at the time of diagnosis and lack of effective therapeutics that target disseminated cells. The fact that the underlying mechanisms driving PDAC cell migration and dissemination are poorly understood have hindered drug development and compounded the lack of clinical success in this disease. Recent evidence indicates that mutational activation of K-Ras up-regulates eIF5A, a component of the cellular translational machinery that is critical for PDAC progression. However, the role of eIF5A in PDAC cell migration and metastasis has not been investigated. We report here that pharmacological inhibition or genetic knockdown of eIF5A reduces PDAC cell migration, invasion, and metastasis in vitro and in vivo. Proteomic profiling and bioinformatic analyses revealed that eIF5A controls an integrated network of cytoskeleton-regulatory proteins involved in cell migration. Functional interrogation of this network uncovered a critical RhoA/ROCK signaling node that operates downstream of eIF5A in invasive PDAC cells. Importantly, eIF5A mediates PDAC cell migration and invasion by modulating RhoA/ROCK protein expression levels. Together our findings implicate eIF5A as a cytoskeletal rheostat controlling RhoA/ROCK protein expression during PDAC cell migration and metastasis. Our findings also implicate the eIF5A/RhoA/ROCK module as a potential new therapeutic target to treat metastatic PDAC cells. PMID:26483550

  12. 4-HPR impairs bladder cancer cell migration and invasion by interfering with the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways

    PubMed Central

    Cao, Yuanfei; Wang, Xiaolong; Xu, Chang; Gao, Zhengyan; Zhou, Haihong; Wang, Yongzhi; Cao, Rui; Liu, Tao; Liu, Tongzu

    2016-01-01

    In order to identify the anti-invasive and anti-metastatic effect of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on the human bladder cancer EJ cell line, and to study its impact on the expression of wingless-type mouse mammary tumor virus integration site family, member 5a (Wnt5a), the phosphorylation of c-Jun N-terminal kinase (JNK), the expression levels of matrix metalloproteinase-2 (MMP-2), and the migration and invasion of EJ cells, migration and Matrigel invasion assays, as well as western blot analyses, were used in the present study. The results of the migration and Matrigel invasion assays indicated that the inhibitor of JNK SP600125 could inhibit the effect of 4-HPR on EJ cells. The expression of Wnt5a and MMP-2, and the phosphorylation of JNK, were analyzed by western blotting. The data revealed that 4-HPR inhibited the migration and invasion of bladder cancer cells through stimulating Wnt5a activation, causing the downregulation of MMP-2 expression and enhancing the phosphorylation of JNK in these cells. However, JNK signaling did not appear to have a direct effect on the expression of MMP-2. The present study demonstrated that 4-HPR may be a potent anti-invasive and anti-metastatic agent that functions via the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways. PMID:27602114

  13. Identification of the Phosphorylated Residues in TveIF5A by Mass Spectrometry

    PubMed Central

    Quintas-Granados, Laura Itzel; López-Camarillo, César; Armas, Jesús Fandiño; Mendoza Hernandez, Guillermo; Alvarez-Sánchez, María Elizbeth

    2013-01-01

    The initiation factor eIF5A in Trichomonas vaginalis (TveIF5A) is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TveIF5A have been reported. The most acidic isoform (pI 5.2) corresponds to the precursor TveIF5A, whereas the mature TveIF5A appears to be the most basic isoform (pI 5.5). In addition, the intermediary isoform (pI 5.3) is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TveIF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TveIF5A contains four phosphorylated residues (S3, T55, T78 and T82). Phosphorylation at S3 and T82 is also identified in the intermediary TveIF5A, while no phosphorylated residues are found in the precursor TveIF5A. It has been demonstrated that eIF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 (CK2). Interestingly, a gene encoding a protein highly similar to CK2 (TvCK2) is found in T. vaginalis, which might be involved in the phosphorylation of TveIF5A in T. vaginalis. PMID:24308916

  14. The effect of hypusine modification on the intracellular localization of eIF5A

    SciTech Connect

    Lee, Seung Bum; Park, Jong Hwan; Kaevel, Joern; Sramkova, Monika; Weigert, Roberto; Park, Myung Hee

    2009-06-12

    Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [N{sup {epsilon}}-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.

  15. The effect of hypusine modification on the intracellular localization of eIF5A

    PubMed Central

    Lee, Seung Bum; Park, Jong Hwan; Kaevel, Jorn; Sramkova, Monika; Weigert, Roberto; Park, Myung Hee

    2009-01-01

    Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [Nε-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the posttranslational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis. PMID:19379712

  16. The translation factor eIF5A and human cancer

    PubMed Central

    Mathews, Michael B.; Hershey, John W. B.

    2015-01-01

    SUMMARY The eukaryotic initiation factor eIF5A is a translation factor that, unusually, has been assigned functions in both initiation and elongation. Additionally, it is implicated in transcription, mRNA turnover and nucleocytoplasmic transport. Two eIF5A isoforms are generated from distinct but related genes. The major isoform, eIF5A1, is considered constitutive, is abundantly expressed in most cells, and is essential for cell proliferation. The second isoform, eIF5A2, is expressed in few normal tissues but is highly expressed in many cancers and has been designated a candidate oncogene. Elevated expression of either isoform carries unfavorable prognostic implications for several cancers, and both have been advanced as cancer biomarkers. The amino acid hypusine, a presumptively unique eIF5A post-translational modification, is required for most known eIF5A functions and it renders eIF5A susceptible to inhibitors of the modification pathway as therapeutic targets. eIF5A has been shown to regulate a number of gene products specifically, termed the eIF5A regulon, and its role in translating proline-rich sequences has recently been identified. A model is advanced that accommodates eIF5A in both the initiation and elongation phases of translation. We review here the biochemical functions of eIF5A, the relationship of its isoforms with human cancer, and evolving clinical applications. PMID:25979826

  17. WNT5A promotes stemness characteristics in nasopharyngeal carcinoma cells leading to metastasis and tumorigenesis.

    PubMed

    Qin, Li; Yin, Yan-Tao; Zheng, Fang-Jing; Peng, Li-Xia; Yang, Chang-Fu; Bao, Ying-Na; Liang, Ying-Ying; Li, Xin-Jian; Xiang, Yan-Qun; Sun, Rui; Li, An-Hua; Zou, Ru-Hai; Pei, Xiao-Qing; Huang, Bi-Jun; Kang, Tie-Bang; Liao, Duan-Fang; Zeng, Yi-Xin; Williams, Bart O; Qian, Chao-Nan

    2015-04-30

    Nasopharyngeal carcinoma (NPC) has the highest metastasis rate among head and neck cancers with unclear mechanism. WNT5A belongs to the WNT family of cysteine-rich secreted glycoproteins. Our previous high-throughput gene expression profiling revealed that WNT5A was up-regulated in highly metastatic cells. In the present study, we first confirmed the elevated expression of WNT5A in metastatic NPC tissues at both the mRNA and protein levels. We then found that WNT5A promoted epithelial-mesenchymal transition (EMT) in NPC cells, induced the accumulation of CD24-CD44+ cells and side population, which are believed to be cancer stem cell characteristics. Moreover, WNT5A promoted the migration and invasion of NPC cells in vitro, while in vivo treatment with recombinant WNT5A promoted lung metastasis. Knocking down WNT5A diminished NPC tumorigenesis in vivo. When elevated expression of WNT5A coincided with the elevated expression of vimentin in the primary NPC, the patients had a poorer prognosis. Among major signaling pathways, protein kinase C (PKC) signaling was activated by WNT5A in NPC cells. A positive feedback loop between WNT5A and phospho-PKC to promote EMT was also revealed. Taken together, these data suggest that WNT5A is an important molecule in promoting stem cell characteristics in NPC, leading to tumorigenesis and metastasis. PMID:25823923

  18. Immunological Effects and Therapeutic Role of C5a in Cancer

    PubMed Central

    Darling, Victoria R.; Hauke, Ralph J.; Tarantolo, Stefano; Agrawal, Devendra K.

    2015-01-01

    The specific role of C5a in cancer, especially in melanoma, has yet to be determined. Differential effects of C5a could be cancer-specific. In the host defense system, C5a functions to protect the body from harmful entities via a plethora of mechanisms. Yet, C5a may also serve to potentiate cancerous process. C5a facilitates cellular proliferation and regeneration by attracting myeloid-derived suppressor cells and supporting tumor promotion. In this article, we critically reviewed the properties, mechanisms of action, and functions of C5a, with particular emphasis on cancer inhibition and promotion, and clinical application of such knowledge in better management of cancer patients. Outstanding questions and future directions in regard to the function of C5a in melanoma and other cancers are discussed. PMID:25387724

  19. Genomic organization of the human SCN5A gene encoding the cardiac sodium channel

    SciTech Connect

    Wang, Qing; Li, Zhizhong; Shen, Jiaxiang; Keating, M.T.

    1996-05-15

    The voltage-gated cardiac sodium channel, SCN5A, is responsible for the initial upstroke of the action potential. Mutations in the human SCN5A gene cause susceptibility to cardiac arrhythmias and sudden death in the long QT syndrome (LQT). In this report we characterize the genomic structure of SCN5A. SCN5A consists of 28 exons spanning approximately 80 kb on chromosome 3p21. We describe the sequences of all intron/exon boundaries and a dinucleotide repeat polymorphism in intron 16. Oligonucleotide primers based on exon-flanking sequences amplify all SCN5A exons by PCR. This work establishes the complete genomic organization of SCN5A and will enable high-resolution analyses of this locus for mutations associated with LQT and other phenotypes for which SCN5A may be a candidate gene. 40 refs., 4 figs., 2 tabs.

  20. 18 CFR 3a.71 - Accountability for classified material.

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  4. 22 CFR 3a.7 - Notification of disapproval and reconsideration.

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  12. 22 CFR 3a.8 - Change in status.

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  13. Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco.

    PubMed

    Liu, Xue-Qin; Liu, Chang-Ying; Guo, Qing; Zhang, Meng; Cao, Bo-Ning; Xiang, Zhong-Huai; Zhao, Ai-Chun

    2015-01-01

    The dehydration responsive element binding (DREB) transcription factors have been reported to be involved in stress responses. Most studies have focused on DREB genes in subgroups A-1 and A-2 in herbaceous plants, but there have been few reports on the functions of DREBs from the A-3-A-6 subgroups and in woody plants. Moreover, mulberry trees are ecologically and economically important perennial woody plants, but there has been little research on its stress physiology, biochemistry and molecular biology. In this study, a DREB gene from the mulberry tree, designated as MnDREB4A, classified into the A-4 subgroup by our previous study, was selected for further characterization. Our results showed that the MnDREB4A protein was localized to the nucleus where it activated transcription. The promoter of MnDREB4A can direct prominent expression downstream of the β-glucuronidase (GUS) gene under heat, cold, drought and salt stress, and GUS staining was deepest after 12 h of stress treatment. The MnDREB4A-overexpression transgenic tobacco showed the improved growth phenotype under untreated conditions, such as greener leaves, longer roots, and lower water loss and senescence rates. Overexpression of MnDREB4A in tobacco can significantly enhance tolerance to heat, cold, drought, and salt stresses in transgenic plants. The leaf discs and seedlings of transgenic plants reduced leaf wilting and senescence rates compared to the wild type plants under the different stress conditions. Further investigation showed that transgenic plants also had higher water contents and proline contents, and lower malondialdehyde contents under untreated condition and stress conditions. Our results indicate that the MnDREB4A protein plays an important role in plant stress tolerance. PMID:26695076

  14. Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development.

    PubMed

    Marra, Amanda N; Wingert, Rebecca A

    2016-03-15

    Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA

  15. 12 CFR Appendix B to Subpart B of... - Article 4A, Funds Transfers

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  16. 12 CFR Appendix B to Subpart B of... - Article 4A, Funds Transfers

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  17. 12 CFR Appendix B to Subpart B of... - Article 4A, Funds Transfers

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  18. 12 CFR Appendix B to Subpart B of... - Article 4A, Funds Transfers

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  19. 12 CFR Appendix B to Subpart B of... - Article 4A, Funds Transfers

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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  20. Molecular cloning of the human proto-oncogene Wnt-5A and mapping of the gene (WNT5A) to chromosome 3p14-p21

    SciTech Connect

    Clark, C.C.; Cohen, I.; Eichstetter, I.; Cannizzaro, L.A.; Iozzo, R.V. ); McPherson, J.D.; Wasmuth, J.J. )

    1993-11-01

    The highly conserved Wnt genes belong to a widely distributed family of presumptive signaling molecules that have been implicated not only in the regulation of normal pattern formation during embryogenesis and differentiation of cell lineages, but also in oncogenic events. All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted growth factors: A hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In this study, the authors report the cloning and sequencing of several overlapping cDNAs encoding [approximately]4.1 kb of the human homologue of Wnt-5A. The mature protein contained 343 residues (M[sub r] [approximately] 38,000 excluding any post-translational modifications) with a >93% homology to the reported sequences of other Wnt-5A proteins (>99% homologous to mouse Wnt-5A). This protein maintained certain features - a hydrophobic signal sequence, the Wnt-1 family [open quotes]signature sequence[close quotes] (CKCHGvSGSC), and a number of other conserved amino acid residues - 24 cysteine residues, 4 asparagine-linked oligosaccharide consensus sequences, and a tyrosine sulfation site - that have been found in all other Wnt-5A proteins. Reverse transcriptase PCR analysis of RNA from a variety of human embryonic, neonatal, and adult cells and/or tissues showed that human Wnt-5A expression was detected only in neonatal heart and lung. It may be relevant, however, that the 3[prime]-untranslated region contained numerous AT-rich motifs that could be involved in the rapid degradation of mRNA. Finally, using a combination of Southern blotting, PCR amplification, and in situ hybridization, the human Wnt-5A (WNT5A) gene was mapped to chromosome 3p14-p21. 36 refs., 7 figs., 2 tabs.

  1. WNT5A Knock-Out Mouse As A New Model of Anorectal Malformation

    PubMed Central

    Tai, Cindy C.; Sala, Frederic G.; Ford, Henri R.; Wang, Kasper S.; Minoo, Parviz; Grikscheit, Tracy C.; Bellusci, Saverio

    2009-01-01

    Background Anorectal malformations (ARM) represent a variety of congenital disorders that involve abnormal termination of the anorectum. Mutations in Shh signaling and Fgf10 produce a variety of ARM phenotypes. Wnt signaling has been shown to be crucial during gastrointestinal development. We therefore hypothesized that Wnt5a may play a role in anorectal development. Methods Wild type (WT), Wnt5a+/-, and Wnt5a-/- embryos were harvested from timed pregnant mice from E15.5 to E18.5 and analyzed for anorectal phenotype. Tissues were processed for whole-mount in situ hybridization and histology. Results Wnt5a is expressed in the embryonic WT colon and rectum. Wnt5a-/- mutants exhibit multiple deformities including anorectal malformation. A fistula between the urinary and intestinal tracts can be identified as early as E15.5. By E18.5, the majority of the Wnt5a-/- mutants display a blind-ending pouch of the distal gut. Conclusions The expression pattern of Wnt5a and the ARM phenotype seen in Wnt5a-/- mutants demonstrate the critical role of Wnt5a during anorectal development. This study establishes a new model of ARM involving the Wnt5a pathway. PMID:19577771

  2. Drosophila SLC5A11 Mediates Hunger by Regulating K(+) Channel Activity.

    PubMed

    Park, Jin-Yong; Dus, Monica; Kim, Seonil; Abu, Farhan; Kanai, Makoto I; Rudy, Bernardo; Suh, Greg S B

    2016-08-01

    Hunger is a powerful drive that stimulates food intake. Yet, the mechanism that determines how the energy deficits that result in hunger are represented in the brain and promote feeding is not well understood. We previously described SLC5A11-a sodium/solute co-transporter-like-(or cupcake) in Drosophila melanogaster, which is required for the fly to select a nutritive sugar over a sweeter nonnutritive sugar after periods of food deprivation. SLC5A11 acts on approximately 12 pairs of ellipsoid body (EB) R4 neurons to trigger the selection of nutritive sugars, but the underlying mechanism is not understood. Here, we report that the excitability of SLC5A11-expressing EB R4 neurons increases dramatically during starvation and that this increase is abolished in the SLC5A11 mutation. Artificial activation of SLC5A11-expresssing neurons is sufficient to promote feeding and hunger-driven behaviors; silencing these neurons has the opposite effect. Notably, SLC5A11 transcript levels in the brain increase significantly when flies are starved and decrease shortly after starved flies are refed. Furthermore, expression of SLC5A11 is sufficient for promoting hunger-driven behaviors and enhancing the excitability of SLC5A11-expressing neurons. SLC5A11 inhibits the function of the Drosophila KCNQ potassium channel in a heterologous expression system. Accordingly, a knockdown of dKCNQ expression in SLC5A11-expressing neurons produces hunger-driven behaviors even in fed flies, mimicking the overexpression of SLC5A11. We propose that starvation increases SLC5A11 expression, which enhances the excitability of SLC5A11-expressing neurons by suppressing dKCNQ channels, thereby conferring the hunger state. PMID:27397890

  3. Clinical and molecular characterization of 40 patients with classic Ehlers–Danlos syndrome: identification of 18 COL5A1 and 2 COL5A2 novel mutations

    PubMed Central

    2013-01-01

    Background Classic Ehlers–Danlos syndrome (cEDS) is a rare autosomal dominant connective tissue disorder that is primarily characterized by skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. A recent study demonstrated that more than 90% of patients who satisfy all of these major criteria harbor a type V collagen (COLLV) defect. Methods This cohort included 40 patients with cEDS who were clinically diagnosed according to the Villefranche nosology. The flowchart that was adopted for mutation detection consisted of sequencing the COL5A1 gene and, if no mutation was detected, COL5A2 analysis. In the negative patients the presence of large genomic rearrangements in COL5A1 was investigated using MLPA, and positive results were confirmed via SNP-array analysis. Results We report the clinical and molecular characterization of 40 patients from 28 families, consisting of 14 pediatric patients and 26 adults. A family history of cEDS was present in 9 patients. The majority of the patients fulfilled all the major diagnostic criteria for cEDS; atrophic scars were absent in 2 females, skin hyperextensibility was not detected in a male and joint hypermobility was negative in 8 patients (20% of the entire cohort). Wide inter- and intra-familial phenotypic heterogeneity was observed. We identified causal mutations with a detection rate of approximately 93%. In 25/28 probands, COL5A1 or COL5A2 mutations were detected. Twenty-one mutations were in the COL5A1 gene, 18 of which were novel (2 recurrent). Of these, 16 mutations led to nonsense-mediated mRNA decay (NMD) and to COLLV haploinsufficiency and 5 mutations were structural. Two novel COL5A2 splice mutations were detected in patients with the most severe phenotypes. The known p. (Arg312Cys) mutation in the COL1A1 gene was identified in one patient with vascular-like cEDS. Conclusions Our findings highlight that the three major criteria for cEDS are useful and sufficient for cEDS clinical

  4. Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway

    PubMed Central

    McDonald, Chloë R.; Cahill, Lindsay S.; Ho, Keith T.; Yang, Jimmy; Kim, Hani; Silver, Karlee L.; Ward, Peter A.; Mount, Howard T.; Liles, W. Conrad; Sled, John G.; Kain, Kevin C.

    2015-01-01

    The in utero environment profoundly impacts childhood neurodevelopment and behaviour. A substantial proportion of pregnancies in Africa are at risk of malaria in pregnancy (MIP) however the impact of in utero exposure to MIP on fetal neurodevelopment is unknown. Complement activation, in particular C5a, may contribute to neuropathology and adverse outcomes during MIP. We used an experimental model of MIP and standardized neurocognitive testing, MRI, micro-CT and HPLC analysis of neurotransmitter levels, to test the hypothesis that in utero exposure to malaria alters neurodevelopment through a C5a-C5aR dependent pathway. We show that malaria-exposed offspring have persistent neurocognitive deficits in memory and affective-like behaviour compared to unexposed controls. These deficits were associated with reduced regional brain levels of major biogenic amines and BDNF that were rescued by disruption of C5a-C5aR signaling using genetic and functional approaches. Our results demonstrate that experimental MIP induces neurocognitive deficits in offspring and suggest novel targets for intervention. PMID:26402732

  5. Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway.

    PubMed

    McDonald, Chloë R; Cahill, Lindsay S; Ho, Keith T; Yang, Jimmy; Kim, Hani; Silver, Karlee L; Ward, Peter A; Mount, Howard T; Liles, W Conrad; Sled, John G; Kain, Kevin C

    2015-09-01

    The in utero environment profoundly impacts childhood neurodevelopment and behaviour. A substantial proportion of pregnancies in Africa are at risk of malaria in pregnancy (MIP) however the impact of in utero exposure to MIP on fetal neurodevelopment is unknown. Complement activation, in particular C5a, may contribute to neuropathology and adverse outcomes during MIP. We used an experimental model of MIP and standardized neurocognitive testing, MRI, micro-CT and HPLC analysis of neurotransmitter levels, to test the hypothesis that in utero exposure to malaria alters neurodevelopment through a C5a-C5aR dependent pathway. We show that malaria-exposed offspring have persistent neurocognitive deficits in memory and affective-like behaviour compared to unexposed controls. These deficits were associated with reduced regional brain levels of major biogenic amines and BDNF that were rescued by disruption of C5a-C5aR signaling using genetic and functional approaches. Our results demonstrate that experimental MIP induces neurocognitive deficits in offspring and suggest novel targets for intervention. PMID:26402732

  6. Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A.

    PubMed

    Sakka, Makiko; Goto, Masayuki; Fujino, Tsuchiyoshi; Fujino, Emi; Karita, Shuichi; Kimura, Tetsuya; Sakka, Kazuo

    2010-01-01

    A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source. PMID:20944403

  7. C5a RECEPTOR (C5aR) CHIMERAS REVEAL THE IMPORTANCE OF LIPID-FACING RESIDUES IN TRANSPORT COMPETENCE

    PubMed Central

    Klco, Jeffery M.; Sen, Saurabh; Hansen, Jakob L.; Lyngsø, Christina; Nikiforovich, Gregory V.; Sheikh, Soren P.; Baranski, Thomas J.

    2009-01-01

    Residues that mediate helix-helix interactions within the seven transmembranes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism. In contrast, the residues directly contacting the lipid bilayer have only recently garnered attention as potential receptor dimerization interfaces. In this study we sought to determine the contributions of these lipid-facing residues to receptor function and oligomerization by systemically generating chimeric C5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angiotensin AT1 receptor. Disulfide-trapping and BRET studies demonstrated robust homodimerization of both C5a receptor and AT1R, but no evidence for heterodimerization. Despite relatively conservative substitutions, the lipid-facing chimeras (TM 1, 2, 4, 5, 6, or 7) were retained in the ER/cis-Golgi network. With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type C5a receptors trapped in the ER with Brefeldin A. These results suggest that the chimeric receptors were properly folded; moreover, native C5a receptors are not fully competent to bind ligand when present in the ER. BRET oligomerization studies demonstrated energy transfer between the wild-type C5aR and the lipid-facing chimeras suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization. These studies highlight the importance of the lipid-facing residues in C5a receptor for transport competence. PMID:19459935

  8. Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides

    PubMed Central

    Chukkapalli, Vineela; Berger, Kristi L.; Kelly, Sean M.; Thomas, Meryl; Deiters, Alexander; Randall, Glenn

    2014-01-01

    Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies are HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation. PMID:25546252

  9. WNT5A Inhibits Metastasis and Alters Splicing of Cd44 in Breast Cancer Cells

    PubMed Central

    Jiang, Wen; Crossman, David K.; Mitchell, Elizabeth H.; Sohn, Philip; Crowley, Michael R.; Serra, Rosa

    2013-01-01

    Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44. PMID:23484019

  10. 46 CFR 30.10-5a - Cargo area-TB/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Cargo area-TB/ALL. 30.10-5a Section 30.10-5a Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS GENERAL PROVISIONS Definitions § 30.10-5a Cargo area—TB/ALL. The term cargo area means that part of a vessel that includes the cargo tanks...

  11. 46 CFR 30.10-5a - Cargo area-TB/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Cargo area-TB/ALL. 30.10-5a Section 30.10-5a Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS GENERAL PROVISIONS Definitions § 30.10-5a Cargo area—TB/ALL. The term cargo area means that part of a vessel that includes the cargo tanks...

  12. 12 CFR 226.5a - Credit and charge card applications and solicitations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 3 2011-01-01 2011-01-01 false Credit and charge card applications and solicitations. 226.5a Section 226.5a Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM TRUTH IN LENDING (REGULATION Z) Open-End Credit § 226.5a Credit and charge card applications and solicitations....

  13. Wnt-3a is critical for caudal embryonic development

    SciTech Connect

    Camper, S.A.; Greco, T.L.; Newhouse, M.M.

    1994-09-01

    Skeletal and neural tube defects represent an important class of birth defects. The majority of mouse mutants with neural tube defects also have malformations of the tail. Vestigial tail (vt) is an autosomal recessive mouse mutation characterized by reduction or absence of the tail, vertebral abnormalities, and reduced fertility. The phenotype has been described as the result of failure of cell migration through the primitive streak, causing abnormalities in the development of the neural tube and a reduction in the ventral ectodermal ridge. Wnt3a is an excellent candidate gene for vt because Wnt3a is expressed in the primitive streak and in the embryonic mesoderm, and it is thought to be involved in cell-to-cell communication and formation of the dorsal-ventral axis in the CNS. A lack of Wnt3a might be expected to result in overdorsalization of the neural tube and reduction of the ventral ectodermal ridge characteristic of vt/vt embryos. In a high resolution backcross segregating vt, we observed no recombination between vt and Wnt3a in 363 individuals analyzed. In vt/vt mice, Southern blot analysis revealed no abnormalities in the Wnt3a gene, and the Wnt3a cDNA sequence does not encode any amino acid changes. Whole mount in situ hybridization analysis demonstrated that Wnt3a expression is severely reduced in the developing tailbud of day 9.5 vt/vt embryos, suggestive of a lesion in the regulation on Wnt3a expression. An alleleism test, carried out by mating vt/vt males with Wnt3a +/Wnt3a- females, demonstrated that vt and Wnt3a are noncomplementing alleles. All of the compound heterozygotes exhibited severe tail defects, including occasional examples of hind limb parlaysis and spina bifida. The vertebral defects are intermediate between those of vt and Wnt3a homozygotes, suggesting that the concentration of Wnt3a correlates with the severity of the defect.

  14. A useful model capable of predicting the clearance of cytochrome 3A4 (CYP3A4) substrates in humans: validity of CYP3A4 transgenic mice lacking their own Cyp3a enzymes.

    PubMed

    Mitsui, Tetsuya; Nemoto, Takayuki; Miyake, Taiji; Nagao, Shunsuke; Ogawa, Kotaro; Kato, Motohiro; Ishigai, Masaki; Yamada, Hideyuki

    2014-09-01

    The accurate prediction for the body clearance of a novel drug candidate by humans during the preclinical stage contributes to its successful development. To improve the predictability of human hepatic clearance, we focused on CYP3A4, which is involved in the metabolism of more than 50% of all currently marketed drugs. In this study, we investigated the validity of the in vivo model using transgenic mice carrying the human CYP3A4 gene and lacking their own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward its substrates in liver microsomes was similar in CYP3A4-Tg mice and humans. As for the clearance, six CYP3A4 substrates (alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and quinidine) were given intravenously to CYP3A4-Tg mice, and their hepatic intrinsic clearance (CLint,h) was evaluated. A regression analysis of the data obtained indicated that the CLint,h values of six substrates in CYP3A4-Tg mice were highly correlated with those in humans (R(2) = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially expressed CYP3A4 to the overall metabolism in the mice. From these findings, it is reasonable to expect that the CLint,h of a particular drug in humans is predictable by applying the CLint,h obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was evaluated and found to be within a range of 2-fold of the regression value. These results suggest that the CYP3A4-Tg mouse model has the potential to accurately predict the human hepatic clearance of CYP3A4 substrates. PMID:25005602

  15. Regulation of drug sensitivity by ribosomal protein S3a.

    PubMed

    Hu, Z B; Minden, M D; McCulloch, E A; Stahl, J

    2000-02-01

    When bcl-2 is immunoprecipitated from (32)P-labeled cell extracts of all-trans retinoic acid (ATRA)-treated acute myeloblastic leukemia (AML) blasts, a phosphorylated protein of approximately 30 kd is coprecipitated. This protein has been identified as ribosomal protein S3a. The biologic effects of S3a include favoring apoptosis and enhancing the malignant phenotype. We sought to determine whether S3a, like bcl-2, influenced the response of cells to chemotherapeutic drugs and ATRA. Cell lines were studied in which S3a was genetically increased or disrupted; increased S3a was regularly associated with increased plating efficiency and increased sensitivity to either cytosine arabinoside (ara-C) or doxorubicin (DNR). S3a did not affect the sensitivity of cells to paclitaxel. Pulse exposures to either (3)HTdR or ara-C showed a greater percentage of clonogenic cells in the S phase of the cell cycle in cells with increased S3a than in controls. Cells with increased S3a responded to ATRA by increased ara-C or DNR sensitivity, whereas cells with reduced S3a protein were either protected by ATRA or not affected. We studied cryopreserved blast cells from patients with AML or chronic myelomonocytic leukemia (CMML). S3a protein levels were heterogeneous in these populations. In 32 cryopreserved blast populations, S3a levels were significantly correlated with both bcl-2 and with cell growth in culture. As in cell lines, high S3a in cryopreserved blasts was associated with ATRA-induced sensitization to ara-C. No significant association was seen between S3a levels and response to treatment. PMID:10648421

  16. Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity

    PubMed Central

    Yang, Jing; Zhao, Haiwei; Qiao, Qinghua; Han, Peijun; Xu, Zhikai; Yin, Wen

    2016-01-01

    Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors. PMID:26943641

  17. Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity.

    PubMed

    Yao, Min; Lu, Xin; Lei, Yingfeng; Yang, Jing; Zhao, Haiwei; Qiao, Qinghua; Han, Peijun; Xu, Zhikai; Yin, Wen

    2016-01-01

    Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors. PMID:26943641

  18. 5-A-DAY: dietary behavior and the fruit and vegetable intake of Latino children.

    PubMed Central

    Basch, C E; Zybert, P; Shea, S

    1994-01-01

    OBJECTIVES. The purpose of the study was to examine children's intake of fruits and vegetables in relation to the recent national "5-A-DAY" campaign. METHODS. Four 24-hour dietary recalls per child collected from 205 mothers of 4- to 5-year-old urban Latino children were used to analyze average 5-A-DAY fruit and vegetable consumption and examine associations between 5-A-DAY consumption, nutrient intakes, and eating patterns. RESULTS. The reported mean servings per day of fruits and vegetables, as defined by 5-A-DAY criteria, were 1.8 and 1.0, respectively, with only 6.8% (n = 14) of the children averaging five or more servings per day. Fruit juice accounted for 36% of 5-A-DAY servings. There were significant linear trends in intake of vitamins A and C, potassium, iron, cholesterol, protein, and fiber across quintiles of 5-A-DAY intake. There were no differences among quintiles in intake of saturated or total fat or in servings from most non-5-A-DAY food groups. CONCLUSIONS. Latino children's intake of fruits and vegetables falls far short of current recommendations. Fruit juice accounted for a disproportionate amount of 5-A-DAY intake in this population. Sensible 5-A-DAY interventions should take into consideration the existing eating patterns of the target population. PMID:8179054

  19. Comparisons of subunit 5A and 5B isoenzymes of yeast cytochrome c oxidase

    PubMed Central

    Dodia, Raksha; Meunier, Brigitte; Kay, Christopher W. M.; Rich, Peter R.

    2014-01-01

    Subunit 5 of Saccharomyces cerevisiae cytochrome c oxidase (CcO) is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions, whereas 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory deficient. Previous studies have reported that respiratory growth could be restored by combining Δcox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants, 5B isoenzyme expression level was 30–50% of wild-type (5A isoenzyme) and exhibited a maximum catalytic activity up to 3-fold faster than that of 5A isoenzyme. To investigate the origin of this effect, we constructed a mutant strain in which COX5B replaced COX5A downstream of the COX5A promoter. This strain expressed wild-type levels of the 5B isoenzyme, without the complication of additional effects caused by mutation of ROX1. When produced this way, the isoenzymes displayed no significant differences in their maximum catalytic activities or in their affinities for oxygen or cytochrome c. Hence the elevated activity of the 5B isoenzyme in the rox1 mutant is not caused simply by exchange of isoforms and must arise from an additional effect that remains to be resolved. PMID:25241981

  20. Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

    PubMed

    Chong, Weng Man; Hsu, Shih-Chin; Kao, Wei-Ting; Lo, Chieh-Wen; Lee, Kuan-Ying; Shao, Jheng-Syuan; Chen, Yi-Hung; Chang, Justin; Chen, Steve S-L; Yu, Ming-Jiun

    2016-02-19

    The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. PMID:26702051

  1. C5a and toll-like receptor 4 crosstalk in retinal pigment epithelial cells

    PubMed Central

    Zhu, Yi; Dai, Bingling; Li, Yongguo

    2015-01-01

    Purpose To investigate the effect of the complement activation product C5a on toll-like receptor (TLR) 4-induced responses in RPE cells. Methods Confluent cultures of human RPE cells (ARPE-19) were stimulated with C5a, lipopolysaccharide (LPS), or a combination of the two. The expression of TLR4 was determined by real-time PCR and flow cytometry. Cytokine profiles were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of p38, ERK 1/2, and JNK was measured by flow cytometry. Results C5a stimulation enhanced the expression of TLR4 in a dose- and time-dependent manner. C5a was able to stimulate the production of TLR4-induced IL-6 and IL-8 by ARPE-19 cells. Blocking experiments showed that the effect of C5a on cytokine production was mediated via C5aR. ERK1/2, but not JNK or p38, were involved in the production of IL-6 and IL-8. Conclusions The results indicate that C5a can induce the TLR4 expression and enhance the production of TLR4-induced IL-6 and IL-8 by ARPE-19. The effect of C5a on cytokine production was mediated by C5aR and the phosphorylation of ERK1/2. PMID:26487798

  2. C5A Protects Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge

    PubMed Central

    Veazey, Ronald S.; Chatterji, Udayan; Bobardt, Michael; Russell-Lodrigue, Kasi E.; Li, Jian; Wang, Xiaolei

    2015-01-01

    A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate. PMID:26552985

  3. C5A Protects Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge.

    PubMed

    Veazey, Ronald S; Chatterji, Udayan; Bobardt, Michael; Russell-Lodrigue, Kasi E; Li, Jian; Wang, Xiaolei; Gallay, Philippe A

    2016-01-01

    A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate. PMID:26552985

  4. Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment.

    PubMed

    Maurelli, Riccardo; Zambruno, Giovanna; Guerra, Liliana; Abbruzzese, Claudia; Dimri, Goberdhan; Gellini, Mara; Bondanza, Sergio; Dellambra, Elena

    2006-07-01

    Replicative senescence of human keratinocytes is determined by a progressive decline of clonogenic and dividing cells, and its timing is controlled by clonal evolution (i.e., the transition from stem cells to transient amplifying and postmitotic cells). Progressive increase of p16INK4a (inhibitor of cyclin-dependent kinase 4A) expression has been shown to correlate with keratinocyte clonal evolution. Thus, the aim of our study is to understand whether p16INK4a accumulation is a triggering mechanism of epidermal clonal evolution or a secondary event. We show that inactivation of p16INK4a, by an antisense strategy, allows primary human keratinocytes to escape replicative senescence. Specifically, p16INK4a inactivation alone blocks clonal evolution and maintains keratinocytes in the stem cell compartment. Antisense excision is followed by keratinocyte senescence, confirming that persistent p16INK4a inactivation is required for maintenance of clonal evolution block. Immortalization is accompanied by resumption of B-Cell Specific Moloney murine leukemia virus site 1 (Bmi-1) expression and telomerase activity, hallmarks of tissue regenerative capacity. In turn, Bmi-1 expression is necessary to maintain the impairment of clonal evolution induced by p16INK4a inactivation. Finally, p16INK4a down-regulation in transient amplifying keratinocytes does not affect clonal evolution, and cells undergo senescence. Thus, p16INK4a inactivation appears to selectively prevent clonal conversion in cells endowed with a high proliferative potential. These data indicate that p16INK4a regulates keratinocyte clonal evolution and that inactivation of p16INK4a in epidermal stem cells is necessary for maintaining stemness. PMID:16754749

  5. Wnt5a Is Associated with Cigarette Smoke-Related Lung Carcinogenesis via Protein Kinase C

    PubMed Central

    Sung, Jae Sook; Ju, Hyun Jung; Kim, Hyun Kyung; Park, Kyong Hwa; Lee, Jong Won; Koh, In Song; Kim, Yeul Hong

    2013-01-01

    Wnt5a is overexpressed during the progression of human non-small cell lung cancer. However, the roles of Wnt5a during smoking-related lung carcinogenesis have not been clearly elucidated. We investigated the associations between Wnt5a and the early development of cigarette smoke related lung cancer using human bronchial epithelial (HBE) cells (NHBE, BEAS-2B, 1799, 1198 and 1170I) at different malignant stages established by exposure to cigarette smoke condensate (CSC). Abnormal up-regulation of Wnt5a mRNA and proteins was detected in CSC-exposed transformed 1198 and tumorigenic 1170I cells as compared with other non-CSC exposed HBE cells. Tumor tissues obtained from smokers showed higher Wnt5a expressions than matched normal tissues. In non-CSC exposed 1799 cells, treatment of recombinant Wnt5a caused the activations of PKC and Akt, and the blockage of Wnt5a and PKC significantly decreased the viabilities of CSC-transformed 1198 cells expressing high levels of Wnt5a. This reduced cell survival rate was associated with increased apoptosis via the down-regulation of Bcl2 and the induction of cleaved poly ADP-ribose polymerase. Moreover, CSC-treated 1799 cells showed induction of Wnt5a expression and enhanced colony-forming capacity. The CSC-induced colony forming efficiency was suppressed by the co-incubation with a PKC inhibitor. In conclusion, these results suggest that cigarette smoke induces Wnt5a-coupled PKC activity during lung carcinogenesis, which causes Akt activity and anti-apoptosis in lung cancer. Therefore, current study provides novel clues for the crucial role of Wnt5a in the smoking-related lung carcinogenesis. PMID:23349696

  6. Bidirectional Crosstalk between C5a Receptors and the NLRP3 Inflammasome in Macrophages and Monocytes

    PubMed Central

    Haggadone, Mikel D.; Grailer, Jamison J.; Fattahi, Fatemeh; Zetoune, Firas S.; Ward, Peter A.

    2016-01-01

    C5a is an inflammatory mediator generated by complement activation that positively regulates various arms of immune defense, including Toll-like receptor 4 (TLR4) signaling. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is activated by pathogen products and cellular/tissue damage products and is a major contributor of IL-1β. In this study, we investigate whether C5a modulates lipopolysaccharide- (LPS-) induced NLRP3 inflammasome activation in myeloid cells. Appearance of plasma IL-1β during endotoxemia was reduced in C5aR1−/− mice when compared to wild-type mice. In vitro, C5a significantly enhanced LPS-induced production of IL-1β in bone marrow Ly6C-high inflammatory monocytes, accompanied by augmented intracellular pro-IL-1β expression. This effect was abolished during p38 blockade by SB 203580 and in the absence of C5aR1. Conversely, C5a suppressed LPS-induced macrophage production of IL-1β, which was accompanied by attenuated levels of pro-IL-1β, NLRP3, and caspase-1 expression. C5a's suppressive effects were negated during phosphoinositide 3-kinase (PI3K) inhibition by wortmannin but were largely preserved in the absence of C5aR1. Thus, C5a bidirectionally amplifies TLR4-mediated NLRP3 inflammasome activation in monocytes while suppressing this pathway in macrophages. However, as C5aR1 deficiency attenuates the IL-1β response to LPS challenge in vivo, our results suggest overall that C5a augments physiologic inflammasome responses. PMID:27382187

  7. Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression.

    PubMed

    Corrales, Leticia; Ajona, Daniel; Rafail, Stavros; Lasarte, Juan J; Riezu-Boj, Jose I; Lambris, John D; Rouzaut, Ana; Pajares, Maria J; Montuenga, Luis M; Pio, Ruben

    2012-11-01

    The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression. PMID:23028051

  8. Inactivation of Human Cytochrome P450 3A4 and 3A5 by Dronedarone and N-Desbutyl Dronedarone.

    PubMed

    Hong, Yanjun; Chia, Yvonne Mei Fen; Yeo, Ray Hng; Venkatesan, Gopalakrishnan; Koh, Siew Kwan; Chai, Christina Li Lin; Zhou, Lei; Kojodjojo, Pipin; Chan, Eric Chun Yong

    2016-01-01

    Dronedarone is an antiarrhythmic agent approved in 2009 for the treatment of atrial fibrillation. An in-house preliminary study demonstrated that dronedarone inhibits cytochrome P450 (CYP) 3A4 and 3A5 in a time-dependent manner. This study aimed to investigate the inactivation of CYP450 by dronedarone. We demonstrated for the first time that both dronedarone and its main metabolite N-desbutyl dronedarone (NDBD) inactivate CYP3A4 and CYP3A5 in a time-, concentration-, and NADPH-dependent manner. For the inactivation of CYP3A4, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 0.87 µM and 0.039 minute(-1), respectively, for dronedarone, and 6.24 µM and 0.099 minute(-1), respectively, for NDBD. For CYP3A5 inactivation, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 2.19 µM and 0.0056 minute(-1) for dronedarone and 5.45 µM and 0.056 minute(-1) for NDBD. The partition ratios for the inactivation of CYP3A4 and CYP3A5 by dronedarone are 51.1 and 32.2, and the partition ratios for the inactivation of CYP3A4 and CYP3A5 by NDBD are 35.3 and 36.6. Testosterone protected both CYP3A4 and CYP3A5 from inactivation by dronedarone and NDBD. Although the presence of Soret peak confirmed the formation of a quasi-irreversible metabolite-intermediate complex between dronedarone/NDBD and CYP3A4/CYP3A5, partial recovery of enzyme activity by potassium ferricyanide illuminated an alternative irreversible mechanism-based inactivation (MBI). MBI of CYP3A4 and CYP3A5 was further supported by the discovery of glutathione adducts derived from the quinone oxime intermediates of dronedarone and NDBD. In conclusion, dronedarone and NDBD inactivate CYP3A4 and CYP3A5 via unique dual mechanisms of MBI and formation of the metabolite-intermediate complex. Our novel findings contribute new knowledge for

  9. Alternative Start Codon Connects eIF5A to Mitochondria.

    PubMed

    Pereira, Karina Danielle; Tamborlin, Letícia; Meneguello, Letícia; de Proença, André Ricardo Gomes; Almeida, Isadora Cristina de Paula Andrade; Lourenço, Rogério Ferreira; Luchessi, Augusto Ducati

    2016-12-01

    Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N-terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N-terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co-purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. J. Cell. Physiol. 231: 2682-2689, 2016. © 2016 Wiley Periodicals, Inc. PMID:27414022

  10. 26 CFR 1.148-5A - Yield and valuation of investments.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 2 2011-04-01 2011-04-01 false Yield and valuation of investments. 1.148-5A..., 1997 § 1.148-5A Yield and valuation of investments. (a) through (b)(2)(ii) . For guidance see § 1.148-5. (b)(2)(iii) Permissive application of single investment rules to certain yield restricted...

  11. Wingless ligand 5a is a critical regulator of placental growth and survival.

    PubMed

    Meinhardt, Gudrun; Saleh, Leila; Otti, Gerlinde R; Haider, Sandra; Velicky, Philipp; Fiala, Christian; Pollheimer, Jürgen; Knöfler, Martin

    2016-01-01

    The maternal uterine environment is likely critical for human placental morphogenesis and development of its different trophoblast subtypes. However, factors controlling growth and differentiation of these cells during early gestation remain poorly elucidated. Herein, we provide evidence that the ligand Wnt5a could be a critical regulator of trophoblast proliferation and survival. Immunofluorescence of tissues and western blot analyses of primary cultures revealed abundant Wnt5a expression and secretion from first trimester decidual and villous stromal cells. The ligand was also detectable in decidual glands, macrophages and NK cells. Wnt5a increased proliferation of villous cytotrophoblasts and cell column trophoblasts, outgrowth on collagen I as well as cyclin A and D1 expression in floating explant cultures, but suppressed camptothecin-induced apoptosis. Similarly, Wnt5a stimulated BrdU incorporation and decreased caspase-cleaved cytokeratin 18 neo-epitope expression in primary cytotrophoblasts. Moreover, Wnt5a promoted activation of the MAPK pathway in the different trophoblast models. Chemical inhibition of p42/44 MAPK abolished cyclin D1 expression and Wnt5a-stimulated proliferation. Compared to controls, MAPK phosphorylation and proliferation of cytotrophoblasts declined upon supplementation of supernatants from Wnt5a gene-silenced decidual or villous stromal cells. In summary, non-canonical Wnt5a signalling could play a role in early human trophoblast development by promoting cell proliferation and survival. PMID:27311852

  12. 40 CFR 60.482-5a - Standards: Sampling connection systems.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... CFR part 63, subpart G, applicable to Group 1 wastewater streams; (B) A treatment, storage, or... (E) A device used to burn off-specification used oil for energy recovery in accordance with 40 CFR.... 60.482-5a Section 60.482-5a Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)...

  13. 26 CFR 1.148-5A - Yield and valuation of investments.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 2 2013-04-01 2013-04-01 false Yield and valuation of investments. 1.148-5A..., 1997 § 1.148-5A Yield and valuation of investments. (a) through (b)(2)(ii) . For guidance see § 1.148-5. (b)(2)(iii) Permissive application of single investment rules to certain yield restricted...

  14. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 5

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. Detail drawings of several assemblies and subassemblies are given. This is the fifth book of volume 4.

  15. Wingless ligand 5a is a critical regulator of placental growth and survival

    PubMed Central

    Meinhardt, Gudrun; Saleh, Leila; Otti, Gerlinde R.; Haider, Sandra; Velicky, Philipp; Fiala, Christian; Pollheimer, Jürgen; Knöfler, Martin

    2016-01-01

    The maternal uterine environment is likely critical for human placental morphogenesis and development of its different trophoblast subtypes. However, factors controlling growth and differentiation of these cells during early gestation remain poorly elucidated. Herein, we provide evidence that the ligand Wnt5a could be a critical regulator of trophoblast proliferation and survival. Immunofluorescence of tissues and western blot analyses of primary cultures revealed abundant Wnt5a expression and secretion from first trimester decidual and villous stromal cells. The ligand was also detectable in decidual glands, macrophages and NK cells. Wnt5a increased proliferation of villous cytotrophoblasts and cell column trophoblasts, outgrowth on collagen I as well as cyclin A and D1 expression in floating explant cultures, but suppressed camptothecin-induced apoptosis. Similarly, Wnt5a stimulated BrdU incorporation and decreased caspase-cleaved cytokeratin 18 neo-epitope expression in primary cytotrophoblasts. Moreover, Wnt5a promoted activation of the MAPK pathway in the different trophoblast models. Chemical inhibition of p42/44 MAPK abolished cyclin D1 expression and Wnt5a-stimulated proliferation. Compared to controls, MAPK phosphorylation and proliferation of cytotrophoblasts declined upon supplementation of supernatants from Wnt5a gene-silenced decidual or villous stromal cells. In summary, non-canonical Wnt5a signalling could play a role in early human trophoblast development by promoting cell proliferation and survival. PMID:27311852

  16. 78 FR 40317 - Highly Migratory Species; Atlantic Shark Management Measures; Amendment 5a

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-03

    ...NMFS publishes this final rule implementing the Final Amendment 5a to the 2006 Consolidated Atlantic Highly Migratory Species (HMS) Fishery Management Plan (FMP). In developing Amendment 5a to the 2006 Consolidated HMS FMP, we examined a full range of management alternatives to maintain rebuilding of sandbar sharks; end overfishing and rebuild scalloped hammerhead and Atlantic blacknose......

  17. From UBE3A to Angelman syndrome: a substrate perspective

    PubMed Central

    Sell, Gabrielle L.; Margolis, Seth S.

    2015-01-01

    Angelman syndrome (AS) is a debilitating neurodevelopmental disorder that is characterized by motor dysfunction, intellectual disability, speech impairment, seizures and common features of autism spectrum disorders (ASDs). Some of these AS related phenotypes can be seen in other neurodevelopmental disorders (Williams, 2011; Tan et al., 2014). AS patients commonly carry mutations that render the maternally inherited UBE3A gene non-functional. Duplication of the chromosomal region containing the UBE3A gene is associated with ASDs. Although the causative role for UBE3A gene mutations in AS is well established, a long-standing challenge in AS research has been to identify neural substrates of UBE3A, an E3 ubiquitin ligase. A prevailing hypothesis is that changes in UBE3A protein levels would alter the levels of a collection of protein substrates, giving rise to the unique phenotypic aspects of AS and possibly UBE3A associated ASDs. Interestingly, proteins altered in AS are linked to additional ASDs that are not previously associated with changes in UBE3A, indicating a possible molecular overlap underlying the broad-spectrum phenotypes of these neurogenetic disorders. This idea raises the possibility that there may exist a “one-size-fits-all” approach to the treatment of neurogenetic disorders with phenotypes overlapping AS. Furthermore, while a comprehensive list of UBE3A substrates and downstream affected pathways should be developed, this is only part of the story. The timing of when UBE3A protein functions, through either changes in UBE3A or possibly substrate expression patterns, appears to be critical for AS phenotype development. These data call for further investigation of UBE3A substrates and their timing of action relevant to AS phenotypes. PMID:26441497

  18. SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

    NASA Astrophysics Data System (ADS)

    Gnedenko, O. V.; Yablokov, E. O.; Usanov, S. A.; Mukha, D. V.; Sergeev, G. V.; Bulko, T. V.; Kuzikov, A. V.; Moskaleva, N. E.; Shumyantseva, V. V.; Ivanov, A. S.; Archakov, A. I.

    2014-02-01

    The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

  19. Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA.

    PubMed

    Ascher, David B; Wielens, Jerome; Nero, Tracy L; Doughty, Larissa; Morton, Craig J; Parker, Michael W

    2014-01-01

    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. PMID:24755925

  20. Discovery of KDM5A inhibitors: Homology modeling, virtual screening and structure-activity relationship analysis.

    PubMed

    Wu, Xiaoai; Fang, Zhen; Yang, Bo; Zhong, Lei; Yang, Qiuyuan; Zhang, Chunhui; Huang, Shenzhen; Xiang, Rong; Suzuki, Takayoshi; Li, Lin-Li; Yang, Sheng-Yong

    2016-05-01

    Herein we report the discovery of a series of new KDM5A inhibitors. A three-dimensional (3D) structure model of KDM5A jumonji domain was firstly established based on homology modeling. Molecular docking-based virtual screening was then performed against commercial chemical databases. A number of hit compounds were retrieved. Further structural optimization and structure-activity relationship (SAR) analysis were carried out to the most active hit compound, 9 (IC50: 2.3μM), which led to the discovery of several new KDM5A inhibitors. Among them, compound 15e is the most potent one with an IC50 value of 0.22μM against KDM5A. This compound showed good selectivity for KDM5A and considerable ability to suppress the demethylation of H3K4me3 in intact cells. Compound 15e could be taken as a good lead compound for further studies. PMID:27020306

  1. Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA

    PubMed Central

    Ascher, David B.; Wielens, Jerome; Nero, Tracy L.; Doughty, Larissa; Morton, Craig J.; Parker, Michael W.

    2014-01-01

    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. PMID:24755925

  2. Can Wnt5a and Wnt non-canonical pathways really mediate adipocyte de-differentiation in a tumour microenvironment?

    PubMed

    Chirumbolo, Salvatore; Bjørklund, Geir

    2016-09-01

    Wnt5a has been recently reported as a possible triggering factor of adipocyte de-differentiation into an adipocyte-derived fibroblast in the tumour microenvironment of pancreas cancer. The Wnt/β-catenin pathway was described in processes involving de-differentiation and epithelial-mesenchymal transition but some Wnt family-belonging molecules exert an adipogenic role on adipocyte, while other ones, such as Wnt10b or Wnt3a, an anti-adipogenic role. Although this ability depends on the different tumoural microenvironments, it is intriguing to ascertain if some Wnt molecules, participating in the non-canonical pathway, may be targeted as fundamental factors able to trigger the desmoplastic reaction of peritumoural white adipose tissue. PMID:27391920

  3. Stabilization/solidification on chromium (III) wastes by C(3)A and C(3)A hydrated matrix.

    PubMed

    Li, Xiangguo; He, Chao; Bai, Yun; Ma, Baoguo; Wang, Guandong; Tan, Hongbo

    2014-03-15

    Hazardous wastes are usually used in the Portland cement production in order to save energy, costs and/or stabilize toxic substances and heavy metals inside the clinker. This work focus on the stabilization/solidification on chromium (III) wastes by C(3)A and C(3)A hydrated matrix. The immobilization rate of chromium in C(3)A and the leaching characteristics of the C(3)A hydrated matrixes containing chromium were investigated by ICP-AES. The results indicated that C(3)A had a good solidifying effect on chromium using the clinkering process, however, the Cr leaching content of Cr-doped C(3)A was higher than that of hydrated C(3)A matrix in Cr(NO(3))3 solution and was lower than that of the hydrated C(3)A matrix in K(2)CrO(4) solution, no matter the leachant was sulphuric acid & nitric acid or water. To explain this, C(3)A formation, chemical valence states of chromium in C(3)A, hydration products and Cr distribution in the C(3)A-gypsum hydrated matrixes were studied by XRD, XPS and FESEM-EDS. The investigation showed that part of Cr(3+) was oxidized to Cr(6+) in the clinkering process and identified as the chromium compounds Ca(4)Al(6)O(12)CrO(4) (3CaO·Al(20O(3)·CaCrO(4)), which resulted in the higher leaching of hydrated matrix of Cr-doped C(3)A. PMID:24468527

  4. Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

    PubMed Central

    Nausch, Henrik; Mischofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active. PMID:23285250

  5. 18 CFR 3a.13 - Classification responsibility and procedure.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Classification responsibility and procedure. 3a.13 Section 3a.13 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  6. 18 CFR 3a.12 - Authority to classify official information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Authority to classify official information. 3a.12 Section 3a.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  7. 18 CFR 3a.11 - Classification of official information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Classification of official information. 3a.11 Section 3a.11 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  8. 18 CFR 3a.31 - Classification markings and special notations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Classification markings and special notations. 3a.31 Section 3a.31 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION...

  9. 18 CFR 3a.13 - Classification responsibility and procedure.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Classification responsibility and procedure. 3a.13 Section 3a.13 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  10. 18 CFR 3a.13 - Classification responsibility and procedure.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Classification responsibility and procedure. 3a.13 Section 3a.13 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  11. 18 CFR 3a.13 - Classification responsibility and procedure.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Classification responsibility and procedure. 3a.13 Section 3a.13 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  12. 18 CFR 3a.11 - Classification of official information.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Classification of official information. 3a.11 Section 3a.11 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  13. 18 CFR 3a.21 - Authority to downgrade and declassify.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Authority to downgrade and declassify. 3a.21 Section 3a.21 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Declassification...

  14. 18 CFR 3a.13 - Classification responsibility and procedure.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Classification responsibility and procedure. 3a.13 Section 3a.13 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification §...

  15. 19 CFR 201.3a - Missing children information.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 3 2012-04-01 2012-04-01 false Missing children information. 201.3a Section 201... Miscellaneous § 201.3a Missing children information. (a) Pursuant to 39 U.S.C. 3220, penalty mail sent by the Commission may be used to assist in the location and recovery of missing children. This section...

  16. 19 CFR 201.3a - Missing children information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 3 2013-04-01 2013-04-01 false Missing children information. 201.3a Section 201... Miscellaneous § 201.3a Missing children information. (a) Pursuant to 39 U.S.C. 3220, penalty mail sent by the Commission may be used to assist in the location and recovery of missing children. This section...

  17. 19 CFR 201.3a - Missing children information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Missing children information. 201.3a Section 201... Miscellaneous § 201.3a Missing children information. (a) Pursuant to 39 U.S.C. 3220, penalty mail sent by the Commission may be used to assist in the location and recovery of missing children. This section...

  18. 19 CFR 201.3a - Missing children information.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 3 2014-04-01 2014-04-01 false Missing children information. 201.3a Section 201... Miscellaneous § 201.3a Missing children information. (a) Pursuant to 39 U.S.C. 3220, penalty mail sent by the Commission may be used to assist in the location and recovery of missing children. This section...

  19. 19 CFR 201.3a - Missing children information.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 3 2011-04-01 2011-04-01 false Missing children information. 201.3a Section 201... Miscellaneous § 201.3a Missing children information. (a) Pursuant to 39 U.S.C. 3220, penalty mail sent by the Commission may be used to assist in the location and recovery of missing children. This section...

  20. CYP3A5 mediates bioactivation and cytotoxicity of tetrandrine.

    PubMed

    Tian, Ye; Shen, Shuijie; Jiang, Yan; Shen, Qi; Zeng, Su; Zheng, Jiang

    2016-07-01

    Tetrandrine is a diaryl ether-type bisbenzylisoquinoline alkaloid and has shown multiple pharmacological activities. Our early work demonstrated that tetrandrine produced acute pulmonary toxicity and that tetrandrine was biotransformed to a quinone methide-derived metabolite mediated by CYP3A enzymes. The formation of the reactive intermediate is suggested to be responsible for the pulmonary toxicity induced by tetrandrine. In the present study, a WI-38-based Cyp3a5 transgenic cell line (WI-38/Cyp3a5) was established to investigate the role of CYP3A5 in tetrandrine-induced cytotoxicity. The transgenic cells were found to be more susceptible to the cytotoxicity of tetrandrine than the wild-type cells (WI-38/Vector). WI-38/Cyp3a5 cells showed higher cellular ROS levels, higher LDH activities in culture media, but lower cellular GSH contents than those observed in WI-38/Vector cells after exposure to tetrandrine. And severer apoptosis were observed in WI-38/Cyp3a5 cells after treatment with tetrandrine: WI-38/Cyp3a5 cells had higher proportion of early and late apoptotic cells, higher expression levels of caspase-3, but lower level of Bcl-2 than WI-38/Vector cells. This study provided strong evidence that CYP3A5 participated in tetrandrine-induced cytotoxicity. PMID:26302866

  1. 17 CFR 270.3a-2 - Transient investment companies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Transient investment companies... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.3a-2 Transient investment companies... which an issuer owns or proposes to acquire investment securities (as defined in section 3(a) of the...

  2. 17 CFR 270.3a-2 - Transient investment companies.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Transient investment companies... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.3a-2 Transient investment companies... which an issuer owns or proposes to acquire investment securities (as defined in section 3(a) of the...

  3. 17 CFR 270.3a-2 - Transient investment companies.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Transient investment companies... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.3a-2 Transient investment companies... which an issuer owns or proposes to acquire investment securities (as defined in section 3(a) of the...

  4. 18 CFR 3a.91 - Data index system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Data index system. 3a..., DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Data Index System § 3a.91 Data index system. A data index system shall be established for Top Secret, Secret, and Confidential information...

  5. 18 CFR 3a.91 - Data index system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Data index system. 3a..., DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Data Index System § 3a.91 Data index system. A data index system shall be established for Top Secret, Secret, and Confidential information...

  6. 18 CFR 3a.91 - Data index system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Data index system. 3a..., DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Data Index System § 3a.91 Data index system. A data index system shall be established for Top Secret, Secret, and Confidential information...

  7. 18 CFR 3a.91 - Data index system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Data index system. 3a..., DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Data Index System § 3a.91 Data index system. A data index system shall be established for Top Secret, Secret, and Confidential information...

  8. 18 CFR 3a.91 - Data index system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Data index system. 3a..., DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Data Index System § 3a.91 Data index system. A data index system shall be established for Top Secret, Secret, and Confidential information...

  9. Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes Cell Proliferation and Motility in Pancreatic Cancer.

    PubMed

    Wang, Shu Qian; Liu, Yu; Yao, Min Ya; Jin, Jing

    2016-10-01

    Identifying a target molecule that is crucially involved in pancreatic tumor growth and metastasis is necessary in developing an effective treatment. The study aimed to investigate the role of the eukaryotic translation initiation factor 3a (eIF3a) in the cell proliferation and motility in pancreatic cancer. Our data showed that the expression of eIF3a was upregulated in pancreatic ductal adenocarcinoma as compared with its expression in normal pancreatic tissues. Knockdown of eIF3a by a specific shRNA caused significant decreases in cell proliferation and clonogenic abilities in pancreatic cancer SW1990 and Capan-1 cells. Consistently, the pancreatic cancer cell growth rates were also impaired in xenotransplanted mice. Moreover, wound-healing assay showed that depletion of eIF3a significantly slowed down the wound recovery processes in SW1990 and Capan-1 cells. Transwell migration and invasion assays further showed that cell migration and invasion abilities were significantly inhibited by knockdown of eIF3a in SW1990 and Capan-1 cells. Statistical analysis of eIF3a expression in 140 cases of pancreatic ductal adenocarcinoma samples revealed that eIF3a expression was significantly associated with tumor metastasis and TNM staging. These analyses suggest that eIF3a contributes to cell proliferation and motility in pancreatic ductal adenocarcinoma. PMID:27550487

  10. Small molecular probes for G-protein-coupled C5a receptors: conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a.

    PubMed

    Wong, A K; Finch, A M; Pierens, G K; Craik, D J; Taylor, S M; Fairlie, D P

    1998-08-27

    Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by 1H NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH. OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH.OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity

  11. The Role Of Semaphorin 3A In The Skeletal System.

    PubMed

    Tang, Peifu; Yin, Pengbin; Lv, Houchen; Zhang, Licheng; Zhang, Lihai

    2015-01-01

    Semaphorin 3A (Sema3A), characterized by a conserved N-terminal "Sema" domain, was originally described as an axon guidance molecule. Recent research indicates that it performs a critical function in the skeletal system. This review highlights recent advances in understanding of the role of Sema3A in the skeletal system as a regulator of bone metabolism and as a potential drug target for bone disease therapy. We summarize Sema3A functions in osteoblastogenesis and osteoclastogenesis, as well as in innervation, and we discuss its multifunctional role in various bone diseases such as osteoporosis and low back pain. Despite limited research in this field, our aim is to promote further understanding of the function of Sema3A in the skeletal system. PMID:25955818

  12. In vitro and pharmacophore insights into CYP3A enzymes.

    PubMed

    Ekins, Sean; Stresser, David M; Williams, J Andrew

    2003-04-01

    The cytochrome P450 3A (CYP3A) enzymes have a major role in the metabolism of drugs in humans. Their wide substrate specificity and induction by a vast array of structurally diverse compounds presents the possibility of metabolic drug-drug interactions. Understanding the enzymes themselves is crucial. Over the past decade, this has occurred mostly with in vitro studies, although more recent approaches incorporate computational models to predict CYP inhibition and substrate potential. The three-dimensional displacement, or pharmacophore, of chemical features in space that are derived from inhibition data have produced pharmacophores for CYP3A4, CYP3A5 and CYP3A7, and provide new insights into ligand binding for each enzyme. PMID:12707001

  13. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    SciTech Connect

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. Black-Right-Pointing-Pointer Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. Black-Right-Pointing-Pointer Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. Black-Right-Pointing-Pointer Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through {beta}-catenin-dependent canonical and {beta}-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent

  14. Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures

    PubMed Central

    Ramakrishnan, Venkat M.; Tien, Kevin T.; McKinley, Thomas R.; Bocard, Braden R.; McCurry, Terry M.; Williams, Stuart K.; Hoying, James B.; Boyd, Nolan L.

    2016-01-01

    Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0–150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt

  15. Characterization of Dengue Virus NS4A and NS4B Protein Interaction

    PubMed Central

    Zou, Jing; Xie, Xuping; Wang, Qing-Yin; Dong, Hongping; Lee, Michelle Yueqi; Kang, Congbao

    2015-01-01

    ABSTRACT Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach. IMPORTANCE Flavivirus NS4A and NS4B proteins are essential components of the ER membrane

  16. SCN1B gene variants in Brugada Syndrome: a study of 145 SCN5A-negative patients.

    PubMed

    Ricci, Maria Teresa; Menegon, Silvia; Vatrano, Simona; Mandrile, Giorgia; Cerrato, Natascia; Carvalho, Paula; De Marchi, Mario; Gaita, Fiorenzo; Giustetto, Carla; Giachino, Daniela Francesca

    2014-01-01

    Brugada syndrome is characterised by a typical ECG with ST segment elevation in the right precordial leads. Individuals with this condition are susceptible to ventricular arrhythmias and sudden cardiac death. The principal gene responsible for this syndrome is SCN5A, which encodes the α-subunit of the Nav1.5 voltage-gated sodium channel. Mutations involving other genes have been increasingly reported, but their contribution to Brugada syndrome has been poorly investigated. Here we focused on the SCN1B gene, which encodes the β1-subunit of the voltage-gated sodium channel and its soluble β1b isoform. SCN1B mutations have been associated with Brugada syndrome as well as with other cardiac arrhythmias and familial epilepsy. In this study, we have analysed SCN1B exons (including the alternatively-spliced exon 3A) and 3'UTR in 145 unrelated SCN5A-negative patients from a single centre. We took special care to report all identified variants (including polymorphisms), following the current nomenclature guidelines and considering both isoforms. We found two known and two novel (and likely deleterious) SCN1B variants. We also found two novel changes with low evidence of pathogenicity. Our findings contribute more evidence regarding the occurrence of SCN1B variants in Brugada syndrome, albeit with a low prevalence, which is in agreement with previous reports. PMID:25253298

  17. Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

    PubMed Central

    Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A.

    2013-01-01

    Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their Ki and IC50 values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC50 values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). PMID:23940536

  18. 32 CFR 1630.40 - Class 4-A: Registrant who has completed military service.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Class 4-A: Registrant who has completed military service. 1630.40 Section 1630.40 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM CLASSIFICATION RULES § 1630.40 Class 4-A: Registrant who has completed military service. (a) In Class 4-A shall be placed...

  19. Genetics Home Reference: COL4A1-related brain small-vessel disease

    MedlinePlus

    ... COL4A1-related brain small-vessel disease COL4A1-related brain small-vessel disease Enable Javascript to view the ... PDF Open All Close All Description COL4A1 -related brain small-vessel disease is part of a group ...

  20. C4.4A as a candidate marker in the diagnosis of colorectal cancer.

    PubMed

    Paret, C; Hildebrand, D; Weitz, J; Kopp-Schneider, A; Kuhn, A; Beer, A; Hautmann, R; Zöller, M

    2007-10-22

    C4.4A is a member of the Ly-6 family with restricted expression in non-transformed tissues. C4.4A expression in human cancer has rarely been evaluated. Thus, it became important to explore C4.4A protein expression in human tumour tissue to obtain an estimate on the frequency of expression and the correlation with tumour progression, the study focusing on colorectal cancer. The analysis of C4.4A in human tumour lines by western blot and immunoprecipitation using polyclonal rabbit antibodies that recognize different C4.4A epitopes revealed C4.4A oligomer and heavily glycosylated C4.4A isoform expression that, in some instances, inhibited antibody binding and interaction with the C4.4A ligand galectin-3. In addition, tumour cell lines released C4.4A by vesicle shedding and proteolytic cleavage. C4.4A was expressed in over 80% of primary colorectal cancer and liver metastasis with negligible expression in adjacent colonic mucosa, inflamed colonic tissue and liver. This compares well with EpCAM and CO-029 expression in over 90% of colorectal cancer. C4.4A expression was only observed in about 50% of pancreatic cancer and renal cell carcinoma. By de novo expression in colonic cancer tissue, we consider C4.4A as a candidate diagnostic marker in colorectal cancer, which possibly can be detected in body fluids. PMID:17912244

  1. C4.4A as a candidate marker in the diagnosis of colorectal cancer

    PubMed Central

    Paret, C; Hildebrand, D; Weitz, J; Kopp-Schneider, A; Kuhn, A; Beer, A; Hautmann, R; Zöller, M

    2007-01-01

    C4.4A is a member of the Ly-6 family with restricted expression in non-transformed tissues. C4.4A expression in human cancer has rarely been evaluated. Thus, it became important to explore C4.4A protein expression in human tumour tissue to obtain an estimate on the frequency of expression and the correlation with tumour progression, the study focusing on colorectal cancer. The analysis of C4.4A in human tumour lines by western blot and immunoprecipitation using polyclonal rabbit antibodies that recognize different C4.4A epitopes revealed C4.4A oligomer and heavily glycosylated C4.4A isoform expression that, in some instances, inhibited antibody binding and interaction with the C4.4A ligand galectin-3. In addition, tumour cell lines released C4.4A by vesicle shedding and proteolytic cleavage. C4.4A was expressed in over 80% of primary colorectal cancer and liver metastasis with negligible expression in adjacent colonic mucosa, inflamed colonic tissue and liver. This compares well with EpCAM and CO-029 expression in over 90% of colorectal cancer. C4.4A expression was only observed in about 50% of pancreatic cancer and renal cell carcinoma. By de novo expression in colonic cancer tissue, we consider C4.4A as a candidate diagnostic marker in colorectal cancer, which possibly can be detected in body fluids. PMID:17912244

  2. 26 CFR 1.668(b)-4A - Information requirements with respect to beneficiary.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... beneficiary. 1.668(b)-4A Section 1.668(b)-4A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Trusts Applicable to Taxable Years Beginning Before January 1, 1969 § 1.668(b)-4A Information... computed under paragraph (c) of § 1.668(b)-1A (the short-cut method) shall be deemed to be the amount...

  3. WNT5A inhibits human dental papilla cell proliferation and migration

    SciTech Connect

    Peng, L.; Ye, L.; Dong, G.; Ren, L.B.; Wang, C.L.; Xu, P.; Zhou, X.D.

    2009-12-18

    WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

  4. C5a Receptor (CD88) Blockade Protects against MPO-ANCA GN

    PubMed Central

    Xiao, Hong; Dairaghi, Daniel J.; Powers, Jay P.; Ertl, Linda S.; Baumgart, Trageen; Wang, Yu; Seitz, Lisa C.; Penfold, Mark E.T.; Gan, Lin; Hu, Peiqi; Lu, Bao; Gerard, Norma P.; Gerard, Craig; Schall, Thomas J.; Jaen, Juan C.; Falk, Ronald J.

    2014-01-01

    Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO–induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO–induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN. PMID:24179165

  5. WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

    PubMed Central

    Anastas, Jamie N.; Kulikauskas, Rima M.; Tamir, Tigist; Rizos, Helen; Long, Georgina V.; von Euw, Erika M.; Yang, Pei-Tzu; Chen, Hsiao-Wang; Haydu, Lauren; Toroni, Rachel A.; Lucero, Olivia M.; Chien, Andy J.; Moon, Randall T.

    2014-01-01

    About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAFV600E/K) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance. PMID:24865425

  6. Novel SLC5A2 mutation contributes to familial renal glucosuria: Abnormal expression in renal tissues

    PubMed Central

    Yu, Lei; Hou, Ping; Liu, Guo-Ping; Zhang, Hong

    2016-01-01

    Familial renal glucosuria (FRG) is characterized by persistent glucosuria in the presence of normal serum glucose concentrations, while other impairments of tubular function are absent. Mutations in the sodium-glucose co-transporter 2 (SLC5A2) gene have been found to be responsible for FRG. However, direct evidence for the presence of SLC5A2 mutant in renal tissues is very rare. In previous studies, a non-sense mutation (c.1320 G>A:p.W440X) that would cause premature termination of the protein was found. However, the effects in the renal tissues were not reported. In the current study, a patient with FRG and a urinary glucose excretion rate of 8.3 g/day is described, for whom a novel missense mutation (c.1319G>A:p.W440X) was revealed by sequencing. Furthermore, in the immunofluorescence examination of a renal biopsy specimen, SLC5A2 was detected in the apical side of the proximal convoluted tubule, discontinuously decreased in comparison with that in normal and disease controls. The results imply that both wild-type SLC5A2 and mutant SLC5A2 with abnormal distribution were expressed in the renal tissues, and that the reduction of SLC5A2 expression and function were due to the c.1319G>A:p.W440X mutation. The current study provides valuable clues regarding the SLC5A2 molecule from genotype to phenotype in families affected by FRG.

  7. Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

    PubMed Central

    Koopmans, Tim; Kumawat, Kuldeep; Halayko, Andrew J; Gosens, Reinoud

    2016-01-01

    A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma. PMID:27468699

  8. Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production.

    PubMed

    Parodi, Jorge; Montecinos-Oliva, Carla; Varas, Rodrigo; Alfaro, Iván E; Serrano, Felipe G; Varas-Godoy, Manuel; Muñoz, Francisco J; Cerpa, Waldo; Godoy, Juan A; Inestrosa, Nibaldo C

    2015-09-01

    Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons. PMID:26311509

  9. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

    PubMed

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  10. STAT5A is regulated by DNA damage via the tumor suppressor p53.

    PubMed

    Mukhopadhyay, Utpal K; Cass, Jamaica; Raptis, Leda; Craig, Andrew W; Bourdeau, Véronique; Varma, Sonal; SenGupta, Sandip; Elliott, Bruce E; Ferbeyre, Gerardo

    2016-06-01

    Here we report that the STAT5A transcription factor is a direct p53 transcriptional target gene. STAT5A is well expressed in p53 wild type cells but not in p53-null cells. Inhibition of p53 reduces STAT5A expression. DNA damaging agents such as doxorubicin also induced STAT5A expression in a p53 dependent manner. Two p53 binding sites were mapped in the STAT5A gene and named PBS1 and PBS2; these sites were sufficient to confer p53 responsiveness in a luciferase reporter gene. Chromatin immunoprecipitation experiments revealed that PBS2 has constitutive p53 bound to it, while p53 binding to PBS1 required DNA damage. In normal human breast lobules, weak p53 staining correlated with regions of intense STAT5A staining. Interestingly, in a cohort of triple negative breast tumor tissues there was little correlation between regions of p53 and STAT5A staining, likely reflecting a high frequency of p53 mutations that stabilize the protein in these tumors. We thus reveal an unexpected connection between cytokine signaling and p53. PMID:26876578

  11. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    PubMed Central

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  12. Cutting Edge: CLEC5A Mediates Macrophage Function and Chronic Obstructive Pulmonary Disease Pathologies.

    PubMed

    Wortham, Brian W; Eppert, Bryan L; Flury, Jennifer L; Garcia, Sara Morgado; Donica, Walter R; Osterburg, Andrew; Joyce-Shaikh, Barbara; Cua, Daniel J; Borchers, Michael T

    2016-04-15

    Chronic obstructive pulmonary disease (COPD) is a devastating disease with no effective therapies. We investigated the role of the C-type lectin receptor, CLEC5A, in macrophage activation and pulmonary pathogenesis in a mouse model of COPD. We demonstrate that CLEC5A is expressed on alveolar macrophages in mice exposed long-term to cigarette smoke (CS), as well as in human smokers. We also show that CLEC5A-mediated activation of macrophages enhanced cytokine elaboration alone, as well as in combination with LPS or GM-CSF in CS-exposed mice. Furthermore, usingClec5a-deficient mice, we demonstrate that CS-induced macrophage responsiveness is mediated by CLEC5A, and CLEC5A is required for the development of inflammation, proinflammatory cytokine expression, and airspace enlargement. These findings suggest a novel mechanism that promotes airway inflammation and pathologies in response to CS exposure and identifies CLEC5A as a novel target for the therapeutic control of COPD pathogenesis. PMID:26927798

  13. The transcription factors STAT5A/B regulate GM-CSF-mediated granulopoiesis.

    PubMed

    Kimura, Akiko; Rieger, Michael A; Simone, James M; Chen, Weiping; Wickre, Mark C; Zhu, Bing-Mei; Hoppe, Philipp S; O'Shea, John J; Schroeder, Timm; Hennighausen, Lothar

    2009-11-19

    Neutrophils play a vital role in the immune defense, which is evident by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. Both the GM-CSF-permitted survival of mature neutrophils and the generation of granulocytes from granulocyte-macrophage progenitors (GMPs) were markedly reduced in Stat5a/b mutants. GMPs showed impaired colony-formation ability with reduced number and size of colonies on GM-CSF stimulation. Moreover, continuous cell fate analyses by time-lapse microscopy and single cell tracking revealed that Stat5a/b-null GMPs showed both delayed cell-cycle progression and increased cell death. Finally, transcriptome analysis indicated that STAT5A/B directs GM-CSF signaling through the regulation of proliferation and survival genes. PMID:19779039

  14. 75 FR 910 - Airworthiness Directives; General Electric Company CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-07

    ...-07R1, Amendment 39-15179 (72 FR 49183, August 28, 2007) and AD 2007-05-16, Amendment 39-14977 (72 FR... FR 17799). That action proposed to require: Replacing certain fan disks installed on regional jets... instructions related to operators who fly a regional jet (RJ) with the CF34-3A1 engine as a business jet...

  15. Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A.

    PubMed

    Sun, Jin-Hua; O'Boyle, Donald R; Fridell, Robert A; Langley, David R; Wang, Chunfu; Roberts, Susan B; Nower, Peter; Johnson, Benjamin M; Moulin, Frederic; Nophsker, Michelle J; Wang, Ying-Kai; Liu, Mengping; Rigat, Karen; Tu, Yong; Hewawasam, Piyasena; Kadow, John; Meanwell, Nicholas A; Cockett, Mark; Lemm, Julie A; Kramer, Melissa; Belema, Makonen; Gao, Min

    2015-11-12

    It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3-4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle. PMID:26536115

  16. Eukaryotic Initiation Factor 5A Plays an Essential Role in Luteinizing Hormone Receptor Regulation

    PubMed Central

    Menon, Bindu; Gulappa, Thippeswamy

    2014-01-01

    Down-regulation of LH receptor (LHR) in the ovary by its ligand is mediated by a specific RNA-binding protein, designated LH receptor mRNA–binding protein (LRBP), through translational suppression and mRNA degradation. Using yeast 2-hybrid screens, we previously identified eukaryotic initiation factor 5A (eIF5A) as one of the proteins that interacts with LRBP during LHR mRNA down-regulation. The present study examined the role of eIF5A and its hypusination in the context of LHR mRNA down-regulation. The association of eIF5A with LRBP or LHR mRNA was determined using immunoprecipitation and RNA immunoprecipitation assays. The results showed that the association of eIF5A with the LHR mRNA-LRBP complex increased significantly during down-regulation. Furthermore, gel fractionation and the hypusination activity assay both showed increased hypusination of eIF5A during LHR mRNA down-regulation. Abolishment of hypusination by pretreatment with the chemical inhibitor GC7 prevented the association of eIF5A with LHR mRNA and LRBP. Inhibition of hypusination also reduced the extent of ligand-induced down-regulation of LHR mRNA as well as the expression of functional LHRs assessed by real-time PCR and 125I-human chorionic gonadotropin (hCG) binding assays, respectively. The loss of human chorionic gonadotropin–mediated downstream signaling during LHR down-regulation was also restored by inhibition of hypusination of eIF5A. Thus, the present study, for the first time, reveals the crucial role of eIF5A and its hypusination in the regulation of LHR expression in the ovary. PMID:25216047

  17. Semaphorin4A Is Cytotoxic to Oligodendrocytes and Is Elevated in Microglia and Multiple Sclerosis.

    PubMed

    Leitner, Dominique F; Todorich, Bozho; Zhang, Xuesheng; Connor, James R

    2015-01-01

    We have previously established that T cell immunoglobulin and mucin domain containing 2 (Tim2) is an H-ferritin receptor on oligodendrocytes (OLs). Tim2 also binds Semaphorin4A (Sema4A). Sema4A is expressed by lymphocytes, and its role in immune activation is known; however, its relationship to diseases that are known to have myelin damage has not been studied. In this study, we demonstrate that Sema4A is cytotoxic to OLs in culture: an effect accompanied by process collapse, membrane blebbing, and phosphatidylserine inversion. We further demonstrate that Sema4A preferentially binds to primary OLs but not astrocytes: an observation consistent with the lack of expression of Tim2 on astrocytes. We found that Sema4A protein levels are increased within multiple sclerosis plaques compared with normal-appearing white matter and that Sema4A induces lactate dehydrogenase release in a human OL cell line. The chief cellular source of Sema4A within the multiple sclerosis plaques appears to be infiltrating lymphocytes and microglia. Macrophages are known to express Sema4A, so we interrogated microglia as a potential source of Sema4A in the brain. We found that rat primary microglia express Sema4A which increased after lipopolysaccharide activation. Because activated microglia accumulate iron, we determined whether iron status influenced Sema4A and found that iron chelation decreased Sema4A and iron loading increased Sema4A in activated microglia. Overall, our data implicate Sema4A in the destruction of OLs and reveal that its expression is sensitive to iron levels. PMID:26024919

  18. Semaphorin4A Is Cytotoxic to Oligodendrocytes and Is Elevated in Microglia and Multiple Sclerosis

    PubMed Central

    Leitner, Dominique F.; Todorich, Bozho; Zhang, Xuesheng

    2015-01-01

    We have previously established that T cell immunoglobulin and mucin domain containing 2 (Tim2) is an H-ferritin receptor on oligodendrocytes (OLs). Tim2 also binds Semaphorin4A (Sema4A). Sema4A is expressed by lymphocytes, and its role in immune activation is known; however, its relationship to diseases that are known to have myelin damage has not been studied. In this study, we demonstrate that Sema4A is cytotoxic to OLs in culture: an effect accompanied by process collapse, membrane blebbing, and phosphatidylserine inversion. We further demonstrate that Sema4A preferentially binds to primary OLs but not astrocytes: an observation consistent with the lack of expression of Tim2 on astrocytes. We found that Sema4A protein levels are increased within multiple sclerosis plaques compared with normal-appearing white matter and that Sema4A induces lactate dehydrogenase release in a human OL cell line. The chief cellular source of Sema4A within the multiple sclerosis plaques appears to be infiltrating lymphocytes and microglia. Macrophages are known to express Sema4A, so we interrogated microglia as a potential source of Sema4A in the brain. We found that rat primary microglia express Sema4A which increased after lipopolysaccharide activation. Because activated microglia accumulate iron, we determined whether iron status influenced Sema4A and found that iron chelation decreased Sema4A and iron loading increased Sema4A in activated microglia. Overall, our data implicate Sema4A in the destruction of OLs and reveal that its expression is sensitive to iron levels. PMID:26024919

  19. Lung Tumorigenesis in a Conditional Cul4A Transgenic Mouse Model

    PubMed Central

    Yang, Yi-Lin; Hung, Ming-Szu; Wang, Yang; Ni, Jian; Mao, Jian-Hua; Hsieh, David; Au, Alfred; Kumar, Atul; Quigley, David; Fang, Li Tai; Yeh, Che-Chung; Xu, Zhidong; Jablons, David M.; You, Liang

    2014-01-01

    Cullin4A (Cul4A) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes and regulates many cellular events, including cell survival, development, growth, and cell cycle control. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Here, we used a Cul4A transgenic mouse model to study the potential oncogenic role of Cul4A in lung tumor development. After Cul4A overexpression was induced in the lungs for 32 weeks, atypical epithelial cells were observed. After 40 weeks, lung tumors were visible and were characterized as Grade I or II adenocarcinomas. Immunohistochemistry (IHC) revealed decreased levels of Cul4A associated proteins p21CIP1 and tumor suppressor p19ARF in the lung tumors, suggesting Cul4A regulated their expression in these tumors. Increased levels of p27KIP1 and p16INK4a were also detected in these tumors. Moreover, protein level of DNA replication licensing factor CDT1 was decreased. Genomic instability in the lung tumors was further analyzed by the results from pericentrin protein expression and array Comparative Genomic Hybridization analysis. Furthermore, knocking down Cul4A expression in lung cancer H2170 cells increased their sensitivity to the chemotherapy drug cisplatin in vitro, suggesting that Cul4A overexpression is associated with cisplatin resistance in the cancer cells. Our findings indicate that Cul4A is oncogenic in vivo, and this Cul4A mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A. PMID:24648314

  20. C5a alters blood-brain barrier integrity in experimental lupus.

    PubMed

    Jacob, Alexander; Hack, Bradley; Chiang, Eddie; Garcia, Joe G N; Quigg, Richard J; Alexander, Jessy J

    2010-06-01

    The blood-brain barrier (BBB) is a crucial anatomic location in the brain. Its dysfunction complicates many neurodegenerative diseases, from acute conditions, such as sepsis, to chronic diseases, such as systemic lupus erythematosus (SLE). Several studies suggest an altered BBB in lupus, but the underlying mechanism remains unknown. In the current study, we observed a definite loss of BBB integrity in MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) lupus mice by IgG infiltration into brain parenchyma. In line with this result, we examined the role of complement activation, a key event in this setting, in maintenance of BBB integrity. Complement activation generates C5a, a molecule with multiple functions. Because the expression of the C5a receptor (C5aR) is significantly increased in brain endothelial cells treated with lupus serum, the study focused on the role of C5a signaling through its G-protein-coupled receptor C5aR in brain endothelial cells, in a lupus setting. Reactive oxygen species production increased significantly in endothelial cells, in both primary cells and the bEnd3 cell line treated with lupus serum from MRL/lpr mice, compared with those treated with control serum from MRL(+/+) mice. In addition, increased permeability monitored by changes in transendothelial electrical resistance, cytoskeletal remodeling caused by actin fiber rearrangement, and increased iNOS mRNA expression were observed in bEnd3 cells. These disruptive effects were alleviated by pretreating cells with a C5a receptor antagonist (C5aRant) or a C5a antibody. Furthermore, the structural integrity of the vasculature in MRL/lpr brain was maintained by C5aR inhibition. These results demonstrate the regulation of BBB integrity by the complement system in a neuroinflammatory setting. For the first time, a novel role of C5a in the maintenance of BBB integrity is identified and the potential of C5a/C5aR blockade highlighted as a promising therapeutic strategy in SLE and other neurodegenerative diseases

  1. Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

    PubMed

    Yu, Wensheng; Coburn, Craig A; Nair, Anilkumar G; Wong, Michael; Rosenblum, Stuart B; Zhou, Guowei; Dwyer, Michael P; Tong, Ling; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Kim, Seong Heon; Zeng, Qingbei; Selyutin, Oleg; Chen, Lei; Masse, Frederic; Agrawal, Sony; Liu, Rong; Xia, Ellen; Zhai, Ying; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Asante-Appiah, Ernest; Lin, Mingxiang; Kozlowski, Joseph A

    2016-08-01

    Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition. PMID:27394665

  2. Inhibitory effects of fruit juices on CYP3A activity.

    PubMed

    Kim, Hyunmi; Yoon, Yune-Jung; Shon, Ji-Hong; Cha, In-June; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2006-04-01

    There have been very limited reports on the effects of commercial fruit juices on human CYP3A activity. Therefore, the inhibitory effects of readily available commercial fruit juices on midazolam 1'-hydroxylase activity, a marker of CYP3A, were evaluated in pooled human liver microsomes. The fruit juices investigated were black raspberry, black mulberry, plum, and wild grape. White grapefruit, pomegranate, and orange juice were used as positive and negative controls. The black mulberry juice showed the most potent inhibition of CYP3A except for grapefruit juice. The inhibition depended on the amount of a fruit juice added to the incubation mixture. The inhibitory potential of human CYP3A was in the order: grapefruit > black mulberry > wild grape > pomegranate > black raspberry. The IC(50) values of all fruit juices tested were reduced after preincubation with microsomes in the presence of the NADPH-generating system, suggesting that a mechanism-based inhibitory component was present in these fruit juices, as in the case of grapefruit. The results suggest that, like grapefruit juice, commercial fruit juices also have the potential to inhibit CYP3A-catalzyed midazolam 1'-hydroxylation. Therefore, in vivo studies investigating the interactions between fruit juices such as black mulberry and wild grape and CYP3A substrates are necessary to determine whether inhibition of CYP3A activity by fruit juices is clinically relevant. PMID:16415112

  3. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  4. Cardiac dysfunction caused by purified human C3a anaphylatoxin.

    PubMed Central

    del Balzo, U H; Levi, R; Polley, M J

    1985-01-01

    The purpose of this investigation was to define the cardiac effects of complement-derived C3a anaphylatoxin, in view of the possibility that cardiac dysfunction may occur as a result of complement activation. Purified human C3a was administered by intracoronary bolus injections into isolated guinea pig hearts. As a function of dose, C3a caused tachycardia, impairment of atrioventricular conduction, left ventricular contractile failure, coronary vasoconstriction, and histamine release. These effects were abolished by cleavage of the COOH-terminal arginine by carboxypeptidase B. The magnitude of C3a-induced tachycardia correlated with the amount of endogenous cardiac histamine released into the coronary effluent. Whereas the tachycardia was markedly reduced by the histamine H2 antagonist cimetidine, the contractile failure and the coronary vasoconstriction caused by C3a were antagonized by the leukotriene antagonist FPL 55712 and by the cyclooxygenase inhibitor indomethacin, respectively. This suggests that histamine, leukotrienes, and vasoactive prostanoates may mediate the various cardiac effects of C3a. Our findings indicate that C3a anaphylatoxin has marked cardiac effects at concentrations that are likely to be attained with a degree of C3 activation commonly seen in various disease states. Thus, our data are compatible with the hypothesis that generation of anaphylatoxins may induce cardiac dysfunction in clinical conditions. PMID:2579381

  5. Proteins Associated with SF3a60 in T. brucei

    PubMed Central

    Nyambega, Benson; Helbig, Claudia; Masiga, Daniel K.; Clayton, Christine; Levin, Mariano J.

    2014-01-01

    Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry. PMID:24651488

  6. Degradation of C3a anaphylatoxins by rat mast cells

    SciTech Connect

    Fukuoka, Y.; Hugli, T.E.

    1986-05-01

    Incubation of /sup 125/I-human C3a with rat peritoneal mast cells (RMC) causes extensive degradation of the ligand. Both cell-bound and free /sup 125/I-C3a (hu) was degraded by RMC, even at 0/sup 0/C, based on SDS-PAGE analysis. The authors examined several protease inhibitors for their ability to prevent degradation of /sup 125/I-C3a (hu). Degradation of /sup 125/I-C3a (hu) by RMC was not inhibited by leupeptin, antipain, elastatinal, pepstatin, ..cap alpha../sub 1/-antitrypsin or EDTA. TPCK and TLCK were only partially effective. PMSF, chymostatin and SBTI were most effective in preventing /sup 125/I-C3a (hu) degradation. These latter compounds are effective inhibitors of the chymotrypsin-like enzyme chymase extracted from RMC, as is TPCK, based on hydrolysis of the substrate BTEE. Degradation of cell-bound ligand is totally prevented only by PMSF (or DFP). Therefore, /sup 125/I-C3a (hu) bound to the RMC appears to be degraded predominantly by chymase; however the cell-bound ligand is attacked by other surface proteases. Degradation of rat C3a by RMC was examined. After incubation with RMC, cell-bound and free /sup 125/I-C3a (rat) showed no evidence of degradation with or without inhibitors present. From these results, the authors conclude that chymase may not play a significant role in regulating anaphylatoxin activity. Furthermore, the authors propose that rat C3a is a preferred ligand for identifying receptors on mast cells because of its resistance to proteolysis.

  7. The RNA Helicase eIF4A Is Required for Sapovirus Translation

    PubMed Central

    Hosmillo, Myra; Sweeney, Trevor R.; Chaudhry, Yasmin; Leen, Eoin; Curry, Stephen

    2016-01-01

    The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation. PMID:26937032

  8. Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase-1, at 1.5 A resolution.

    PubMed

    Yokota, Takehiro; Nara, Yukinori; Kashima, Akiko; Matsubara, Keiko; Misawa, Satoru; Kato, Ryohei; Sugio, Shigetoshi

    2007-02-01

    Human JNK stimulatory phosphatase-1 (JSP-1) is a novel member of dual specificity phosphatases. A C-terminus truncated JSP-1 was expressed in Escherichia coli and was crystallized using the sitting-drop vapor diffusion method. Thin-plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit-cell parameters a = 84.0 A, b = 49.3 A, c = 47.3 A, and beta = 119.5 degrees , and diffract up to 1.5 A resolution at 100 K. The structure of JSP-1 has a single compact (alpha/beta) domain, which consists of six alpha-helices and five beta-strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP-loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP-1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP-1 has no loop corresponding to the Lys120-loop of PTP1B, and tryptophan residue corresponding to the substrate-stacking in PTP1B is substituted by alanine residue in JSP-1. PMID:17068812

  9. Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

    SciTech Connect

    Yang, Yi -Lin; Ni, Jian; Hsu, Ping -Chih; Mao, Jian -Hua; Hsieh, David; Xu, Angela; Chan, Geraldine; Au, Alfred; Xu, Zhidong; Jablons, David M.; You, Liang

    2015-07-27

    Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.

  10. Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

    DOE PAGESBeta

    Yang, Yi -Lin; Ni, Jian; Hsu, Ping -Chih; Mao, Jian -Hua; Hsieh, David; Xu, Angela; Chan, Geraldine; Au, Alfred; Xu, Zhidong; Jablons, David M.; et al

    2015-07-27

    Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressedmore » and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.« less

  11. Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma.

    PubMed

    Yang, Yi-Lin; Ni, Jian; Hsu, Ping-Chih; Mao, Jian-Hua; Hsieh, David; Xu, Angela; Chan, Geraldine; Au, Alfred; Xu, Zhidong; Jablons, David M; You, Liang

    2015-10-01

    Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma. PMID:26218750

  12. Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

    PubMed Central

    Yang, Yi-Lin; Ni, Jian; Hsu, Ping-Chih; Mao, Jian-Hua; Hsieh, David; Xu, Angela; Chan, Geraldine; Au, Alfred; Xu, Zhidong; Jablons, David M; You, Liang

    2015-01-01

    Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma. PMID:26218750

  13. CUL4A facilitates hepatocarcinogenesis by promoting cell cycle progression and epithelial-mesenchymal transition

    PubMed Central

    Pan, Yingfang; Wang, Bo; Yang, Xiaoyun; Bai, Fuxiang; Xu, Qun; Li, Xueen; Gao, Lifen; Ma, Chunhong; Liang, Xiaohong

    2015-01-01

    CUL4A, a member of the CULLIN family, functions as a scaffold protein for an E3 ubiquitin ligase. It was reported that the CUL4A gene showed amplification in some human primary hepatocellular carcinomas (HCC). However, the exact role of CUL4A in HCC remains unknown. Here, we aimed to investigate the expression and function of CUL4A in HCC development. Through immunohistochemistry study, we showed increased CUL4A expression in HCC tissues. Statistical analysis disclosed an inverse correlation between CUL4A expression and tumor differentiation grade, and patient survival, but a positive correlation with hepatocyte proliferation as well as lymphatic and venous invasion. CUL4A expression in HCC tissues was associated with HBeAg status in patients and upregulated by HBV in HCC cell lines. Further functional assay showed that CUL4A overexpression significantly promoted growth of H22 tumor homografts in BALB/c mice. Consistently, CUL4A knockdown inhibited the proliferation of established HCC cells, accompanied by S-phase reduction and Cyclin A and Cyclin B1 repression. Furthermore, CUL4A siRNA ameliorated the motility of HCC cell lines with altered expression of epithelial-mesenchymal transition (EMT)-associated molecules. Taken together, our findings indicate that CUL4A plays a pivotal role in HCC progression and may serve as a potential marker for clinical diagnosis and target for therapy. PMID:26593394

  14. Facility 5A, interior oblique view from the west end. View ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Facility 5A, interior oblique view from the west end. View facing east-northeast - U.S. Naval Base, Pearl Harbor, Forge Shop & Galvanizing Shop, Sixth Street between Avenues D & E, Pearl City, Honolulu County, HI

  15. Facility 5A, interior oblique view from the east end. View ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Facility 5A, interior oblique view from the east end. View facing west-northwest - U.S. Naval Base, Pearl Harbor, Forge Shop & Galvanizing Shop, Sixth Street between Avenues D & E, Pearl City, Honolulu County, HI

  16. KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification

    PubMed Central

    Tetreault, Marie-Pier; Weinblatt, Daniel; Shaverdashvili, Khvaramze; Yang, Yizeng; Katz, Jonathan P.

    2016-01-01

    Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases. PMID:27184424

  17. Matched and mixed cap derivatives in the tetracyclic indole class of HCV NS5A inhibitors.

    PubMed

    Dwyer, Michael P; Keertikar, Kerry M; Chen, Lei; Tong, Ling; Selyutin, Oleg; Nair, Anilkumar G; Yu, Wensheng; Zhou, Guowei; Lavey, Brian J; Yang, De-Yi; Wong, Michael; Kim, Seong Heon; Coburn, Craig A; Rosenblum, Stuart B; Zeng, Qingbei; Jiang, Yueheng; Shankar, Bandarpalle B; Rizvi, Razia; Nomeir, Amin A; Liu, Rong; Agrawal, Sony; Xia, Ellen; Kong, Rong; Zhai, Ying; Ingravallo, Paul; Asante-Appiah, Ernest; Kozlowski, Joseph A

    2016-08-15

    A matched and mixed capping SAR study was conducted on the tetracyclic indole class of HCV NS5A inhibitors to examine the influence of modifications of this region on the overall HCV virologic resistance profiles. PMID:27423481

  18. VIEW OF SOUTH FRONT WITH BUILDING 5A ATTACHED TO LEFT, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF SOUTH FRONT WITH BUILDING 5A ATTACHED TO LEFT, FACING NORTHWEST. - Douglas Aircraft Company Long Beach Plant, Aircraft Parts Receiving & Storage Building, 3855 Lakewood Boulevard, Long Beach, Los Angeles County, CA

  19. Human aldehyde dehydrogenase 3A1 (ALDH3A1): biochemical characterization and immunohistochemical localization in the cornea.

    PubMed Central

    Pappa, Aglaia; Estey, Tia; Manzer, Rizwan; Brown, Donald; Vasiliou, Vasilis

    2003-01-01

    ALDH3A1 (aldehyde dehydrogenase 3A1) is expressed at high concentrations in the mammalian cornea and it is believed that it protects this vital tissue and the rest of the eye against UV-light-induced damage. The precise biological function(s) and cellular distribution of ALDH3A1 in the corneal tissue remain to be elucidated. Among the hypotheses proposed for ALDH3A1 function in cornea is detoxification of aldehydes formed during UV-induced lipid peroxidation. To investigate in detail the biochemical properties and distribution of this protein in the human cornea, we expressed human ALDH3A1 in Sf9 insect cells using a baculovirus vector and raised monoclonal antibodies against ALDH3A1. Recombinant ALDH3A1 protein was purified to homogeneity with a single-step affinity chromatography method using 5'-AMP-Sepharose 4B. Human ALDH3A1 demonstrated high substrate specificity for medium-chain (6 carbons and more) saturated and unsaturated aldehydes, including 4-hydroxy-2-nonenal, which are generated by the peroxidation of cellular lipids. Short-chain aliphatic aldehydes, such as acetaldehyde, propionaldehyde and malondialdehyde, were found to be very poor substrates for human ALDH3A1. In addition, ALDH3A1 metabolized glyceraldehyde poorly and did not metabolize glucose 6-phosphate, 6-phosphoglucono-delta-lactone and 6-phosphogluconate at all, suggesting that this enzyme is not involved in either glycolysis or the pentose phosphate pathway. Immunohistochemistry in human corneas, using the monoclonal antibodies described herein, revealed ALDH3A1 expression in epithelial cells and stromal keratocytes, but not in endothelial cells. Overall, these cumulative findings support the metabolic function of ALDH3A1 as a part of a corneal cellular defence mechanism against oxidative damage caused by aldehydic products of lipid peroxidation. Both recombinant human ALDH3A1 and the highly specific monoclonal antibodies described in the present paper may prove to be useful in probing

  20. Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a

    PubMed Central

    McCaffary, David; Iqbal, Asif Jilani; Greaves, David R.

    2016-01-01

    Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of

  1. Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a.

    PubMed

    Taylor, Lewis; Brodermann, Maximillian Hugo; McCaffary, David; Iqbal, Asif Jilani; Greaves, David R

    2016-01-01

    Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of

  2. A Possible Role for WNT5A Hypermethylation in Pediatric Acute Lymphoblastic Leukemia

    PubMed Central

    Hatırnaz Ng, Özden; Fırtına, Sinem; Can, İsmail; Karakaş, Zeynep; Ağaoğlu, Leyla; Doğru, Ömer; Celkan, Tiraje; Akçay, Arzu; Yıldırmak, Yıldız; Timur, Çetin; Özbek, Uğur; Sayitoğlu, Müge

    2015-01-01

    Objective: WNT5A is one of the most studied noncanonical WNT ligands and is shown to be deregulated in different tumor types. Our aim was to clarify whether hypermethylation might be the cause of low WNT5A mRNA levels and whether we could restore this downregulation by reversing the event. Materials and Methods: The expression of WNT5A mRNA was studied in a large acute lymphoblastic leukemia (ALL) patient group (n=86) by quantitative real-time PCR. The methylation status was detected by methylation-specific PCR (MSPCR) and bisulphate sequencing. In order to determine whether methylation has a direct effect on WNT5A expression, disease-representative cell lines were treated by 5’-aza-20-deoxycytidine. Results: Here we designed a validation experiment of the WNT5A gene, which was previously examined and found to be differentially expressed by microarray study in 31 T-cell ALL patients. The expression levels were confirmed by quantitative real-time PCR and the expression levels were significantly lower in T-cell ALL patients than in control thymic subsets (p=0.007). MSPCR revealed that 86% of the patients were hypermethylated in the WNT5A promoter region. Jurkat and RPMI cell lines were treated with 5’-aza-20-deoxycytidine and WNT5A mRNA expression was restored after treatment. Conclusion: According to our results, WNT5A hypermethylation does occur in ALL patients and it has a direct effect on mRNA expression. Our findings show that epigenetic changes of WNT signaling can play a role in ALL pathogenesis and reversing methylation might be useful as a possible treatment of leukemia. PMID:26316480

  3. Is the COL5A1 rs12722 gene polymorphism associated with running economy?

    PubMed

    Bertuzzi, Rômulo; Pasqua, Leonardo A; Bueno, Salomão; Lima-Silva, Adriano Eduardo; Matsuda, Monique; Marquezini, Monica; Saldiva, Paulo H

    2014-01-01

    The COL5A1 rs12722 polymorphism is considered to be a novel genetic marker for endurance running performance. It has been postulated that COL5A1 rs12722 may influence the elasticity of tendons and the energetic cost of running. To date, there are no experimental data in the literature supporting the relationship between range of motion, running economy, and the COL5A1 rs12722 gene polymorphism. Therefore, the main purpose of the current study was to analyze the influence of the COL5A1rs12722 polymorphism on running economy and range of motion. One hundred and fifty (n = 150) physically active young men performed the following tests: a) a maximal incremental treadmill test, b) two constant-speed running tests (10 km · h(-1)) and 12 km · h(-1)) to determine the running economy, and c) a sit-and-reach test to determine the range of motion. All of the subjects were genotyped for the COL5A1 rs12722 single-nucleotide polymorphism. The genotype frequencies were TT = 27.9%, CT = 55.8%, and CC = 16.3%. There were no significant differences between COL5A1 genotypes for running economy measured at 10 km · h(-1) (p = 0.232) and 12 km · h(-1) (p = 0.259). Similarly, there were no significant differences between COL5A1 genotypes for range of motion (p = 0.337). These findings suggest that the previous relationship reported between COL5A1 rs12722 genotypes and running endurance performance might not be mediated by the energetic cost of running. PMID:25188268

  4. A Wnt5a signaling pathway in the pathogenesis of HIV-1 gp120-induced pain

    PubMed Central

    Yuan, Su-Bo; Ji, Guangchen; Li, Bei; Andersson, Tommy; Neugebauer, Volker; Tang, Shao-Jun

    2015-01-01

    Pathological pain is one of the most common neurological complications in HIV-1/AIDS patients. However, the pathogenic process is unclear. Our recent studies show that Wnt5a is up-regulated in the spinal cord dorsal horn of the HIV patients who develop pain and that HIV-1 gp120, a potential causal factor of the HIV-associated pain, rapidly up-regulates Wnt5a in the mouse SDH. Using a mouse model, we show here that a specific Wnt5a antagonist, Box-5, attenuated gp120-induced mechanical allodynia. Conversely, a Wnt5a agonist, Foxy5, facilitated the allodynia. To elucidate the molecular mechanism by which Wnt5a regulates gp120-induced allodynia, we tested the role of the JNK/TNF-α pathway. We observed that the JNK-specific inhibitor SP600125 blocked either gp120- or Foxy5-induced allodynia. Similarly, the TNF-α-specific antagonist Enbrel also reversed either gp120- or Foxy5-induced allodynia. These data suggest that JNK and TNF-α mediate the biological effects of Wnt5a in regulating gp120-induced allodynia. To investigate the cellular mechanism, we performed extracellular single-unit recording from SDH neurons in anesthetized mice. Both Box5 and SP600125 negated gp120-induced potentiation of SDH neuron spiking evoked by mechanical stimulation of the hindpaw. Furthermore, while Foxy5 potentiated spike frequency of SDH neurons, either SP600125 or Enbrel blocked the potentiation. The data indicate that Wnt5a potentiates the activity of SDH neurons via the JNK-TNF-α pathway. Collectively, our findings suggest that Wnt5a regulates the pathogenesis of gp120-induced pain, likely by sensitizing pain-processing SDH neurons via JNK/TNF-α signaling. PMID:25840108

  5. Is the COL5A1 rs12722 Gene Polymorphism Associated with Running Economy?

    PubMed Central

    Bertuzzi, Rômulo; Pasqua, Leonardo A.; Bueno, Salomão; Lima-Silva, Adriano Eduardo; Matsuda, Monique; Marquezini, Monica; Saldiva, Paulo H.

    2014-01-01

    The COL5A1 rs12722 polymorphism is considered to be a novel genetic marker for endurance running performance. It has been postulated that COL5A1 rs12722 may influence the elasticity of tendons and the energetic cost of running. To date, there are no experimental data in the literature supporting the relationship between range of motion, running economy, and the COL5A1 rs12722 gene polymorphism. Therefore, the main purpose of the current study was to analyze the influence of the COL5A1rs12722 polymorphism on running economy and range of motion. One hundred and fifty (n = 150) physically active young men performed the following tests: a) a maximal incremental treadmill test, b) two constant-speed running tests (10 km•h−1 and 12 km•h−1) to determine the running economy, and c) a sit-and-reach test to determine the range of motion. All of the subjects were genotyped for the COL5A1 rs12722 single-nucleotide polymorphism. The genotype frequencies were TT = 27.9%, CT = 55.8%, and CC = 16.3%. There were no significant differences between COL5A1 genotypes for running economy measured at 10 km•h−1 (p = 0.232) and 12 km•h−1 (p = 0.259). Similarly, there were no significant differences between COL5A1 genotypes for range of motion (p = 0.337). These findings suggest that the previous relationship reported between COL5A1 rs12722 genotypes and running endurance performance might not be mediated by the energetic cost of running. PMID:25188268

  6. C5L2: a controversial receptor of complement anaphylatoxin, C5a.

    PubMed

    Li, Rui; Coulthard, Liam G; Wu, M C L; Taylor, Stephen M; Woodruff, Trent M

    2013-03-01

    C5a is the paramount proinflammatory mediator of the complement cascade, and has been previously thought to act only through a single, G-protein-coupled, C5a receptor (C5aR; also termed CD88). In 2000, a second C5a receptor, C5L2 (previously known as GPR77), was discovered; yet, despite 12 yr of intensive research, its biological, or pathophysiological, function is both enigmatic and controversial. Unlike C5aR, this receptor does not couple to G proteins, and early studies promoted the hypothesis that C5L2 functions as a decoy receptor. However, recent data have provided other evidence for more complicated and conflicting interactions between C5L2 and other inflammatory mediators. C5L2 has been recently demonstrated to physically interact with both C5aR and β-arrestin to negatively regulate C5aR signaling toward an anti-inflammatory manner, and to reduce pathology, in several disease models in vivo. In direct contrast, other groups have demonstrated that C5L2 stimulation caused release of HMGB1 both in vitro and in vivo, and enhanced pathology in sepsis models, suggesting a clear proinflammatory signaling role. These astoundingly contradictory data challenge our precepts and complicate the foundational bases for the possible targeting of C5L2 as a therapeutic option in inflammatory disease. C5L2 may be the great masquerader in complement biology; its function dependent on the cell type, species, and disease context. Because of these unusual and unforeseen complexities, we present the current state of knowledge on C5L2 structure, expression and, most controversially, its putative functions.-Li, R., Coulthard, L.G., Wu, M. C. L., Taylor, S. M., Woodruff, T. M. C5L2: a controversial receptor of complement anaphylatoxin, C5a. PMID:23239822

  7. Wnt-5a signaling restores tamoxifen sensitivity in estrogen receptor-negative breast cancer cells

    PubMed Central

    Ford, Caroline E.; Ekström, Elin J.; Andersson, Tommy

    2009-01-01

    One third of all breast cancers are estrogen receptor alpha (ERα) negative, carry a poor overall prognosis, and do not respond well to currently available endocrine therapies. New treatment strategies are therefore required. Loss of Wnt-5a has previously been correlated with loss of ERα in clinical breast cancer samples, and we sought to investigate this association further. Three breast cancer cell lines (MDA-MB-231, MDA-MB-468, and 4T1) lacking expression of ERα and Wnt-5a, and one breast cancer cell line (T47D) expressing both proteins were used in this study. Wnt-5a signaling was generated in ERα-negative cell lines via stimulation with either recombinant Wnt-5a protein or a Wnt-5a-derived hexapeptide (Foxy-5) possessing Wnt-5a signaling properties. ERα expression was restored at both mRNA and protein level, after treatment with recombinant Wnt-5a or Foxy-5. This restoration of expression occurred in parallel with a reduction in methylation of the ERα promoter. Up-regulated ERα could be activated, initiate transcription of progesterone receptor and pS2, and activate an estrogen response element reporter construct. Significantly, breast cancer cells re-expressing ERα responded to treatment with the selective estrogen receptor modulator tamoxifen, as measured by induction of apoptosis and cell growth inhibition. Finally, Foxy-5 also increased ERα expression in an in vivo model of ERα-negative breast cancer. This represents the first evidence that Wnt-5a signaling acts to re-establish ERα expression in ERα-negative breast cancer cells. Our data suggest that combinatorial therapy with Foxy-5 and tamoxifen should be considered as a future treatment possibility for ERα-negative breast cancer patients. PMID:19237581

  8. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

    SciTech Connect

    Jensen, Taylor J.; Wozniak, Ryan J.; Eblin, Kylee E.; Wnek, Sean M.; Gandolfi, A. Jay; Futscher, Bernard W.

    2009-02-15

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.

  9. Wnt5a and Ror2 expression associate with the disease progress of primary thyroid lymphoma.

    PubMed

    Wang, Lei; Yang, Dong; Wang, Ying-Hou; Li, Xi; Gao, Hong-Ming; Lv, Jun-Yuan; Wang, Lei; Xin, Shi-Jie

    2016-05-01

    Primary thyroid lymphoma (PTL) is a rare malignant thyroid tumor; its pathogenesis is closely related to chronic lymphocytic thyroiditis. The different pathological subtypes and stages of PTL have distinct clinical characteristics and prognosis, but the specific reasons are not clear. Wnt5a is a representative protein of non-canonical Wnt signaling. It plays an important role in many different types of tumors. This study is to explore the changes of Wnt5a and its receptor Ror2 in PTL development process and the clinical significance of their represent. We collected 22 PTL patient tumor specimens and clinical data. We observed the expression of Wnt5a and Ror2 in PTL tumor tissues by immunohistochemistry. Wnt5a was expressed positively in 12 (54.5 %) cases, and Ror2 was expressed positively in 18 (81.8 %) cases. The expression of Wnt5a had a significant difference in different pathological subtypes of PTL (P < 0.05). Wnt5a and Ror2 expression were associated with local invasion and clinical stage, respectively (P < 0.05), and had no significant correlation with age, gender, and tumor size. Although, no significant difference in overall survival was found between positive and negative groups of Wnt5a (P = 0.416) or Ror2 (P = 0.256), respectively. We still consider that Wnt5a and Ror2 play a complex and subtle role in the pathogenesis and progression of PTL and may become potential biomarkers and therapeutic targets of PTL. PMID:26614433

  10. Wnt5a and Ror2 expression associate with the disease progress of primary thyroid lymphoma.

    PubMed

    Wang, Lei; Yang, Dong; Wang, Ying-Hou; Li, Xi; Gao, Hong-Ming; Lv, Jun-Yuan; Wang, Lei; Xin, Shi-Jie

    2016-05-01

    Primary thyroid lymphoma (PTL) is a rare malignant thyroid tumor; its pathogenesis is closely related to chronic lymphocytic thyroiditis. The different pathological subtypes and stages of PTL have distinct clinical characteristics and prognosis, but the specific reasons are not clear. Wnt5a is a representative protein of non-canonical Wnt signaling. It plays an important role in many different types of tumors. This study is to explore the changes of Wnt5a and its receptor Ror2 in PTL development process and the clinical significance of their represent. We collected 22 PTL patient tumor specimens and clinical data. We observed the expression of Wnt5a and Ror2 in PTL tumor tissues by immunohistochemistry. Wnt5a was expressed positively in 12 (54.5 %) cases, and Ror2 was expressed positively in 18 (81.8 %) cases. The expression of Wnt5a had a significant difference in different pathological subtypes of PTL (P < 0.05). Wnt5a and Ror2 expression were associated with local invasion and clinical stage, respectively (P < 0.05), and had no significant correlation with age, gender, and tumor size. Although, no significant difference in overall survival was found between positive and negative groups of Wnt5a (P = 0.416) or Ror2 (P = 0.256), respectively. We still consider that Wnt5a and Ror2 play a complex and subtle role in the pathogenesis and progression of PTL and may become potential biomarkers and therapeutic targets of PTL. PMID:26608372

  11. SSF Terra-FM1 Ed3A

    Atmospheric Science Data Center

    2016-07-13

    SSF Terra-FM1 Ed3A Project Title:  CERES Discipline:  Clouds Radiation Budget ...   Reverb Tutorial Subset/Visualization Tool: CERES Order Tool Subset Data:  CERES Search and Subset Tool (HDF4 & ...

  12. SSF Aqua-FM3 Ed3A

    Atmospheric Science Data Center

    2016-07-13

    SSF Aqua-FM3 Ed3A Project Title:  CERES Discipline:  Clouds Radiation Budget ...   Reverb Tutorial Subset/Visualization Tool: CERES Order Tool Subset Data:  CERES Search and Subset Tool (HDF4 & ...

  13. Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

    NASA Astrophysics Data System (ADS)

    Martínez-González, José Ángel; Rodríguez, Alex; Puyuelo, María Pilar; González, Miguel; Martínez, Rodrigo

    2015-01-01

    The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Møller-Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.

  14. 27 CFR 21.35 - Formula No. 3-A.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 3-A. 21.35 Section 21.35 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.35 Formula No. 3-A....

  15. Semaphorin 3A: A Potential Target for Low Back Pain

    PubMed Central

    Yin, Pengbin; Lv, Houchen; Zhang, Lihai; Zhang, Licheng; Tang, Peifu

    2015-01-01

    Low back pain is a common disorder. Pathological innervation and intervertebral disc degeneration are two major factors associated with this disease. Semaphorin 3A, originally known for its potent inhibiting effect on axonal outgrowth, is recently found to correlate with disease activity and histological features in some skeletal disorders. Based on its effects on innervation and vascularization, as well as enzyme secretion, we presume that semaphorin 3A may act as a potential target for low back pain. PMID:26635602

  16. 27 CFR 21.35 - Formula No. 3-A.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 3-A. 21.35 Section 21.35 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.35 Formula No. 3-A....

  17. Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence

    PubMed Central

    Martinez-Rocha, Ana Lilia; Woriedh, Mayada; Chemnitz, Jan; Willingmann, Peter; Kröger, Cathrin; Hadeler, Birgit; Hauber, Joachim; Schäfer, Wilhelm

    2016-01-01

    Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A. PMID:27098988

  18. The COL5A1 genotype is associated with range of motion

    PubMed Central

    Lim, Seung-Taek; Kim, Chang-Sun; Kim, Woo-Nam; Min, Seok-Ki

    2015-01-01

    [Purpose] The aim of our study was to investigate the association between COL5A1 genotype and Range of Motion as measured by the passive straight leg raise (SLR) and whole body join laxity (WBJL) in Asian population. [Methods] One hundred and seventy seven participants including Korean and Japanese college students (male = 109, female = 68) participated in the study. Each subject performed the passive straight leg raise and whole body join laxity test. Genotyping for the COL5A1 (rs 12722) polymorphism was performed using the TaqMan approach. The COL5A1 genotype exhibited a Hardy-Weinberg equilibrium distribution in our population. [Results] The physical parameters including height, weight, and BMI were higher in < 90° group than > 90° group. The SLR exhibited significant difference among the COL5A1 group. However, the WBJL did not differ significantly among the COL5A1 genotype, but significant difference was seen in CC genotype when compared to CT (2.99 ± 1.72) or TT (2.70 ± 1.52) genotype. [Conclusion] We concluded that COL5A1 gene polymorphism is associated with increased SLR ROM in Asian population. PMID:26244122

  19. MOD-5A wind turbine generator program design report: Volume 1: Executive Summary

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator covering work performed between July 1980 and June 1984 is discussed. The report is divided into four volumes: Volume 1 summarizes the entire MOD-5A program, Volume 2 discusses the conceptual and preliminary design phases, Volume 3 describes the final design of the MOD-5A, and Volume 4 contains the drawings and specifications developed for the final design. Volume 1, the Executive Summary, summarizes all phases of the MOD-5A program. The performance and cost of energy generated by the MOD-5A are presented. Each subsystem - the rotor, drivetrain, nacelle, tower and foundation, power generation, and control and instrumentation subsystems - is described briefly. The early phases of the MOD-5A program, during which the design was analyzed and optimized, and new technologies and materials were developed, are discussed. Manufacturing, quality assurance, and safety plans are presented. The volume concludes with an index of volumes 2 and 3.

  20. Regulation of lipid synthesis by the RNA helicase Mov10 controls Wnt5a production

    PubMed Central

    Wang, W; Snyder, N; Worth, A J; Blair, I A; Witze, E S

    2015-01-01

    Expression of the Wnt ligand Wnt5a is frequently elevated in melanoma and is thought to be a critical regulator of cell movement during metastasis. However, the mechanisms regulating its expression are unknown. We find that the level of secreted Wnt5a varies by as much as 10-fold between cell lines and correlates more strongly with invasion than total cellular levels. Our results indicate that the RNA helicase Mov10 plays a role in Wnt5a synthesis and secretion. Inhibition of Mov10 increases secreted Wnt5a levels in melanoma cells by increasing Wnt5a synthesis and acylation. This is achieved by increasing fatty acid synthase (FASN) and stearoyl-CoA desaturase expression, leading to elevated levels of palmitoleoyl-CoA, required for Wnt ligand lipid modification and secretion. Melanoma tumors exhibit reduced expression of Mov10 compared with benign nevi and Mov10 levels inversely correlate with FASN levels in primary tumors. These results reveal a previously unappreciated role for aberrant lipid metabolism in regulating Wnt5a signaling that may be a critical step in melanoma progression. PMID:26029828

  1. The role of C5a in acute lung injury induced by highly pathogenic viral infections

    PubMed Central

    Wang, Renxi; Xiao, He; Guo, Renfeng; Li, Yan; Shen, Beifen

    2015-01-01

    The complement system, an important part of innate immunity, plays a critical role in pathogen clearance. Unregulated complement activation is likely to play a crucial role in the pathogenesis of acute lung injury (ALI) induced by highly pathogenic virus including influenza A viruses H5N1, H7N9, and severe acute respiratory syndrome (SARS) coronavirus. In highly pathogenic virus-induced acute lung diseases, high levels of chemotactic and anaphylatoxic C5a were produced as a result of excessive complement activaiton. Overproduced C5a displays powerful biological activities in activation of phagocytic cells, generation of oxidants, and inflammatory sequelae named “cytokine storm”, and so on. Blockade of C5a signaling have been implicated in the treatment of ALI induced by highly pathogenic virus. Herein, we review the literature that links C5a and ALI, and review our understanding of the mechanisms by which C5a affects ALI during highly pathogenic viral infection. In particular, we discuss the potential of the blockade of C5a signaling to treat ALI induced by highly pathogenic viruses. PMID:26060601

  2. Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis.

    PubMed

    Okamoto, Masanori; Udagawa, Nobuyuki; Uehara, Shunsuke; Maeda, Kazuhiro; Yamashita, Teruhito; Nakamichi, Yuko; Kato, Hiroyuki; Saito, Naoto; Minami, Yasuhiro; Takahashi, Naoyuki; Kobayashi, Yasuhiro

    2014-01-01

    Wnt regulates bone formation through β-catenin-dependent canonical and -independent noncanonical signaling pathways. However, the cooperation that exists between the two signaling pathways during osteoblastogenesis remains to be elucidated. Here, we showed that the lack of Wnt5a in osteoblast-lineage cells impaired Wnt/β-catenin signaling due to the reduced expression of Lrp5 and Lrp6. Pretreatment of ST2 cells, a stromal cell line, with Wnt5a enhanced canonical Wnt ligand-induced Tcf/Lef transcription activity. Short hairpin RNA-mediated knockdown of Wnt5a, but not treatment with Dkk1, an antagonist of Wnt/β-catenin signaling, reduced the expression of Lrp5 and Lrp6 in osteoblast-lineage cells under osteogenic culture conditions. Osteoblast-lineage cells from Wnt5a-deficient mice exhibited reduced Wnt/β-catenin signaling, which impaired osteoblast differentiation and enhanced adipocyte differentiation. Adenovirus-mediated gene transfer of Lrp5 into Wnt5a-deficient osteoblast-lineage cells rescued their phenotypic features. Therefore, Wnt5a-induced noncanonical signaling cooperates with Wnt/β-catenin signaling to achieve proper bone formation. PMID:24670389

  3. Genetically engineered SCN5A mutant pig hearts exhibit conduction defects and arrhythmias.

    PubMed

    Park, David S; Cerrone, Marina; Morley, Gregory; Vasquez, Carolina; Fowler, Steven; Liu, Nian; Bernstein, Scott A; Liu, Fang-Yu; Zhang, Jie; Rogers, Christopher S; Priori, Silvia G; Chinitz, Larry A; Fishman, Glenn I

    2015-01-01

    SCN5A encodes the α subunit of the major cardiac sodium channel Na(V)1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5A(E558X/+) pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5A(E558X/+) hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5A(E558X/+) pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias. PMID:25500882

  4. Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia.

    PubMed

    Amir, Mohd; Wahiduzzaman; Dar, Mohammad Aasif; Haque, Md Anzarul; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-01-01

    Ras related protein (Rab5a) is one of the most important member of the Rab family which regulates the early endosome fusion in endocytosis, and it also helps in the regulation of the budding process. Here, for the first time we report a simple and reproducible method for the purification of the Rab5a from a medicinal plant Tinospora cordifolia. We have used weak cation-exchange (CM-Sepharose-FF) followed by gel-filtration chromatography. A purified protein of 22-kDa was observed on SDS-PAGE which was identified as Rab5a using MALDI-TOF/MS. Our purification procedure is fast and simple with high yield. The purified protein was characterized using circular dichroism for the measurement of secondary structure followed by GdmCl- and urea-induced denaturation to calculate the values of Gibbs free energy change (ΔGD), ΔGD°, midpoint of the denaturation Cm, i.e. molar GdmCl [GdmCl] and molar urea [Urea] concentration at which ΔGD=0; and m, the slope (=∂ΔGD/∂[d]) values. Furthermore, thermodynamic properties of Rab5a were also measured by differential scanning calorimeter. Here, using isothermal calorimeteric measurements we further showed that Rab5a binds with the GTP. This is a first report on the purification and biophysical characterization of Rab5a protein from T. cordifolia. PMID:26517959

  5. Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence.

    PubMed

    Martinez-Rocha, Ana Lilia; Woriedh, Mayada; Chemnitz, Jan; Willingmann, Peter; Kröger, Cathrin; Hadeler, Birgit; Hauber, Joachim; Schäfer, Wilhelm

    2016-01-01

    Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A. PMID:27098988

  6. Inhibitory effects of citrus fruits on cytochrome P450 3A (CYP3A) activity in humans.

    PubMed

    Fujita, Ken-Ichi; Hidaka, Muneaki; Takamura, Norito; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Yamaguchi, Masatoshi; Ikenoue, Tsuyomu; Arimori, Kazuhiko

    2003-09-01

    The capacities of citrus fruits to inhibit midazolam 1'-hydroxylase activity of cytochrome P450 3A (CYP3A) expressed in human liver microsomes were evaluated. Eight citrus fruits such as ama-natsu, banpeiyu, Dekopon, hassaku, hyuga-natsu, completely matured kinkan (Tamatama), takaoka-buntan and unshu-mikan were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and grapefruit juice (white, Tropicana-Kirin). The addition of a fruit juice prepared from banpeiyu, hassaku, takaoka-buntan or Tamatama caused the inhibition of the microsomal CYP3A activity. The inhibition depended on the amount of a fruit juice added to the incubation mixture (2.5 and 5.0%, v/v). The fruit juice from banpeiyu showed the most potent inhibition of CYP3A. The addition of a banpeiyu juice (5.0%, v/v) resulted in the inhibition of midazolam 1'-hydroxylase activity to about 20% of control without a fruit juice. The elongation of the preincubation period of a fruit juice from banpeiyu (5.0%, v/v) with the microsomal fraction (5 to 15 min) led to the enhancement of the CYP3A inhibition (5% of control). Thus, we discovered ingredients of banpeiyu to be inhibitor(s) or mechanism-based inhibitor(s) of human CYP3A activity, but the inhibitory effects of them were somewhat lower than those of grapefruit. PMID:12951492

  7. Isolation and characterization of recombinant murine Wnt3a

    PubMed Central

    Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A.; Ryan, Robert O.

    2014-01-01

    Wnt proteins are a family of morphogens that possess potent biological activity. Structure – function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30 mM methyl-β-cyclodextrin (MβCD). Wnt3a recovered in MβCD-containing buffer was soluble and biologically active. Insofar as MβCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. PMID:25448592

  8. Isolation and characterization of recombinant murine Wnt3a.

    PubMed

    Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A; Ryan, Robert O

    2015-02-01

    Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. PMID:25448592

  9. Interactions between CYP3A4 and Dietary Polyphenols

    PubMed Central

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  10. Dnmt3a2: a hub for enhancing cognitive functions.

    PubMed

    Oliveira, A M M; Hemstedt, T J; Freitag, H E; Bading, H

    2016-08-01

    The mechanisms responsible for fear memory formation and extinction are far from being understood. Uncovering the molecules and mechanisms regulating these processes is vital for identifying molecular targets for the development of novel therapeutic strategies for anxiety and fear disorders. Cognitive abilities require the activation of gene expression necessary to the consolidation of lasting changes in neuronal function. In this study we established a key role for an epigenetic factor, the de novo DNA methyltransferase, Dnmt3a2, in memory formation and extinction. We found that Dnmt3a2 overexpression in the hippocampus of young adult mice induced memory enhancements in a variety of situations; it converted a weak learning experience into long-term memory, enhanced fear memory formation and facilitated fear memory extinction. Dnmt3a2 overexpression was also associated with the increased expression of plasticity-related genes. Furthermore, the knockdown of Dnmt3a2 expression impaired the animals' ability to extinguish memories, identifying Dnmt3a2 as a key player in extinction. Thus, Dnmt3a2 is at the core of memory processes and represents a novel target for cognition-enhancing therapies to ameliorate anxiety and fear disorders and boost memory consolidation. PMID:26598069

  11. Interactions between CYP3A4 and Dietary Polyphenols.

    PubMed

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  12. Structural requirements of chromokinesin Kif4A for its proper function in mitosis

    SciTech Connect

    Wu Guikai; Chen, P.-L.

    2008-08-01

    Human Kif4A is a member of the Kinesin-4 family of kinesins. Kif4A is thought to be a bona fide chromokinesin because it possesses a motor domain and associates with condensed chromosomes during mitosis. Genetic deletion of Kif4A promotes tumorigenic phenotypes in mouse embryonic cells. Kif4A is critical for mitotic regulation including chromosome condensation, spindle organization and cytokinesis. However, the precise chromatin-binding domain of Kif4A has not been characterized. Herein, we report the identification of two conserved motifs critical for chromatin-binding: the first leucine Zip motif (Zip1) of a leucine Zip/Basic/leucine Zip region (ZBZ) previously thought to be a nuclear localization signal (NLS), and a cysteine-rich (CR) motif within the C-terminal region of Kif4A. Furthermore, by depleting endogenous Kif4A via RNAi and concurrently expressing RNAi-resistant Kif4A versions, we observed that wild type Kif4A, but not the mutants deficient in DNA-binding (Zip1 or CR deleted) or ATPase activity (K94A point mutant), was able to rescue the RNAi-elicited abnormal mitotic profile. Taken together, our results show that both the Zip1 and CR motifs are important for Kif4A chromatin-binding and its mitotic function.

  13. Col4a1 mutations cause progressive retinal neovascular defects and retinopathy

    PubMed Central

    Alavi, Marcel V.; Mao, Mao; Pawlikowski, Bradley T.; Kvezereli, Manana; Duncan, Jacque L.; Libby, Richard T.; John, Simon W. M.; Gould, Douglas B.

    2016-01-01

    Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1+/Δex41mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes. PMID:26813606

  14. Disruption of Slc4a10 augments neuronal excitability and modulates synaptic short-term plasticity

    PubMed Central

    Sinning, Anne; Liebmann, Lutz; Hübner, Christian A.

    2015-01-01

    Slc4a10 is a Na+-coupled Cl−-HCO3− exchanger, which is expressed in principal and inhibitory neurons as well as in choroid plexus epithelial cells of the brain. Slc4a10 knockout (KO) mice have collapsed brain ventricles and display an increased seizure threshold, while heterozygous deletions in man have been associated with idiopathic epilepsy and other neurological symptoms. To further characterize the role of Slc4a10 for network excitability, we compared input-output relations as well as short and long term changes of evoked field potentials in Slc4a10 KO and wildtype (WT) mice. While responses of CA1 pyramidal neurons to stimulation of Schaffer collaterals were increased in Slc4a10 KO mice, evoked field potentials did not differ between genotypes in the stratum radiatum or the neocortical areas analyzed. Paired pulse facilitation was diminished in the hippocampus upon disruption of Slc4a10. In the neocortex paired pulse depression was increased. Though short term plasticity is modulated via Slc4a10, long term potentiation appears independent of Slc4a10. Our data support that Slc4a10 dampens neuronal excitability and thus sheds light on the pathophysiology of SLC4A10 associated pathologies. PMID:26136660

  15. Evidence for a SULT4A1 haplotype correlating with baseline psychopathology and atypical antipsychotic response

    PubMed Central

    Ramsey, Timothy L; Meltzer, Herbert Y; Brock, Guy N; Mehrotra, Bharat; Jayathilake, Karu; Bobo, William V; Brennan, Mark D

    2011-01-01

    Aim This study evaluated the impact of SULT4A1 gene variation on psychopathology and antipsychotic drug response in Caucasian subjects from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study and a replication sample. Patients & methods SULT4A1 haplotypes were determined using SNP data. The relationship to baseline psychopathology was evaluated using linear regression of Positive and Negative Syndrome Scale (PANSS) total score. Drug response was evaluated using Mixed Model Repeat Measures (MMRM) for change in PANSS. Results For the CATIE sample, patients carrying a haplotype designated SULT4A1-1(+) displayed higher baseline PANSS (p = 0.03) and, when treated with olanzapine, demonstrated a significant interaction with time (p = 0.009) in the MMRM. SULT4A1-1(+) patients treated with olanzapine displayed improved response compared with SULT4A1-1(−) patients treated with olanzapine (p = 0.008) or to SULT4A1-1(+) patients treated with risperidone (p = 0.006). In the replication sample, SULT4A1-1(+) patients treated with olanzapine demonstrated greater improvement than SULT4A1-1(−) patients treated with olanzapine (p = 0.05) or than SULT4A1-1(+) patients treated with risperidone (p = 0.05). Conclusion If validated, determination of SULT4A1-1 haplotype status might be useful for identifying patients who show an enhanced response to long-term olanzapine treatment. PMID:21521020

  16. Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis.

    PubMed Central

    Jagels, M A; Travis, J; Potempa, J; Pike, R; Hugli, T E

    1996-01-01

    The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils. Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsullonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at

  17. Role of CYB5A in Pancreatic Cancer Prognosis and Autophagy Modulation

    PubMed Central

    2014-01-01

    Background Loss of 18q22.3 is a prognostic marker in pancreatic ductal adenocarcinoma (PDAC). This study investigated genes encoded by this cytoband. Methods We studied mRNA/protein expression in radically resected (n = 130) and metastatic patients (n = 50). The role of CYB5A was tested in 11 PDAC cell lines and five primary cultures through retrovirus-mediated upregulation and small interfering RNA using wound-healing, invasion, annexin-V, electron microscopy, and autophagic assays, as well as autophagy genes and kinases arrays. CYB5A+ orthotopic models (n = 6 mice/group) were monitored by Firefly and Gaussia-luciferase bioluminescence, magnetic resonance imaging, and high-frequency ultrasound. Data were analyzed by t test, Fisher exact-test, log-rank test and Cox proportional hazards models. All statistical tests were two-sided. Results Both resected and metastatic patients with low mRNA or protein expression of CYB5A had statistically significantly shorter survival (eg, median = 16.7 months, 95% confidence interval [CI] = 13.5 to 19.9; vs median = 24.8 months, 95% CI = 12.8 to 36.9; P = .02, two-sided log-rank test; n = 82 radically resected PDACs), and multivariable analyses confirmed prognostic relevance. Moreover, we characterized a novel function to CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of PDAC cells. Network analysis of proautophagic pathways suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 downregulation after CYB5A reconstitution (−69% in SU.86.86-CYB5A+; P = .005, two-sided t test). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound healing. In vivo studies showed that CYB5A induced autophagy while inhibiting tumor growth/metastasis and increasing survival (median = 57 days, 95% CI = 52 to 61; vs median = 44 days, 95% CI = 21 to 57; P = .03, two-sided log-rank test). Conclusions These results define CYB5A as a novel prognostic factor for PDAC that exerts its

  18. Histone acetylation regulates orphan nuclear receptor NR4A1 expression in hypercholesterolaemia.

    PubMed

    Xie, Xina; Song, Xuhong; Yuan, Song; Cai, Haitao; Chen, Yequn; Chang, Xiaolan; Liang, Bin; Huang, Dongyang

    2015-12-01

    Hypercholesterolaemia and inflammation are correlated with atherogenesis. Orphan nuclear receptor NR4A1, as a key regulator of inflammation, is closely associated with lipid levels in vivo. However, the mechanism by which lipids regulate NR4A1 expression remains unknown. We aimed to elucidate the underlying mechanism of NR4A1 expression in monocytes during hypercholesterolaemia, and reveal the potential role of NR4A1 in hypercholesterolaemia-induced circulating inflammation. Circulating leucocytes were collected from blood samples of 139 patients with hypercholesterolaemia and 139 sex- and age-matched healthy subjects. We found that there was a low-grade inflammatory state and higher expression of NR4A1 in patients. Both total cholesterol and low-density lipoprotein cholesterol levels in plasma were positively correlated with NR4A1 mRNA level. ChIP revealed that acetylation of histone H3 was enriched in the NR4A1 promoter region in patients. Human mononuclear cell lines THP-1 and U937 were treated with cholesterol. Supporting our clinical observations, cholesterol enhanced p300 acetyltransferase and decreased HDAC7 (histone deacetylase 7) recruitment to the NR4A1 promoter region, resulting in histone H3 hyperacetylation and further contributing to NR4A1 up-regulation in monocytes. Moreover, cytosporone B, an NR4A1 agonist, completely reversed cholesterol-induced IL-6 (interleukin 6) and MCP-1 (monocyte chemoattractant protein 1) expression to below basal levels, and knockdown of NR4A1 expression by siRNA not only mimicked, but also exaggerated the effects of cholesterol on inflammatory biomarker up-regulation. Thus we conclude that histone acetylation contributes to the regulation of NR4A1 expression in hypercholesterolaemia, and that NR4A1 expression reduces hypercholesterolaemia-induced inflammation. PMID:26396259

  19. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.

    PubMed

    Noll, Elisa M; Eisen, Christian; Stenzinger, Albrecht; Espinet, Elisa; Muckenhuber, Alexander; Klein, Corinna; Vogel, Vanessa; Klaus, Bernd; Nadler, Wiebke; Rösli, Christoph; Lutz, Christian; Kulke, Michael; Engelhardt, Jan; Zickgraf, Franziska M; Espinosa, Octavio; Schlesner, Matthias; Jiang, Xiaoqi; Kopp-Schneider, Annette; Neuhaus, Peter; Bahra, Marcus; Sinn, Bruno V; Eils, Roland; Giese, Nathalia A; Hackert, Thilo; Strobel, Oliver; Werner, Jens; Büchler, Markus W; Weichert, Wilko; Trumpp, Andreas; Sprick, Martin R

    2016-03-01

    Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. PMID:26855150

  20. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

    PubMed Central

    Noll, Elisa M.; Eisen, Christian; Stenzinger, Albrecht; Espinet, Elisa; Muckenhuber, Alexander; Klein, Corinna; Vogel, Vanessa; Klaus, Bernd; Nadler, Wiebke; Rösli, Christoph; Lutz, Christian; Kulke, Michael; Engelhardt, Jan; Zickgraf, Franziska M.; Espinosa, Octavio; Schlesner, Matthias; Jiang, Xiaoqi; Kopp-Schneider, Annette; Neuhaus, Peter; Bahra, Marcus; Sinn, Bruno V.; Eils, Roland; Giese, Nathalia A.; Hackert, Thilo; Strobel, Oliver; Werner, Jens; Büchler, Markus W.; Weichert, Wilko; Trumpp, Andreas; Sprick, Martin R.

    2016-01-01

    Although subtypes of pancreatic ductal adenocarcinoma (PDAC) were described, this malignancy is clinically still treated as a single disease. Here, we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Individuals bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4 alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. PMID:26855150

  1. Cloning, expression, and functional characterization of the rat Pax6 5a orthologous splicing variant.

    PubMed

    Wei, Fei; Li, Min; Cheng, Sai-Yu; Wen, Liang; Liu, Ming-Hua; Shuai, Jie

    2014-08-15

    Pax6 functions as a pleiotropic regulator in eye development and neurogenesis. Its splice variant Pax6 5a has been cloned in many vertebrate species including human and mouse, but never in rat. This study focused on the cloning and characterization of the Pax6 5a orthologous splicing variant in rat. It was cloned from Sprague-Dawley rats 10 days post coitum (E10) by RT-PCR and was sequenced for comparison with Pax6 sequences in the GenBank by BLAST. The rat Pax6 5a was revealed to contain an additional 42 bp insertion at the paired domain. At the nucleotide level, the rat Pax6 5a coding sequence (1,311 bp) had a higher degree of homology to the mouse (96% identical) than to the human (93% identical) sequence. At the amino acid (aa) level, rat PAX6 5a shares 99.8% identity with the mouse sequence and 99.5% with the human sequence. The splice variant is preferentially expressed in the rat E10 embryonic headfolds and not in the trunk of neurula. Its effects on the proliferation of rat mesenchymal stem cells (rMSCs) were preliminarily evaluated by the MTT assay. Both pLEGFP-Pax6 5a-transfected cells and pLEGFP-Pax6-transfected cells exhibited a similar growth curve (P>0.05), suggesting that the Pax6 5a has a similar effect on the proliferation of rMSCs as Pax6. PMID:24952136

  2. Wnt5a and Wnt11 regulate mammalian anterior-posterior axis elongation

    PubMed Central

    Andre, Philipp; Song, Hai; Kim, Wantae; Kispert, Andreas; Yang, Yingzi

    2015-01-01

    Mesoderm formation and subsequent anterior-posterior (A-P) axis elongation are fundamental aspects of gastrulation, which is initiated by formation of the primitive streak (PS). Convergent extension (CE) movements and epithelial-mesenchymal transition (EMT) are important for A-P axis elongation in vertebrate embryos. The evolutionarily conserved planar cell polarity (PCP) pathway regulates CE, and Wnts regulate many aspects of gastrulation including CE and EMT. However, the Wnt ligands that regulate A-P axis elongation in mammalian development remain unknown. Wnt11 and Wnt5a regulate axis elongation in lower vertebrates, but only Wnt5a, not Wnt11, regulates mammalian PCP signaling and A-P axis elongation in development. Here, by generating Wnt5a; Wnt11 compound mutants, we show that Wnt11 and Wnt5a play redundant roles during mouse A-P axis elongation. Both genes regulate trunk notochord extension through PCP-controlled CE of notochord cells, establishing a role for Wnt11 in mammalian PCP. We show that Wnt5a and Wnt11 are required for proper patterning of the neural tube and somites by regulating notochord formation, and provide evidence that both genes are required for the generation and migration of axial and paraxial mesodermal precursor cells by regulating EMT. Axial and paraxial mesodermal precursors ectopically accumulate in the PS at late gastrula stages in Wnt5a−/−; Wnt11−/− embryos and these cells ectopically express epithelial cell adhesion molecules. Our data suggest that Wnt5a and Wnt11 regulate EMT by inducing p38 (Mapk14) phosphorylation. Our findings provide new insights into the role of Wnt5a and Wnt11 in mouse early development and also in cancer metastasis, during which EMT plays a crucial role. PMID:25813538

  3. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

    PubMed Central

    Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

    2016-01-01

    Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

  4. Overexpression of p16(INK4a) in Mastocytosis (Urticarial Pigmentosa).

    PubMed

    Tsujita, Jun; Doi, Kazuko; Nakahara, Makiko; Nakahara, Takeshi; Kaku, Yumiko; Nishio, Kiichiro; Kan, Nagisa; Sato, Yuki; Nagata, Shoko; Nakao, Asako; Yoshida, Maiko; Uchi, Hiroshi; Furue, Masutaka

    2016-01-01

    The expression of p16(INK4a) has been reported to induce cell-cycle arrest and cellular senescence. The p16(INK4a) expression has never been examined in human mast cells and mastocytosis. We immunohistologically examined the expression of p16(INK4a) and tryptase in 5 normal human skin and 4 mastocytosis. In normal mast cells, only 5.9 ± 3.4 (mean ± standard deviation) % of tryptase-positive mast cells coexpressed p16(INK4a). However, significantly higher percentage (86.0 ± 14.1%) of tryptase-positive tumor cells was immunoreactive to p16(INK4a) in all of 4 mastocytosis. The p16(INK4a) overexpression may induce the senescence of neoplastic mast cells to undergo spontaneous regression of mastocytosis. PMID:27333655

  5. FoxO3a and disease progression

    PubMed Central

    Nho, Richard Seonghun; Hergert, Polla

    2014-01-01

    The Forkhead box O (FoxO) family has recently been highlighted as an important transcriptional regulator of crucial proteins associated with the many diverse functions of cells. So far, FoxO1, FoxO3a, FoxO4 and FoxO6 proteins have been identified in humans. Although each FoxO family member has its own role, unlike the other FoxO families, FoxO3a has been extensively studied because of its rather unique and pivotal regulation of cell proliferation, apoptosis, metabolism, stress management and longevity. FoxO3a alteration is closely linked to the progression of several types of cancers, fibrosis and other types of diseases. In this review, we will examine the function of FoxO3a in disease progression and also explore FoxO3a’s regulatory mechanisms. We will also discuss FoxO3a as a potential target for the treatment of several types of disease. PMID:25225602

  6. Hydration of C{sub 3}A-gypsum systems

    SciTech Connect

    Quennoz, Alexandra Scrivener, Karen L.

    2012-07-15

    Hydration of C{sub 3}A-gypsum systems with different gypsum additions was investigated in terms of the phase assemblage, kinetics and microstructural development. The second stage of the reaction, which begins after the depletion of gypsum, was of particular interest. From in-situ X-ray diffraction results, it was seen that the dissolution of ettringite and C{sub 3}A to form monosulfoaluminate and/or hydroxy-AFm phases is a rapid reaction that occurs right after the depletion of gypsum. The observation of the calorimetric curves obtained for the different gypsum additions leads us to the conclusion that the mechanism controlling the hydration rate during this period is the nucleation and growth of the AFm phases. The microstructural study showed that the formation of AFm phases occurs in the space between the C{sub 3}A grains but also within the boundaries of the original C{sub 3}A grains. Hydrogarnet was observed growing as a shell around the C{sub 3}A grains.

  7. PDE3A mutations cause autosomal dominant hypertension with brachydactyly.

    PubMed

    Maass, Philipp G; Aydin, Atakan; Luft, Friedrich C; Schächterle, Carolin; Weise, Anja; Stricker, Sigmar; Lindschau, Carsten; Vaegler, Martin; Qadri, Fatimunnisa; Toka, Hakan R; Schulz, Herbert; Krawitz, Peter M; Parkhomchuk, Dmitri; Hecht, Jochen; Hollfinger, Irene; Wefeld-Neuenfeld, Yvette; Bartels-Klein, Eireen; Mühl, Astrid; Kann, Martin; Schuster, Herbert; Chitayat, David; Bialer, Martin G; Wienker, Thomas F; Ott, Jürg; Rittscher, Katharina; Liehr, Thomas; Jordan, Jens; Plessis, Ghislaine; Tank, Jens; Mai, Knut; Naraghi, Ramin; Hodge, Russell; Hopp, Maxwell; Hattenbach, Lars O; Busjahn, Andreas; Rauch, Anita; Vandeput, Fabrice; Gong, Maolian; Rüschendorf, Franz; Hübner, Norbert; Haller, Hermann; Mundlos, Stefan; Bilginturan, Nihat; Movsesian, Matthew A; Klussmann, Enno; Toka, Okan; Bähring, Sylvia

    2015-06-01

    Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension. PMID:25961942

  8. Nutlin-3a Decreases Male Fertility via UQCRC2

    PubMed Central

    Shukla, Kamla Kant; Kwon, Woo-Sung; Rahman, Md Saidur; Park, Yoo-Jin; You, Young-Ah; Pang, Myung-Geol

    2013-01-01

    Ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) is a component of ubiquinol-cytochrome c reductase complex that is known to correlate with male fertility via spermatogenesis. Simultaneously, nutlin-3a is a small molecule antagonist of mouse double minute 2 repressor (MDM2), activate p53 and induce apoptosis responsible for spermatogenesis. To date, however there are no known effects of nutlin-3a on reproduction. Therefore, present study was designed to investigate the effect of nutlin-3a on male fertility via UQCRC2. In this in vitro trial with mice spermatozoa, we utilized CASA, CTC staining, ATP assay, western blotting, and IVF to measure the main study outcome. The short-term exposure of spermatozoa in nutlin-3a decreases sperm motion kinematics, intracellular ATP production, capacitation, the acrosome reaction, UQCRC2, and tyrosine phosphorylation (TYP) of sperm proteins in a dose-dependent manner. Notably, the decreased UQCRC2 and TYP were associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately led to adverse effects on male fertility such as poor fertilization rates and embryo development. Thus, nutlin-3a may be considered as a potential male contraceptive agent due to its ability to decrease fertility secondary to changes in overall sperm physiology and embryonic development. However, the results of this preliminary study have to be confirmed by additional independent trial. PMID:24130818

  9. Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication

    PubMed Central

    de la Torre, Beatriz G.; Valle, Javier; Andreu, David; Sobrino, Francisco

    2015-01-01

    Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7) sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM) were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target. PMID:26505190

  10. ERM proteins regulate growth cone responses to Sema3A

    PubMed Central

    Mintz, C. David; Carcea, Ioana; McNickle, Daniel G.; Dickson, Tracey C.; Ge, Yongchao; Salton, Stephen R.J.; Benson, Deanna L.

    2008-01-01

    Axonal growth cones initiate and sustain directed growth in response to cues in their environment. A variety of events such as receptor internalization, kinase activation, and actin rearrangement can be stimulated by guidance cues and are essential for mediating targeted growth cone behavior. Surprisingly little is known about how such disparate actions are coordinated. Our data suggest that ezrin, radixin, and moesin (ERMs), a family of highly homologous, multifunctional proteins may be able to coordinate growth cone responses to the guidance cue, Sema3A. We show that active ERMs concentrate asymmetrically in neocortical growth cones, are rapidly and transiently inactivated by Sema3A, and are required for Sema3A-mediated growth cone collapse and guidance. The FERM domain of active ERMs regulates internalization of the Sema3A receptor, Npn1 and its co-receptor, L1CAM, while the ERM C-terminal domain binds and caps F-actin. Our data support a model in which ERMs can coordinate membrane and actin dynamics in response to Sema3A. PMID:18651636

  11. MiRNA-1469 promotes lung cancer cells apoptosis through targeting STAT5a

    PubMed Central

    Xu, Chengshan; Zhang, Ling; Li, Hengheng; Liu, Zhihua; Duan, Lianning; Lu, Chengrong

    2015-01-01

    MicroRNAs play key roles in cell growth, differentiation, and apoptosis. In this study, we described the regulation and function of miR-1469 in apoptosis of lung cancer cells (A549 and NCI-H1650). Expression analysis verified that miR-1469 expression significantly increased in apoptotic cells. Overexpression of miR-1469 in lung cancer cells increased cell apoptosis induced by etoposide. Additionally, we identified that Stat5a is a downstream target of miR-1469, which can bind directly to the 3’-untranslated region of the Stat5a, subsequently reducing both the mRNA and protein levels of Stat5a. Finally, co-expression of miR-1469 and Stat5a in A549 and NCI-H1650 cells partially abrogated the effect of miR-1469 on cell apoptosis. Our results show that miR-1469 functions as an apoptosis enhancer to regulate lung cancer apoptosis through targeting Stat5a and may become a critical therapeutic target in lung cancer. PMID:26045996

  12. Orai1 controls C5a-induced neutrophil recruitment in inflammation.

    PubMed

    Sogkas, Georgios; Vögtle, Timo; Rau, Eduard; Gewecke, Britta; Stegner, David; Schmidt, Reinhold E; Nieswandt, Bernhard; Gessner, J Engelbert

    2015-07-01

    Stromal interaction molecule 1 (STIM1)-dependent store operated calcium-entry (SOCE) through Orai1-mediated calcium (Ca(2+) ) influx is considered a major pathway of Ca(2+) signaling, serving T-cell, mast cell, and platelet responses. Here, we show that Orai1 is critical for neutrophil function. Orai1-deficient neutrophils present defects in fMLP and complement C5a-induced Ca(2+) influx and migration, although they respond normally to another chemoattractant, CXCL2. Up until now, no specific contribution of Orai1 independent from STIM1 or SOCE has been recognized in immune cells. Here, we observe that Orai1-deficient neutrophils exhibit normal STIM1-dependent SOCE and STIM1-deficient neutrophils respond to fMLP and C5a efficiently. Despite substantial cytokine production, Orai1(-/-) chimeric mice show impaired neutrophil recruitment in LPS-induced peritonitis. Moreover, Orai1 deficiency results in profoundly defective C5a-triggered neutrophil lung recruitment in hypersensitivity pneumonitis. Comparative evaluation of inflammation in Stim1(-/-) chimeras reveals a distinct pathogenic contribution of STIM1, including its involvement in IgG-induced C5a production. Our data establish Orai1 as key signal mediator of C5aR activation, contributing to inflammation by a STIM1-independent pathway of Ca(2+) -influx in neutrophils. PMID:25912155

  13. Nonlinear magnetoelectric behavior of Terfenol-D/PZT-5A laminate composites

    NASA Astrophysics Data System (ADS)

    Wang, Yezuo; Atulasimha, Jayasimha; Prasoon, Ruchir

    2010-12-01

    In this paper, a comprehensive experimental study and modeling of the nonlinear behavior of Terfenol-D/PZT-5A magnetoelectric laminate composites is reported. Magnetostriction versus magnetic field of an individual Terfenol-D sample of dimensions length = 22 mm, width = 19 mm, thickness = 0.683 mm, and polarization versus electric field as well as strain versus electric field of an individual PZT-5A sample of dimensions length = 22 mm, width = 19 mm, thickness = 0.127 mm were characterized. These samples were bonded to form a symmetric PZT-5A/Terfenol-D/PZT-5A laminate composite to avoid bending-extension coupling. Electric response of this composite to magnetic input was comprehensively characterized to include major loop and minor loop behavior. A modeling approach that structurally couples the nonlinear magnetostrictive Terfenol-D behavior and linear PZT-5A behavior to predict the magnetoelectric response was developed and validated against experimental results. This analysis, with further refinements, could prove to be a useful tool to model and design magnetoelectric sensors.

  14. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  15. Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

    PubMed Central

    Liu, Hong-Xiang; Grosse, Ann S.; Iwatsuki, Ken; Mishina, Yuji; Gumucio, Deborah L.; Mistretta, Charlotte M.

    2012-01-01

    Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor. PMID:22024319

  16. Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity.

    PubMed

    Coburn, Craig A; Meinke, Peter T; Chang, Wei; Fandozzi, Christine M; Graham, Donald J; Hu, Bin; Huang, Qian; Kargman, Stacia; Kozlowski, Joseph; Liu, Rong; McCauley, John A; Nomeir, Amin A; Soll, Richard M; Vacca, Joseph P; Wang, Dahai; Wu, Hao; Zhong, Bin; Olsen, David B; Ludmerer, Steven W

    2013-12-01

    The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection. PMID:24127258

  17. Wnt5a functions in planar cell polarity regulation in mice

    PubMed Central

    Qian, Dong; Jones, Chonnettia; Rzadzinska, Agnieszka; Mark, Sharayne; Zhang, Xiaohui; Steel, Karen P; Dai, Xing; Chen, Ping

    2007-01-01

    Planar cell polarity (PCP) refers to the polarization of cells within the plane of a cell sheet. A distinctive epithelial PCP in vertebrates is the uniform orientation of stereociliary bundles of the sensory hair cells in the mammalian cochlea. In addition to establishing epithelial PCP, planar polarization is also required for convergent extension (CE), a polarized cellular movement that occurs during neural tube closure and cochlear extension. Studies in Drosophila and vertebrates have revealed a conserved PCP pathway, including Frizzled (Fz) receptors. Here we use the cochlea as a model system to explore the involvement of known ligands of Fz, Wnt morphogens, in PCP regulation. We show that Wnt5a forms a reciprocal expression pattern with a Wnt antagonist, the secreted frizzled-related protein 3 (Sfrp3 or Frzb), along the axis of planar polarization in the cochlear epithelium. We further demonstrate that Wnt5a antagonizes Frzb in regulating cochlear extension and stereociliary bundle orientation in vitro, and that Wnt5a−/− animals have a shortened and widened cochlea. Finally, we show that Wnt5a is required for proper subcellular distribution of a PCP protein, Ltap/Vangl2, and that Wnt5a interacts genetically with Ltap/Vangl2 for uniform orientation of stereocilia, cochlear extension, and neural tube closure. Together, these findings demonstrate that Wnt5a functions in PCP regulation in mice. PMID:17433286

  18. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.

    PubMed

    Schmidt, Christian; Becker, Thomas; Heuer, André; Braunger, Katharina; Shanmuganathan, Vivekanandan; Pech, Markus; Berninghausen, Otto; Wilson, Daniel N; Beckmann, Roland

    2016-02-29

    During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis. PMID:26715760

  19. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

    PubMed Central

    Dass, Randall A.; Sarshad, Aishe A.; Feenstra, Jennifer M.; Kaur, Amanpreet; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C.; Percipalle, Piergiorgio; Brown, Anthony M. C.; Vincent, C. Theresa

    2016-01-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  20. Nuclear receptor NR5A2 controls neural stem cell fate decisions during development

    PubMed Central

    Stergiopoulos, Athanasios; Politis, Panagiotis K.

    2016-01-01

    The enormous complexity of mammalian central nervous system (CNS) is generated by highly synchronized actions of diverse factors and signalling molecules in neural stem/progenitor cells (NSCs). However, the molecular mechanisms that integrate extrinsic and intrinsic signals to control proliferation versus differentiation decisions of NSCs are not well-understood. Here we identify nuclear receptor NR5A2 as a central node in these regulatory networks and key player in neural development. Overexpression and loss-of-function experiments in primary NSCs and mouse embryos suggest that NR5A2 synchronizes cell-cycle exit with induction of neurogenesis and inhibition of astrogliogenesis by direct regulatory effects on Ink4/Arf locus, Prox1, a downstream target of proneural genes, as well as Notch1 and JAK/STAT signalling pathways. Upstream of NR5a2, proneural genes, as well as Notch1 and JAK/STAT pathways control NR5a2 endogenous expression. Collectively, these observations render NR5A2 a critical regulator of neural development and target gene for NSC-based treatments of CNS-related diseases. PMID:27447294

  1. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    SciTech Connect

    Wang Zuguang; Chen Hong

    2009-01-23

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear {beta}-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  2. MiRNA-1469 promotes lung cancer cells apoptosis through targeting STAT5a.

    PubMed

    Xu, Chengshan; Zhang, Ling; Li, Hengheng; Liu, Zhihua; Duan, Lianning; Lu, Chengrong

    2015-01-01

    MicroRNAs play key roles in cell growth, differentiation, and apoptosis. In this study, we described the regulation and function of miR-1469 in apoptosis of lung cancer cells (A549 and NCI-H1650). Expression analysis verified that miR-1469 expression significantly increased in apoptotic cells. Overexpression of miR-1469 in lung cancer cells increased cell apoptosis induced by etoposide. Additionally, we identified that Stat5a is a downstream target of miR-1469, which can bind directly to the 3'-untranslated region of the Stat5a, subsequently reducing both the mRNA and protein levels of Stat5a. Finally, co-expression of miR-1469 and Stat5a in A549 and NCI-H1650 cells partially abrogated the effect of miR-1469 on cell apoptosis. Our results show that miR-1469 functions as an apoptosis enhancer to regulate lung cancer apoptosis through targeting Stat5a and may become a critical therapeutic target in lung cancer. PMID:26045996

  3. Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.

    PubMed

    Anderson, Elizabeth T; Wetherell, Michael G; Winter, Laurie A; Olmsted, Stephen B; Cleary, Patrick P; Matsuka, Yury V

    2002-10-01

    A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate. PMID:12354115

  4. Adipocytes WNT5a mediated dedifferentiation: a possible target in pancreatic cancer microenvironment

    PubMed Central

    Zoico, Elena; Darra, Elena; Rizzatti, Vanni; Budui, Simona; Franceschetti, Guido; Mazzali, Gloria; Rossi, Andrea P; Fantin, Francesco; Menegazzi, Marta; Cinti, Saverio; Zamboni, Mauro

    2016-01-01

    A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known. After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes. Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture. These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment. PMID:26958939

  5. Wnt5a uses CD146 as a receptor to regulate cell motility and convergent extension

    NASA Astrophysics Data System (ADS)

    Ye, Zhongde; Zhang, Chunxia; Tu, Tao; Sun, Min; Liu, Dan; Lu, Di; Feng, Jing; Yang, Dongling; Liu, Feng; Yan, Xiyun

    2013-12-01

    Dysregulation of Wnt signalling leads to developmental defects and diseases. Non-canonical Wnt signalling via planar cell polarity proteins regulates cell migration and convergent extension; however, the underlying mechanisms are poorly understood. Here we report that Wnt5a uses CD146 as a receptor to regulate cell migration and zebrafish embryonic convergent extension. CD146 binds to Wnt5a with the high affinity required for Wnt5a-induced activation of Dishevelled (Dvl) and c-jun amino-terminal kinase (JNK). The interaction between CD146 and Dvl2 is enhanced on Wnt5a treatment. Mutation of the Dvl2-binding region impairs its ability to activate JNK, promote cell migration and facilitate the formation of cell protrusions. Knockdown of Dvls impairs CD146-induced cell migration. Interestingly, CD146 inhibits canonical Wnt signalling by promoting β-catenin degradation. Our results suggest a model in which CD146 acts as a functional Wnt5a receptor in regulating cell migration and convergent extension, turning off the canonical Wnt signalling branch.

  6. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

    PubMed Central

    Schmidt, Christian; Becker, Thomas; Heuer, André; Braunger, Katharina; Shanmuganathan, Vivekanandan; Pech, Markus; Berninghausen, Otto; Wilson, Daniel N.; Beckmann, Roland

    2016-01-01

    During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis. PMID:26715760

  7. Increased expression of C5a receptor (CD88) mRNA in canine mammary tumors.

    PubMed

    Hezmee, Mohd Noor Mohd; Kyaw-Tanner, Myat; Lee, Jia Yu Peppermint; Shiels, Ian A; Rolfe, Barbara; Woodruff, Trent; Mills, Paul C

    2011-01-01

    Mammary tumors are among the most common neoplastic conditions in dogs, and there is evidence that inflammation plays a role in the development of some tumor types in dogs. The complement system is a major participant in the inflammatory process and the complement activation component, C5a, is a potent inflammatory peptide. This study investigated the mRNA expression of the major receptor for C5a (C5aR; CD88) in histopathological samples of canine mammary tumors by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using canine-specific primers for CD88. A total of seven canine mammary tumors (four malignant carcinomas, two benign mixed mammary tumors, and one myoepithelioma) and eight normal mammary glands were analysed. All the tumor samples expressed low levels of CD88 mRNA, while none of the normal mammary tissues showed any detectable expression. These preliminary results suggest that C5a-CD88 interaction may play a contributory role in the inflammatory response associated with mammary tumor development in dogs. Further studies investigating the mechanisms behind complement activation and C5a receptor expression in canine mammary tumors are warranted. PMID:20846729

  8. Structural disorganization of pronephric glomerulus in zebrafish mpp5a/nagie oko mutant

    PubMed Central

    Ichimura, Koichiro; Fukuyo, Yayoi; Nakamura, Tomomi; Powell, Rebecca; Sakai, Tatsuo; Obara, Tomoko

    2012-01-01

    Background The podocyte slit diaphragm (SD) is an essential component of the selective filtration barrier in the glomerulus. Several structural proteins required for formation and maintenance of SD have been identified; however, molecular mechanisms regulating these proteins are still limited. Results Here, we demonstrate that MAGUK p55 subfamily member 5a (Mpp5a)/Nagie oko, a component of the Crb multi-protein complex, was colocalized with an SD-associated protein ZO-1 in the zebrafish pronephric glomerulus. To characterize the function of Mpp5a, zebrafish mpp5am520 mutant embryos, which are known to have defects in cardiac and neuronal morphogenesis, were analyzed. These mutants failed to merge the bilateral glomerular primordia and to form the glomerular capillary and mesangium, but the foot processes and SD showed normal appearance. The structural disorganization in the mpp5am520 mutant glomerulus was quite similar to that of a cardiac troponin T2a/tnnt2a/silent heart knockdown zebrafish, which exhibited circulatory failure due to lack of heart beating. Conclusions Mpp5a is not prerequisite to form podocyte slit diaphragm in the pronephric glomerular development in zebrafish. The structural disorganization of the pronephric glomerulus in the mpp5am520 mutant is likely to result from circulatory failure, rather than the anomaly of Mpp5a protein in the glomerulus. PMID:23027442

  9. Adipocytes WNT5a mediated dedifferentiation: a possible target in pancreatic cancer microenvironment.

    PubMed

    Zoico, Elena; Darra, Elena; Rizzatti, Vanni; Budui, Simona; Franceschetti, Guido; Mazzali, Gloria; Rossi, Andrea P; Fantin, Francesco; Menegazzi, Marta; Cinti, Saverio; Zamboni, Mauro

    2016-04-12

    A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known.After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes.Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture.These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment. PMID:26958939

  10. SLC4A11 and the Pathophysiology of Congenital Hereditary Endothelial Dystrophy.

    PubMed

    Patel, Sangita P; Parker, Mark D

    2015-01-01

    Congenital hereditary endothelial dystrophy (CHED) is a rare autosomal recessive disorder of the corneal endothelium characterized by nonprogressive bilateral corneal edema and opacification present at birth. Here we review the current knowledge on the role of the SLC4A11 gene, protein, and its mutations in the pathophysiology and clinical presentation of CHED. Individuals with CHED have mutations in SLC4A11 which encodes a transmembrane protein in the SLC4 family of bicarbonate transporters. The expression of SLC4A11 in the corneal endothelium and inner ear patterns the deficits seen in CHED with corneal edema and hearing loss (Harboyan syndrome). slc4a11-null-mouse models recapitulate the CHED disease phenotype, thus establishing a functional role for SLC4A11 in CHED. However, the transport function of SLC4A11 remains unsettled. Some of the roles that have been attributed to SLC4A11 include H(+) and NH4 (+) permeation, electrogenic Na(+)-H(+) exchange, and water transport. Future studies of the consequences of SLC4A11 dysfunction as well as further understanding of corneal endothelial ion transport will help clarify the involvement of SLC4A11 in the pathophysiology of CHED. PMID:26451371

  11. SLC4A11 and the Pathophysiology of Congenital Hereditary Endothelial Dystrophy

    PubMed Central

    Patel, Sangita P.; Parker, Mark D.

    2015-01-01

    Congenital hereditary endothelial dystrophy (CHED) is a rare autosomal recessive disorder of the corneal endothelium characterized by nonprogressive bilateral corneal edema and opacification present at birth. Here we review the current knowledge on the role of the SLC4A11 gene, protein, and its mutations in the pathophysiology and clinical presentation of CHED. Individuals with CHED have mutations in SLC4A11 which encodes a transmembrane protein in the SLC4 family of bicarbonate transporters. The expression of SLC4A11 in the corneal endothelium and inner ear patterns the deficits seen in CHED with corneal edema and hearing loss (Harboyan syndrome). slc4a11-null-mouse models recapitulate the CHED disease phenotype, thus establishing a functional role for SLC4A11 in CHED. However, the transport function of SLC4A11 remains unsettled. Some of the roles that have been attributed to SLC4A11 include H+ and NH4+ permeation, electrogenic Na+-H+ exchange, and water transport. Future studies of the consequences of SLC4A11 dysfunction as well as further understanding of corneal endothelial ion transport will help clarify the involvement of SLC4A11 in the pathophysiology of CHED. PMID:26451371

  12. NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration.

    PubMed

    Hedrick, Erik; Lee, Syng-Ook; Doddapaneni, Ravi; Singh, Mandip; Safe, Stephen

    2016-05-01

    Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a relatedp-carboxymethylphenyl [1,1-bis(3'-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis. PMID:26929200

  13. Kinesin Family member 4A: A Potential Predictor for Progression of Human Oral Cancer

    PubMed Central

    Minakawa, Yasuyuki; Kasamatsu, Atsushi; Koike, Hirofumi; Higo, Morihiro; Nakashima, Dai; Kouzu, Yukinao; Sakamoto, Yosuke; Ogawara, Katsunori; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

    2013-01-01

    Background Kinesin family member 4A (KIF4A), a microtubule-based motor protein, was implicated in regulation of chromosomal structure and kinetochore microtubule dynamics. Considering the functions of KIF4A, we assumed that KIF4A is involved in progression of oral squamous cell carcinomas (OSCCs) via activation of the spindle assembly checkpoint (SAC). However, little is known about the relevance of KIF4A in the behavior of OSCC. We investigated the KIF4A expression status and its functional mechanisms in OSCC. Methods The KIF4A expression levels in seven OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using a KIF4A knockdown model, we assessed the expression of (SAC)-related molecules (BUB1, MAD2, CDC20, and cyclin B1), cell-cycle, and cellular proliferation. In addition to in vitro data, the clinical correlation between the KIF4A expression levels in primary OSCCs (n = 106 patients) and the clinicopathologic status by immunohistochemistry (IHC) also were evaluated. Results KIF4A mRNA and protein were up-regulated significantly (P < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. In the KIF4A knockdown cells, SAC activation was observed via increased BUB1 expression on the kinetochores, appropriate kinetochore localization of MAD2, down-regulation of CDC20, up-regulation of cyclin B1, and cell-cycle arrested at G2/M phase. The results showed that cellular proliferation of KIF4A knockdown cells decreased significantly (P < 0.05) compared with control cells. IHC showed that KIF4A expression in primary OSCCs was significantly (P < 0.05) greater than in the normal oral counterparts and that KIF4A-positive OSCCs were correlated closely (P < 0.05) with tumoral size. Conclusions Our results proposed for the first time that KIF4A controls cellular proliferation via SAC activation. Therefore, KIF4A might be a key regulator for tumoral progression in OSCCs. PMID

  14. LNG Safety Research: FEM3A Model Development

    SciTech Connect

    2006-09-30

    The initial scope of work for this project included: 1) Improving the FEM3A advanced turbulence closure module, 2) Adaptation of FEM3A for more general applications, and 3) Verification of dispersion over rough surfaces, with and without obstacle using the advanced turbulence closure module. These work elements were to be performed by Chemical Hazards Research Center (CHRC), Department of Chemical Engineering, University of Arkansas as a subcontractor to Gas Technology Institute (GTI). The tasks for GTI included establishment of the scientific support base for standardization of the FEM3A model, project management, technology transfer, and project administration. Later in the course of the project, the scope of work was modified by the National Energy Technology Laboratories (NETL) to remove the emphasis on FEM3A model and instead, develop data in support of NETL’s FLUENT modeling. With this change, GTI was also instructed to cease activities relative to FEM3A model. GTI’s technical activities through this project included the initial verification of FEM3A model, provision of technical inputs to CHRC researchers regarding the structure of the final product, and participation in technical discussion sessions with CHRC and NETL technical staff. GTI also began the development of a Windows-based front end for the model but the work was stopped due to the change in scope of work. In the meantime, GTI organized a workshop on LNG safety in Houston, Texas. The workshop was very successful and 75 people from various industries participated. All technical objectives were met satisfactorily by Dr. Jerry Havens and Dr. Tom Spicer of CHRC and results are presented in a stand-alone report included as Appendix A to this report.

  15. LNG Safety Research: FEM3A Model Development

    SciTech Connect

    Iraj A. Salehi; Jerry Havens; Tom Spicer

    2006-09-30

    The initial scope of work for this project included: (1) Improving the FEM3A advanced turbulence closure module, (2) Adaptation of FEM3A for more general applications, and (3) Verification of dispersion over rough surfaces, with and without obstacle using the advanced turbulence closure module. These work elements were to be performed by Chemical Hazards Research Center (CHRC), Department of Chemical Engineering, University of Arkansas as a subcontractor to Gas Technology Institute (GTI). The tasks for GTI included establishment of the scientific support base for standardization of the FEM3A model, project management, technology transfer, and project administration. Later in the course of the project, the scope of work was modified by the National Energy Technology Laboratories (NETL) to remove the emphasis on FEM3A model and instead, develop data in support of NETL's FLUENT modeling. With this change, GTI was also instructed to cease activities relative to FEM3A model. GTI's technical activities through this project included the initial verification of FEM3A model, provision of technical inputs to CHRC researchers regarding the structure of the final product, and participation in technical discussion sessions with CHRC and NETL technical staff. GTI also began the development of a Windows-based front end for the model but the work was stopped due to the change in scope of work. In the meantime, GTI organized a workshop on LNG safety in Houston, Texas. The workshop was very successful and 75 people from various industries participated. All technical objectives were met satisfactorily by Dr. Jerry Havens and Dr. Tom Spicer of CHRC and results are presented in a stand-alone report included as Appendix A to this report.

  16. Inhibition of STAT5: A therapeutic option in BCR-ABL1-driven leukemia

    PubMed Central

    Berger, Angelika; Sexl, Veronika; Valent, Peter; Moriggl, Richard

    2014-01-01

    The two transcription factors STAT5A and STAT5B are central signaling molecules in leukemias driven by Abelson fusion tyrosine kinases and they fulfill all criteria of drug targets. STAT5A and STAT5B display unique nuclear shuttling mechanisms and they have a key role in resistance of leukemic cells against treatment with tyrosine kinase inhibitors (TKI). Moreover, STAT5A and STAT5B promote survival of leukemic stem cells. We here discuss the possibility of targeting up-stream kinases with TKI, direct STAT5 inhibition via SH2 domain obstruction and blocking nuclear translocation of STAT5. All discussed options will result in a stop of STAT5 transport to the nucleus to block STAT5-mediated transcriptional activity. In summary, recently described shuttling functions of STAT5 are discussed as potentially druggable pathways in leukemias. PMID:25333255

  17. 2-5A ligands--a new concept for the treatment of prostate cancer.

    PubMed

    Cramer, Hagen; Okicki, James R; Rho, Taikyun; Wang, Xinning; Silverman, Robert H; Heston, Warren D W

    2007-01-01

    Several potent prostate specific membrane antigen (PSMA) inhibitors have been described recently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI 1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50 = 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hydrolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 = 1974 nM). Also, RBI 1033's affinity to PSMA was found to be 10 times higher than ZJ-24 itself. PMID:18066809

  18. Molecular evolution of the cytochrome c oxidase subunit 5A gene in primates

    PubMed Central

    2008-01-01

    Background Many electron transport chain (ETC) genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains. Results In a dataset comprising 29 vertebrate taxa, including representatives from all major groups of primates, there is nearly 100% conservation of the COX5A amino acid sequence among extant, non-anthropoid placental mammals. The most recent common ancestor of these species lived about 100 million years (MY) ago. In contrast, anthropoid primates show markedly elevated rates of nonsynonymous evolution. In particular, branch site tests identify five positively selected codons in anthropoids, and ancestral reconstructions infer that substitutions in these codons occurred predominantly on stem lineages (anthropoid, ape and New World monkey) and on the human terminal branch. Examination of catarrhine brain samples by immunohistochemistry characterizes for the first time COX5A protein distribution in the primate neocortex, and suggests that the protein is most abundant in the mitochondria of large-size projection neurons. Real time quantitative PCR supports previous microarray results showing COX5A is expressed in cerebral cortical tissue at a higher level in human than in chimpanzee or gorilla. Conclusion Taken together, these results suggest that both protein structural and gene regulatory changes contributed to COX5A evolution during humankind's ancestry. Furthermore, these findings are consistent with the hypothesis that adaptations in ETC genes contributed to the emergence of the energetically expensive anthropoid neocortex. PMID:18197981

  19. AB070. Mutations of SRD5A2 in Vietnamese patients: phenotype and genotype

    PubMed Central

    Dung, Vu Chi; Thao, Bui Phuong; Khanh, Nguyen Ngoc; Ngoc, Can Thi Bich; Fukami, Maki

    2015-01-01

    A rare form of the 46,XY disorders of sex development (DSD), 5α-reductase deficiency was first described in patients with pseudovaginal perineoscrotal hypospadias, microphallus, and cryptorchid testes in 1974 by Imperato-McGinley et al. and Walsh et al. This undervirilization in the male is due to an alteration in the 5α-reductase type 2 gene (SRD5A2), which encodes for 5α-reductase activity. Our registry of 750 patients with DSD showed no definitive diagnosis in 80% of cases with 46,XY DSD. Our aim is to identify mutations in SRD5A2 gene and to describe phenotype of detected mutative cases. Mutation analysis was performed for genomic DNA extracted from WBC of 10 patients with 46,XY DSD using PCR and direct sequencing. We identified mutations of SRD5A2 gene in two cases. The first case presented with isolated micropenis at birth, two palpable testes in the normal scrotum. Pelvic ultrasound showed no ovaries and uterus, karyotype was 46,XY and SRY was positive. Serum FSH level was 2.4 UI/L; LH level was 0.9 UI/L and testosterone level was 0.4 nmol/L at 8 years of age. A homozygous missense mutation (p.R237G) was identified in the SRD5A2 gene. The second case presented with microphallus, penoscrotal hypospadias, and gonad bilateral in labioscrotal folds. No uterus and ovaries were found by pelvic ultrasound. Karyotype was 46,XY and SRY was positive. A novel homozygous missense mutation (c.659C>T; p.S220L) was identified in the SRD5A2 gene. Mutation analysis of SRD5A2 gene helps to make definitive diagnosis for patients with 46,XY DSD.

  20. COL5A1 haploinsufficiency is a common molecular mechanism underlying the classical form of EDS.

    PubMed Central

    Wenstrup, R J; Florer, J B; Willing, M C; Giunta, C; Steinmann, B; Young, F; Susic, M; Cole, W G

    2000-01-01

    We have identified haploinsufficiency of the COL5A1 gene that encodes the proalpha1(V) chain of type V collagen in the classical form of the Ehlers-Danlos syndrome (EDS), a heritable connective-tissue disorder that severely alters the collagen-fibrillar structure of the dermis, joints, eyes, and blood vessels. Eight of 28 probands with classical EDS who were heterozygous for expressed polymorphisms in COL5A1 showed complete or nearly complete loss of expression of one COL5A1 allele. Reduced levels of proalpha1(V) mRNA relative to the levels of another type V collagen mRNA, proalpha2(V), were also observed in the cultured fibroblasts from EDS probands. Products of the two COL5A1 alleles were approximately equal after the addition of cycloheximide to the fibroblast cultures. After harvesting of mRNAs from cycloheximide-treated cultured fibroblasts, heteroduplex analysis of overlapping reverse transcriptase-PCR segments spanning the complete proalpha1(V) cDNA showed anomalies in four of the eight probands that led to identification of causative mutations, and, in the remaining four probands, targeting of CGA-->TGA mutations in genomic DNA revealed a premature stop at codon in one of them. We estimate that approximately one-third of individuals with classical EDS have mutations of COL5A1 that result in haploinsufficiency. These findings indicate that the normal formation of the heterotypic collagen fibrils that contain types I, III, and V collagen requires the expression of both COL5A1 alleles. PMID:10777716

  1. Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

    PubMed Central

    Park, Chorong; Min, Saehong; Park, Eun-Mee; Lim, Yun-Sook; Kang, Sangmin; Suzuki, Tetsuro; Shin, Eui-Cheol

    2015-01-01

    ABSTRACT The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCE Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell

  2. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 2

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the second book of volume four. Some of the items it contains are specs for the emergency shutdown panel, specs for the simulator software, simulator hardware specs, site operator terminal requirements, control data system requirements, software project management plan, elastomeric teeter bearing requirement specs, specs for the controls electronic cabinet, and specs for bolt pretensioning.

  3. Discovery of fused tricyclic core containing HCV NS5A inhibitors with pan-genotype activity.

    PubMed

    Yu, Wensheng; Coburn, Craig A; Yang, De-Yi; Meinke, Peter T; Wong, Michael; Rosenblum, Stuart B; Chen, Kevin X; Njoroge, George F; Chen, Lei; Dwyer, Michael P; Jiang, Yueheng; Nair, Anilkumar G; Selyutin, Oleg; Tong, Ling; Zeng, Qingbei; Zhong, Bin; Ji, Tao; Hu, Bin; Agrawal, Sony; Xia, Ellen; Zhai, Ying; Liu, Rong; Kong, Rong; Ingravallo, Paul; Asante-Appiah, Ernest; Nomeir, Amin; Fells, James; Kozlowski, Joseph A

    2016-07-01

    HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat. PMID:27180013

  4. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 4

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator are documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This volume contains 5 books of which this is the fourth, providing drawings 47A380128 through 47A387125. In addition to the parts listing and where-used list, the logic design of the controller software and the code listing of the controller software are provided. Also given are the aerodynamic profile coordinates.

  5. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 2

    NASA Astrophysics Data System (ADS)

    1984-08-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the second book of volume four. Some of the items it contains are specs for the emergency shutdown panel, specs for the simulator software, simulator hardware specs, site operator terminal requirements, control data system requirements, software project management plan, elastomeric teeter bearing requirement specs, specs for the controls electronic cabinet, and specs for bolt pretensioning.

  6. Mod-5A Wind Turbine Generator Program Design Report. Volume 4: Drawings and Specifications, Book 1

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. Volume 4 contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the first of five books of volume four. It contains structural design criteria, generator step-up transformer specs, specs for design, fabrication and testing of the system, specs for the ground control enclosure, systems specs, slip ring specs, and control system specs.

  7. Impaired Neurite Contact Guidance in Ubiquitin Ligase E3a (Ube3a)-Deficient Hippocampal Neurons on Nanostructured Substrates.

    PubMed

    Tonazzini, I; Meucci, S; Van Woerden, G M; Elgersma, Y; Cecchini, M

    2016-04-01

    Recent discoveries indicate that during neuronal development the signaling processes that regulate extracellular sensing (e.g., adhesion, cytoskeletal dynamics) are important targets for ubiquitination-dependent regulation, in particular through E3 ubiquitin ligases. Among these, Ubiquitin E3a ligase (UBE3A) has a key role in brain functioning, but its function and how its deficiency results in the neurodevelopmental disorder Angelman syndrome is still unclear. Here, the role of UBE3A is investigated in neurite contact guidance during neuronal development, in vitro. The microtopography sensing of wild-type and Ube3a-deficient hippocampal neurons is studied by exploiting gratings with different topographical characteristics, with the aim to compare their capabilities to read and follow physical directional stimuli. It is shown that neuronal contact guidance is defective in Ube3a-deficient neurons, and this behavior is linked to an impaired activation of the focal adhesion signaling pathway. Taken together, the results suggest that the neuronal contact sensing machinery might be affected in Angelman syndrome. PMID:26845073

  8. 18 CFR 3a.31 - Classification markings and special notations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Classification markings... REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification Markings and Special Notations § 3a.31 Classification markings and special notations. (a) After...

  9. 3. A LONG VIEW, LOOKING SOUTHEAST FROM THE SAME LOCATION ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. A LONG VIEW, LOOKING SOUTHEAST FROM THE SAME LOCATION AS THE PREVIOUS PHOTO, SHOWING THE EAST HALF OF THE NORTH SIDE OF THE BRIDGE - Putnam County Bridge No. 111, Spanning Little Walnut Creek on County Road 50, Greencastle, Putnam County, IN

  10. 18 CFR 3a.11 - Classification of official information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Classification of... REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION Classification § 3a.11 Classification of official information. (a) Security Classification Categories. Information...

  11. 16 CFR 1105.3 - A more satisfactory standard.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false A more satisfactory standard. 1105.3 Section 1105.3 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION CONSUMER PRODUCT SAFETY ACT REGULATIONS CONTRIBUTIONS TO COSTS OF PARTICIPANTS IN DEVELOPMENT OF CONSUMER PRODUCT SAFETY STANDARDS § 1105.3 A...

  12. 16 CFR 1105.3 - A more satisfactory standard.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false A more satisfactory standard. 1105.3 Section 1105.3 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION CONSUMER PRODUCT SAFETY ACT REGULATIONS CONTRIBUTIONS TO COSTS OF PARTICIPANTS IN DEVELOPMENT OF CONSUMER PRODUCT SAFETY STANDARDS § 1105.3 A...

  13. Hairy Math: Add Wnt-3a to Multiply Bulge Cells

    PubMed Central

    Hossain, M. Zulfiquer; Garza, Luis A.

    2015-01-01

    Canonical Wnt signals are important for activation of epithelial skin stem cells, but the role of individual Wnt ligands remains uncertain. Ouji et al. demonstrate a key role for Wnt-3a in partial maintenance and long-term expansion of epithelial skin stem cells in vitro. They also report a method for expanding these cells in vitro without feeder cells. PMID:25964269

  14. 29. SITE BUILDING 002 SCANNER BUILDING FLOOR 3A ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    29. SITE BUILDING 002 - SCANNER BUILDING - FLOOR 3A ("A" FACE) AT SYSTEM LAYOUT GRID 17. GENERAL OBLIQUE VIEW OF "A" FACE INTERIOR SHOWING RADAR EMITTER/ANTENNA INTERFACE ELECTRONICS. - Cape Cod Air Station, Technical Facility-Scanner Building & Power Plant, Massachusetts Military Reservation, Sandwich, Barnstable County, MA

  15. 3. A VIEW TAKEN FROM THE SIDEWALK ON THE SOUTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. A VIEW TAKEN FROM THE SIDEWALK ON THE SOUTH APPROACH TO THE BRIDGE, LOOKING NORTH, SHOWING THE TRUCK BODY WORKS AND A PORTION OF DOWNTOWN DELPHI. - Delphi Bridge on U.S. Route 421, Spanning Deer Creek at U.S. Route 421, Delphi, Carroll County, IN

  16. 40 CFR 60.482-4a - Standards: Pressure relief devices in gas/vapor service.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Standards: Pressure relief devices in gas/vapor service. (a) Except during pressure releases, each pressure... 40 Protection of Environment 7 2014-07-01 2014-07-01 false Standards: Pressure relief devices in gas/vapor service. 60.482-4a Section 60.482-4a Protection of Environment ENVIRONMENTAL...

  17. 40 CFR 60.482-4a - Standards: Pressure relief devices in gas/vapor service.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Standards: Pressure relief devices in gas/vapor service. (a) Except during pressure releases, each pressure... 40 Protection of Environment 7 2012-07-01 2012-07-01 false Standards: Pressure relief devices in gas/vapor service. 60.482-4a Section 60.482-4a Protection of Environment ENVIRONMENTAL...

  18. 40 CFR 60.482-4a - Standards: Pressure relief devices in gas/vapor service.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Standards: Pressure relief devices in gas/vapor service. (a) Except during pressure releases, each pressure... 40 Protection of Environment 6 2011-07-01 2011-07-01 false Standards: Pressure relief devices in gas/vapor service. 60.482-4a Section 60.482-4a Protection of Environment ENVIRONMENTAL...

  19. 40 CFR 60.482-4a - Standards: Pressure relief devices in gas/vapor service.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 7 2013-07-01 2013-07-01 false Standards: Pressure relief devices in gas/vapor service. 60.482-4a Section 60.482-4a Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY SOURCES Standards of Performance for Equipment Leaks of VOC...

  20. 40 CFR 60.482-4a - Standards: Pressure relief devices in gas/vapor service.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Standards: Pressure relief devices in gas/vapor service. (a) Except during pressure releases, each pressure... 40 Protection of Environment 6 2010-07-01 2010-07-01 false Standards: Pressure relief devices in gas/vapor service. 60.482-4a Section 60.482-4a Protection of Environment ENVIRONMENTAL...

  1. NR4A1 promotes PDGF-BB-induced cell colony formation in soft agar.

    PubMed

    Eger, Glenda; Papadopoulos, Natalia; Lennartsson, Johan; Heldin, Carl-Henrik

    2014-01-01

    The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth. PMID:25269081

  2. NR4A1 Promotes PDGF-BB-Induced Cell Colony Formation in Soft Agar

    PubMed Central

    Eger, Glenda; Papadopoulos, Natalia; Lennartsson, Johan; Heldin, Carl-Henrik

    2014-01-01

    The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth. PMID:25269081

  3. Synthesis Gas Demonstration Plant, Baskett, Kentucky: environmental report. [Contains chapter 4 and appendix 4A

    SciTech Connect

    1980-01-01

    This volume contains chapter 4 and Appendix 4A which include descriptions of use of adjacent land and water (within miles of the proposed site), baseline ecology, air quality, meteorology, noise, hydrology, water quality, geology, soils and socio-economic factors. Appendix 4A includes detailed ecological surveys made in the area including the methods used. (LTN)

  4. 26 CFR 1.668(b)-4A - Information requirements with respect to beneficiary.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Information requirements with respect to beneficiary. 1.668(b)-4A Section 1.668(b)-4A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning...

  5. Sulfotransferase 4A1 Haplotype 1 (SULT4A1-1) Is Associated With Decreased Hospitalization Events in Antipsychotic-Treated Patients With Schizophrenia

    PubMed Central

    Liu, Qian; Ramsey, Timothy L.; Meltzer, Herbert Y.; Massey, Bill W.; Padmanabhan, Saranya

    2012-01-01

    Objective: To evaluate a common genetic variant, sulfotransferase 4A1 haplotype 1 (SULT4A1-1), as a predictor of hospitalization events due to the exacerbation of schizophrenia for patients treated with antipsychotic medications. Haplotypes were determined using single nucleotide polymorphism data. Method: The study included 417 white subjects from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study with a DSM-IV diagnosis of schizophrenia. Patients were assigned to 1 of 4 atypical antipsychotics (olanzapine, quetiapine, risperidone, or ziprasidone) or to the first-generation antipsychotic perphenazine. Kaplan-Meier survival analysis and Cox proportional hazards regression models were used to measure if haplotype status impacted hospitalization events for these 5 treatments. Haplotype status was evaluated for its relationship to hospitalization events regardless of treatment and for the individual treatments, with or without previous exacerbation. Data for the CATIE study were collected from January 2001 to December 2004. The current post hoc analysis was performed between May 2011 and August 2011. Results: In phase 1 of the trial, considering only the first hospitalization events, the haplotype had a significant impact on hospitalization events, with a hazard ratio for SULT4A1-1(−) versus SULT4A1-1(+) of 2.54 (P = .048). When prior exacerbation was included in the model for phase 1, the hazard ratio was 2.34 (P = .072) considering only the first hospitalization event and 2.71 (P = .039) considering all hospitalization events in the phase. When data for all phases were evaluated, SULT4A1-1(−) status was associated with increased hospitalization risk for subjects treated with olanzapine, with a hazard ratio of 8.26 (P = .041), and possibly for subjects treated with quetiapine, with a hazard ratio of 6.80 (P = .070). Conclusions: The SULT4A1-1 haplotype may be an important predictor of risk of hospitalization. SULT4A1-1(+) status was

  6. Rough order of magnitude cost estimate for immobilization of 18.2 MT of plutonium using existing facilities at the Savannah River site: alternatives 3A/5A/6A/6B/7A/9A

    SciTech Connect

    DiSabatino, A., LLNL

    1998-06-01

    The purpose of this Cost Estimate Report is to identify preliminary capital and operating costs for a facility to immobilize 18.2 metric tons (nominal) of plutonium using ceramic in a new facility at Savannah River Site (SRS).

  7. Identification of quantitative trait locus for abscisic acid responsiveness on chromosome 5A and association with dehydration tolerance in common wheat seedlings.

    PubMed

    Iehisa, Julio C M; Matsuura, Takakazu; Mori, Izumi C; Takumi, Shigeo

    2014-01-15

    The phytohormone abscisic acid (ABA) plays important roles in response to environmental stress as well as in seed maturation and dormancy. In common wheat, quantitative trait loci (QTLs) for ABA responsiveness at the seedling stage have been reported on chromosomes 1B, 2A, 3A, 6D and 7B. In this study, we identified a novel QTL for ABA responsiveness on chromosome 5A using an F2 population derived from a cross between the common wheat cultivar Chinese Spring (CS) and a chromosome substitution line of CS with chromosome 5A of cultivar Hope (Hope5A). This QTL was found in a similar chromosomal region to previously reported QTLs for drought tolerance and seed dormancy. Physiological characterization of the QTL revealed a small effect on dehydration tolerance and seed dormancy. The rate of water loss from leaves during dehydration was lower, and transcript accumulation of the cold responsive (COR)/late embryogenesis abundant (LEA) genes Wrab18 and Wdhn13 tended to be higher under dehydration stress in F2 individuals carrying the Hope allele of the QTL, which also showed higher ABA responsiveness than the CS allele-carrying individuals. Seed dormancy of individuals carrying the Hope allele also tended to be lower than those carrying the CS allele. Our results suggest that variation in ABA responsiveness among common wheat cultivars is at least partly determined by the 5A QTL, and that this QTL contributes to development of dehydration and preharvest sprouting tolerance. PMID:24331416

  8. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 CYP4A15.

    PubMed

    Ngo, Suong Ngoc Thi; McKinnon, Ross Allan; Stupans, Ieva

    2006-07-01

    In the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala (Phascolarctos cinereus), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not ingest eucalyptus leaves as food [Ngo, S., Kong, S., Kirlich, A., Mckinnon, R.A., Stupans, I., 2000. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comp. Biochem. Physiol., C 127, 327-334]. A 1544 bp koala liver CYP4A cDNA, designated CYP4A15, was cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CYP4A15 cDNA encodes a protein of 500 amino acids and shares 69% nucleotide and 65% amino acid sequence identity to human CYP4A11. Transfection of the koala CYP4A15 cDNA into Cos-7 cells resulted in the expression of a protein with lauric acid hydroxylase activity. The koala CYP4A15 cDNA-expressed enzyme catalysed lauric acid hydroxylation at the rates of 0.45+/-0.18 nmol/min/mg protein and 4.79+/-1.91 nmol/min/nmol CYP (mean+/-SD, n=3), which were comparable to that of rat CYP4A subfamilies. Total CYP content for koala CYP4A15-expressed protein in Cos-7 cells was 0.094+/-0.001 nmol/mg protein (mean+/-SD, n=3) with negligible CYP content in untransfected Cos-7 cells lysate. Immunoblot analysis, using a sheep anti-rat CYP4A polyclonal antibody, detected multiple CYP4A immunoreactive bands in the liver from all species studied. The koala bands were found to be fainter and less confined but appeared much broader as compared to rat, human and wallaby. Northern blot analysis, utilising the koala CYP4A15 cDNA 417 bp probe, detected a mRNA species of approximately 2.6 kb in the koala liver and a mRNA species of approximately 2.4 kb in other species studied. Relative to the intensity of the beta

  9. Characterization of the novel T4-like Salmonella enterica bacteriophage STP4-a and its endolysin.

    PubMed

    Li, Meng; Li, Mengzhe; Lin, Hong; Wang, Jingxue; Jin, Yanqiu; Han, Feng

    2016-02-01

    While screening for new antimicrobial agents for multidrug-resistant Salmonella enterica, the novel lytic bacteriophage STP4-a was isolated and characterized. Phage morphology revealed that STP4-a belongs to the family Myoviridae. Bacterial challenge assays showed that different serovars of Salmonella enterica were susceptible to STP4-a infection. The genomic characteristics of STP4-a, containing 159,914 bp of dsDNA with an average GC content of 36.86 %, were determined. Furthermore, the endolysin of STP4-a was expressed and characterized. The novel endolysin, LysSTP4, has hydrolytic activity towards outer-membrane-permeabilized S. enterica and Escherichia coli. These results provide essential information for the development of novel phage-based biocontrol agents against S. enterica. PMID:26563319

  10. A single base mutation in COL5A2 causes Ehlers-Danlos syndrome type II.

    PubMed

    Richards, A J; Martin, S; Nicholls, A C; Harrison, J B; Pope, F M; Burrows, N P

    1998-10-01

    Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders. Recently mutations have been found in the genes for type V collagen in a small number of people with the most common forms of EDS, types I and II. Here we characterise a COL5A2 mutation in an EDS II family. Cultured dermal fibroblasts obtained from an affected subject synthesised abnormal type V collagen. Haplotype analysis excluded COL5A1 but was concordant with COL5A2 as the disease locus. The entire open reading frame of the COL5A2 cDNA was directly sequenced and a single base mutation detected. It substituted a glycine residue within the triple helical domain (G934R) of alpha2(V) collagen, typical of the dominant negative changes in other collagens, which cause various other inherited connective tissue disorders. All three affected family members possessed the single base change, which was absent in 50 normal chromosomes. PMID:9783710

  11. Fragmentation of silicon nuclei at 4. 5 A GeV/c

    SciTech Connect

    Ahmad, T.; Irfan, M. )

    1991-11-30

    1024 interactions caused by 4.5 A GeV/c {sup 28}Si in emulsion are studied. In this paper result on the mean free path is discussed and compared with the corresponding results obtained for the interactions initiated by other projectiles. Multiplicity distributions for Z = 1, 2 and {ge}3 fragments are also investigated.

  12. The 5 A day Virtual Classroom: an on-line strategy to promote healthful eating.

    PubMed

    DiSogra, L; Glanz, K

    2000-03-01

    Communications technology can help stimulate youth to become involved in health promotion. This article reports on an innovative, Internet-based nutrition program that encouraged children to be advocates for policies that promote eating more fruits and vegetables, the 5 A Day Virtual Classroom. Through this program, students from across the United States discussed the recommendation of 5 A Day at the same time in a classroom without walls. In September 1997 children were asked, "If you were President Clinton, how would you get kids across the country to eat 5 A Day?" Based on content analysis of responses, this article suggests strategies that policymakers could use to encourage children to consume more fruits and vegetables. Approximately 2,600 students participated; 635 entries and 910 suggestions were received. The suggestion categories cited most often were mass media (19.8%), economic issues (15.4%), and social influence (13.8%). The most frequently mentioned specific ideas were to reward children for eating fruits and vegetables and to use presidential authority. Some regional, age, and gender patterns were found. Findings support the potential impact on health education of the 5 A Day Virtual Classroom and of interventions based on communications technology. PMID:10719410

  13. 5 A DAY ACHIEVEMENT BADGE FOR AFRICAN AMERICAN BOY SCOUTS: PILOT OUTCOME RESULTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Boy Scouts are an important channel to complement school-based programs to enable boys to eat more fruit, 100% juice, and vegetables (FJV) for chronic disease prevention. The "5 a Day Achievement Badge" program was presented on a pilot study basis to African-American Boy Scout troops in Houston. Tro...

  14. Investigation of Northrop F-5A wing buffet intensity in transonic flight

    NASA Technical Reports Server (NTRS)

    Chintsun, H.; Pi, W. S.

    1974-01-01

    A flight test and data processing program utilizing a Northrop F-5A aircraft instrumented to acquire buffet pressures and response data during transonic maneuvers is discussed. The data are presented in real-time format followed by spectral and statistical analyses. Also covered is a comparison of the aircraft response data with computed responses based on the measured buffet pressures.

  15. [Involvement of nonstructural protein 5A and lipids on production of hepatitis C virus particles].

    PubMed

    Suzuki, Tetsuro; Masaki, Takahiro; Aizaki, Hideki

    2008-12-01

    A robust system for production of recombinant infectious hepatitis C virus (HCV) has been established in 2005 and classical virological techniques are now able to be applied to the HCV research, especially regarding molecular mechanisms on virion assembly and maturation. We recently demonstrated that the C-terminal serine cluster of NS5A is a determinant of NS5A interaction with Core and the subcellular localization of NSSA. Mutation of this cluster blocks the NS5A-Core interaction, resulting in perturbation of association between Core and HCV RNA. It is thus tempting to consider that NS5A plays a key role in transporting the viral genome RNA synthesized by the replication complex to the surface of lipid droplets (LDs) or LD-associated membranes, where Core localizes, leading to facilitation of nucleocapsid formation. We also demonstrated an important role of cholesterol and sphingolipid in HCV infection and virion maturation. Specifically, mature HCV particles are rich in cholesterol. Depletion of cholesterol from HCV or hydrolysis of virion-associated sphingomyelin results in a loss of infectivity, and the addition of exogenous cholesterol restores infectivity. In addition, cholesterol and sphingolipid on the HCV membrane play a key role in virus internalization. Finally, inhibitors of the sphingolipid biosynthetic pathway efficiently block virion production. PMID:19374198

  16. The hypusine-containing translation factor eIF5A

    PubMed Central

    Dever, Thomas E.; Gutierrez, Erik; Shin, Byung-Sik

    2014-01-01

    In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a βlysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues. PMID:25029904

  17. Conceptual design of the 6 MW Mod-5A wind turbine generator

    NASA Technical Reports Server (NTRS)

    Barton, R. S.; Lucas, W. C.

    1982-01-01

    The General Electric Company, Advanced Energy Programs Department, is designing under DOE/NASA sponsorship the MOD-5A wind turbine system which must generate electricity for 3.75 cent/KWH (1980) or less. During the Conceptual Design Phase, completed in March, 1981, the MOD-5A WTG system size and features were established as a result of tradeoff and optimization studies driven by minimizing the system cost of energy (COE). This led to a 400' rotor diameter size. The MOD-5A system which resulted is defined in this paper along with the operational and environmental factors that drive various portions of the design. Development of weight and cost estimating relationships (WCER's) and their use in optimizing the MOD-5A are discussed. The results of major tradeoff studies are also presented. Subsystem COE contributions for the 100th unit are shown along with the method of computation. Detailed descriptions of the major subsystems are given, in order that the results of the various trade and optimization studies can be more readily visualized.

  18. Notice of release of RMUP-C5, a random mated population of Upland cotton germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Agricultural Research Service of the United States Department of Agriculture, Mississippi Agricultural and Forestry Experiment Station, North Carolina State University, and Cotton Incorporated, Cary, NC, announce the release of RMUP-C5, a population of upland cotton that has been random mated fo...

  19. Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense*

    PubMed Central

    Jones, Alexis; Kainz, Danielle; Khan, Faatima; Lee, Cara; Carrithers, Michael D.

    2014-01-01

    Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon β. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation. PMID:25368329

  20. Targeting complement component 5a promotes vascular integrity and limits airway remodeling.

    PubMed

    Khan, Mohammad A; Maasch, Christian; Vater, Axel; Klussmann, Sven; Morser, John; Leung, Lawrence L; Atkinson, Carl; Tomlinson, Stephen; Heeger, Peter S; Nicolls, Mark R

    2013-04-01

    Increased microvascular dilatation and permeability is observed during allograft rejection. Because vascular integrity is an important indicator of transplant health, we have sought to limit injury to blood vessels by blocking complement activation. Although complement component 3 (C3) inhibition is known to be vasculoprotective in transplantation studies, we recently demonstrated the paradoxical finding that, early in rejection, C3(-/-) transplant recipients actually exhibit worse microvascular injury than controls. In the genetic absence of C3, thrombin-mediated complement component 5 (C5) convertase activity leads to the generation of C5a (anaphylatoxin), a promoter of vasodilatation and permeability. In the current study, we demonstrated that microvessel thrombin deposition is significantly increased in C3(-/-) recipients during acute rejection. Thrombin colocalization with microvessels is closely associated with remarkably elevated plasma levels of C5a, vasodilatation, and increased vascular permeability. Administration of NOX-D19, a specific C5a inhibitor, to C3(-/-) recipients of airway transplants significantly improved tissue oxygenation, limited microvascular leakiness, and prevented airway ischemia, even in the absence of conventional T-cell-directed immunosuppression. As C3 inhibitors enter the clinics, the simultaneous targeting of this thrombin-mediated complement activation pathway and/or C5a itself may confer significant clinical benefit. PMID:23530212

  1. Issues in NASA Program and Project Management. Special Edition: A Collection of Papers on NASA Procedures and Guidance 7120.5A. Volume 14

    NASA Technical Reports Server (NTRS)

    Hoffman, Edward J. (Editor); Lawbaugh, William M. (Editor)

    1998-01-01

    A key aspect of NASA's new Strategic Management System is improving the way we plan, approve, execute and evaluate our programs and projects. To this end, NASA has developed the NASA Program and Project Management processes and Requirements-NASA Procedures and Guidelines (NPG) 7120.5A, which formally documents the "Provide Aerospace Products and Capabilities" crosscutting process, and defines the processes and requirements that are responsive to the Program/Project Management-NPD 7120.4A. The Program/Project Management-NPD 7120.4A, issued November 14, 1996, provides the policy for managing programs and projects in a new way that is aligned with the new NASA environment. An Agencywide team has spent thousands of hours developing the NASA Program and Project Management Processes and Requirements-NPG 7120.5A. We have created significant flexibility, authority and discretion for the program and project managers to exercise and carry out their duties, and have delegated the responsibility and the accountability for their programs and projects.

  2. Thermoluminescence solid-state nanodosimetry—the peak 5A/5 dosemeter

    PubMed Central

    Fuks, E.; Horowitz, Y. S.; Horowitz, A.; Oster, L.; Marino, S.; Rainer, M.; Rosenfeld, A.; Datz, H.

    2011-01-01

    The shape of composite peak 5 in the glow curve of LiF:Mg,Ti (TLD-100) following 90Sr/90Y beta irradiation, previously demonstrated to be dependent on the cooling rate used in the 400°C pre-irradiation anneal, is shown to be dependent on ionisation density in both naturally cooled and slow-cooled samples. Following heavy-charged particle high-ionisation density (HID) irradiation, the temperature of composite peak 5 decreases by ∼5°C and the peak becomes broader. This behaviour is attributed to an increase in the relative intensity of peak 5a (a low-temperature satellite of peak 5). The relative intensity of peak 5a is estimated using a computerised glow curve deconvolution code based on first-order kinetics. The analysis uses kinetic parameters for peaks 4 and 5 determined from ancillary measurements resulting in nearly ‘single-glow peak’ curves for both the peaks. In the slow-cooled samples, owing to the increased relative intensity of peak 5a compared with the naturally cooled samples, the precision of the measurement of the 5a/5 intensity ratio is found to be ∼15 % (1 SD) compared with ∼25 % for the naturally cooled samples. The ratio of peak 5a/5 in the slow-cooled samples is found to increase systematically and gradually through a variety of radiation fields from a minimum value of 0.13±0.02 for 90Sr/90Y low-ionisation density irradiations to a maximum value of ∼0.8 for 20 MeV Cu and I ion HID irradiations. Irradiation by low-energy electrons of energy 0.1–1.5 keV results in values between 1.27 and 0.95, respectively. The increasing values of the ratio of peak 5a/5 with increasing ionisation density demonstrate the viability of the concept of the peak 5a/5 nanodosemeter and its potential in the measurement of average ionisation density in a ‘nanoscopic’ mass containing the trapping centre/luminescent centre spatially correlated molecule giving rise to composite peak 5. PMID:21149323

  3. Thermoluminescence solid-state nanodosimetry--the peak 5A/5 dosemeter.

    PubMed

    Fuks, E; Horowitz, Y S; Horowitz, A; Oster, L; Marino, S; Rainer, M; Rosenfeld, A; Datz, H

    2011-02-01

    The shape of composite peak 5 in the glow curve of LiF:Mg,Ti (TLD-100) following (90)Sr/(90)Y beta irradiation, previously demonstrated to be dependent on the cooling rate used in the 400°C pre-irradiation anneal, is shown to be dependent on ionisation density in both naturally cooled and slow-cooled samples. Following heavy-charged particle high-ionisation density (HID) irradiation, the temperature of composite peak 5 decreases by ∼5°C and the peak becomes broader. This behaviour is attributed to an increase in the relative intensity of peak 5a (a low-temperature satellite of peak 5). The relative intensity of peak 5a is estimated using a computerised glow curve deconvolution code based on first-order kinetics. The analysis uses kinetic parameters for peaks 4 and 5 determined from ancillary measurements resulting in nearly 'single-glow peak' curves for both the peaks. In the slow-cooled samples, owing to the increased relative intensity of peak 5a compared with the naturally cooled samples, the precision of the measurement of the 5a/5 intensity ratio is found to be ∼15% (1 SD) compared with ∼25% for the naturally cooled samples. The ratio of peak 5a/5 in the slow-cooled samples is found to increase systematically and gradually through a variety of radiation fields from a minimum value of 0.13±0.02 for (90)Sr/(90)Y low-ionisation density irradiations to a maximum value of ∼0.8 for 20 MeV Cu and I ion HID irradiations. Irradiation by low-energy electrons of energy 0.1-1.5 keV results in values between 1.27 and 0.95, respectively. The increasing values of the ratio of peak 5a/5 with increasing ionisation density demonstrate the viability of the concept of the peak 5a/5 nanodosemeter and its potential in the measurement of average ionisation density in a 'nanoscopic' mass containing the trapping centre/luminescent centre spatially correlated molecule giving rise to composite peak 5. PMID:21149323

  4. Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways

    SciTech Connect

    Samarzija, Ivana; Sini, Patrizia; Schlange, Thomas; MacDonald, Gwen; Hynes, Nancy E.

    2009-08-28

    Untangling the signaling pathways involved in endothelial cell biology is of central interest for the development of antiangiogenesis based therapies. Here we report that Wnt3a induces the proliferation and migration of HUVECs, but does not affect their survival. Wnt3a-induced proliferation was VEGFR signaling independent, but reduced upon CamKII inhibition. In a search for the downstream mediators of Wnt3a's effects on HUVEC biology, we found that Wnt3a treatment leads to phosphorylation of DVL3 and stabilization of {beta}-catenin. Moreover, under the same conditions we observed an upregulation in c-MYC, TIE-2 and GJA1 mRNA transcripts. Although treatment of HUVECs with Wnt5a induced DVL3 phosphorylation, we did not observe any of the other effects seen upon Wnt3a stimulation. Taken together, our data indicate that Wnt3a induces canonical and non-canonical Wnt signaling in HUVECs, and stimulates their proliferation and migration.

  5. Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development

    PubMed Central

    Belda-Palazón, Borja; Almendáriz, Carla; Martí, Esmeralda; Carbonell, Juan; Ferrando, Alejandro

    2016-01-01

    One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of eIF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development. PMID:26973686

  6. Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation

    PubMed Central

    Yu, Jian; Chen, Liguang; Cui, Bing; Widhopf, George F.; Shen, Zhouxin; Wu, Rongrong; Zhang, Ling; Zhang, Suping; Briggs, Steven P.; Kipps, Thomas J.

    2015-01-01

    Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation. PMID:26690702