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Sample records for 6p isoforms inhibit

  1. The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity.

    PubMed

    Megarity, Clare F; Looi, Hong Keat; Timson, David J

    2014-08-01

    Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p. PMID:24866129

  2. Isoform-selective Inhibition of Facilitative Glucose Transporters

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Tzekov, Anatoly; Wildman, Scott A.; Hruz, Paul W.

    2014-01-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  3. Nitric oxide synthases activation and inhibition by metallacarborane cluster-based isoform-specific affectors

    PubMed Central

    Kaplánek, Robert; Martásek, Pavel; Grüner, Bohumír; Panda, Satya; Rak, Jakub; Masters, Bettie Sue Siler; Král, Vladimír; Roman, Linda J.

    2012-01-01

    A small library of boron cluster and metallacarborane cluster-based ligands was designed, prepared and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. Based on the concept of creating a hydrophobic analog of a natural substrate, a stable and non-toxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and non-classical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms. PMID:23075390

  4. Inhibition of various isoforms of rat liver glutathione S-transferases by tannic acid and butein.

    PubMed

    Zhang, K; Mack, P; Wong, K P

    1997-07-01

    Glutathione S-transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S-hexylglutathione affinity chromatography and six isoforms, namely C-1, C-2, C-3, C-4, A-2 and A-1, were isolated by CM-cellulose and DEAE-cellulose ion-exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C-2, C-3 and C-4 with IC50 values of 6.8, 8.5 and 8.0 muM respectively. All the isoforms showed lower activity towards p-nitrobenzyt chloride when compared to CDNB and inhibition of the p-nitrobenzyl chloride-activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5. PMID:19856286

  5. Comparison of inhibition capability of scutellarein and scutellarin towards important liver UDP-glucuronosyltransferase (UGT) isoforms.

    PubMed

    Ma, Guang-You; Cao, Yun-Feng; Hu, Cui-Min; Fang, Zhong-Ze; Sun, Xiao-Yu; Hong, Mo; Zhu, Zhi-Tu

    2014-03-01

    Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. PMID:23620377

  6. Characterization of a saporin isoform with lower ribosome-inhibiting activity.

    PubMed Central

    Fabbrini, M S; Rappocciolo, E; Carpani, D; Solinas, M; Valsasina, B; Breme, U; Cavallaro, U; Nykjaer, A; Rovida, E; Legname, G; Soria, M R

    1997-01-01

    We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem. 232, 165-171]. Here we report that SAP-C bound to alpha2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands. PMID:9148741

  7. Mitochondrial Complex 1 Inhibition Increases 4-Repeat Isoform Tau by SRSF2 Upregulation

    PubMed Central

    De Andrade, Anderson; Höglinger, Günter

    2014-01-01

    Progressive Supranuclear Palsy (PSP) is a neurodegenerative disorder characterised by intracellular aggregation of the microtubule-associated protein tau. The tau protein exists in 6 predominant isoforms. Depending on alternative splicing of exon 10, three of these isoforms have four microtubule-binding repeat domains (4R), whilst the others only have three (3R). In PSP there is an excess of the 4R tau isoforms, which are thought to contribute significantly to the pathological process. The cause of this 4R increase is so far unknown. Several lines of evidence link mitochondrial complex I inhibition to the pathogenesis of PSP. We demonstrate here for the first time that annonacin and MPP+, two prototypical mitochondrial complex I inhibitors, increase the 4R isoforms of tau in human neurons. We show that the splicing factor SRSF2 is necessary to increase 4R tau with complex I inhibition. We also found SRSF2, as well as another tau splicing factor, TRA2B, to be increased in brains of PSP patients. Thereby, we provide new evidence that mitochondrial complex I inhibition may contribute as an upstream event to the pathogenesis of PSP and suggest that splicing factors may represent an attractive therapeutic target to intervene in the disease process. PMID:25402454

  8. Isoform-specific inhibition of ROR alpha-mediated transcriptional activation by human FOXP3.

    PubMed

    Du, Jianguang; Huang, Chunjian; Zhou, Baohua; Ziegler, Steven F

    2008-04-01

    FOXP3 is a forkhead family transcriptional repressor important for the development and function of CD4(+)CD25(+) regulatory T cells. In humans, FOXP3 is expressed as two isoforms, a full-length form and a smaller form lacking exon 2. These two isoforms are expressed in approximately equal amounts in circulating regulatory T cells, and are induced equally in freshly activated CD4(+)CD25(-) T cells. Herein, we show that FOXP3 interacts with retinoic acid receptor-related orphan receptor (ROR)alpha, and that this interaction inhibits transcriptional activation mediated by RORalpha. Full-length FOXP3, but not the isoform lacking exon 2, interacts with RORalpha, and the region of FOXP3 involved in the interaction is encoded by exon 2. Mutation of the LxxLL motif in FOXP3, located in exon 2, abolished interaction and repression by FOXP3. Additionally, the inhibition of RORalpha by FOXP3 does not require an intact forkhead domain, demonstrating a mode of FOXP3 function that is independent of DNA binding. Interestingly, expression of RORalpha in T cells leads to the expression of genes that define Th17 cells, and the expression of each of these gene was inhibited by coexpression of full-length, but not DeltaEx2, FOXP3. These data expand the possible targets of FOXP3-mediated repression and demonstrate functional differences between FOXP3 isoforms. PMID:18354202

  9. VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

    SciTech Connect

    Gu, Fang; Li, Xiuli; Kong, Jian; Pan, Bing; Sun, Min; Zheng, Lemin; Yao, Yuanqing

    2013-11-08

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA{sub 3.1} empty vector, pcDNA{sub 3.1}-VEGF111b or pcDNA{sub 3.1}-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

  10. In vitro inhibition of multiple cytochrome P450 isoforms by xanthone derivatives from mangosteen extract.

    PubMed

    Foti, Robert S; Pearson, Josh T; Rock, Dan A; Wahlstrom, Jan L; Wienkers, Larry C

    2009-09-01

    Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9. PMID:19541824

  11. Inhibition of mammalian carbonic anhydrase isoforms I-XIV with a series of phenolic acid esters.

    PubMed

    Maresca, Alfonso; Akyuz, Gulay; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T

    2015-11-15

    A series of phenolic acid esters incorporating caffeic, ferulic, and p-coumaric acid, and benzyl, m/p-hydroxyphenethyl- as well as p-hydroxy-phenethoxy-phenethyl moieties were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Many of the mammalian isozymes of human (h) or murine (m) origin, hCA I-hCA XII, mCA XIII and hCA XIV, were inhibited in the submicromolar range by these derivatives (with KIs of 0.31-1.03 μM against hCA VA, VB, VI, VII, IX and XIV). The off-target, highly abundant isoforms hCA I and II, as well as hCA III, IV and XII were poorly inhibited by many of these esters, although the original phenolic acids were micromolar inhibitors. These phenols, like others investigated earlier, possess a CA inhibition mechanism distinct of the sulfonamides/sulfamates, clinically used drugs for the treatment of a multitude of pathologies, but with severe side effects due to hCA I/II inhibition. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule, binding more externally within the active site cavity, and making contacts with amino acid residues at the entrance of the active site. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds shows isoform-selective inhibitory profiles, which may be exploited for obtaining pharmacological agents with less side effects compared to other classes of inhibitors. PMID:26498394

  12. A splicing isoform of TEAD4 attenuates the Hippo–YAP signalling to inhibit tumour proliferation

    PubMed Central

    Qi, Yangfan; Yu, Jing; Han, Wei; Fan, Xiaojuan; Qian, Haili; Wei, Huanhuan; Tsai, Yi-hsuan S.; Zhao, Jinyao; Zhang, Wenjing; Liu, Quentin; Meng, Songshu; Wang, Yang; Wang, Zefeng

    2016-01-01

    Aberrant splicing is frequently found in cancer, yet the biological consequences of such alterations are mostly undefined. Here we report that the Hippo–YAP signalling, a key pathway that regulates cell proliferation and organ size, is under control of a splicing switch. We show that TEAD4, the transcription factor that mediates Hippo–YAP signalling, undergoes alternative splicing facilitated by the tumour suppressor RBM4, producing a truncated isoform, TEAD4-S, which lacks an N-terminal DNA-binding domain, but maintains YAP interaction domain. TEAD4-S is located in both the nucleus and cytoplasm, acting as a dominant negative isoform to YAP activity. Consistently, TEAD4-S is reduced in cancer cells, and its re-expression suppresses cancer cell proliferation and migration, inhibiting tumour growth in xenograft mouse models. Furthermore, TEAD4-S is reduced in human cancers, and patients with elevated TEAD4-S levels have improved survival. Altogether, these data reveal a splicing switch that serves to fine tune the Hippo–YAP pathway. PMID:27291620

  13. Distinct CD55 Isoform Synthesis and Inhibition of Complement-Dependent Cytolysis by Hepatitis C Virus.

    PubMed

    Kwon, Young-Chan; Kim, Hangeun; Meyer, Keith; Di Bisceglie, Adrian M; Ray, Ranjit

    2016-08-15

    CD55/DAF, one of the regulators of complement activation, is known to limit excess complement activation on the host cell surface by accelerating the decay of C3 convertase. We reported previously that hepatitis C virus (HCV) infection or virus core protein expression upregulates CD55 expression. CD55 associates with HCV particles, potentially protecting HCV from lysis in circulation. An increase in CD55 on the surface of HCV-infected cells may inhibit complement-mediated cell killing. In this study, we show that Abs against cancer cell surface proteins induce complement-dependent cytolysis or Ab-dependent cell-mediated cytotoxicity of immortalized human hepatocytes in the presence of CD55-blocking Ab. CD55 has a secreted isoform (sCD55) that is generated by alternative splicing. We observed that sCD55 is induced in HCV-infected or HCV replicon-harboring cells, as well as in liver biopsy samples from chronically HCV-infected patients. Conditioned medium from HCV-infected hepatoma cells (Huh7.5 cells) or immortalized human hepatocytes inhibited C3 convertase activity and complement-dependent cytolysis of sheep blood erythrocytes. Chronically HCV-infected patient sera inhibited C3 convertase activity, further implicating HCV-specific impairment of complement function in infected humans. CD55-blocking Ab inhibited erythrocyte lysis by conditioned medium, suggesting that CD55/sCD55 impairs convertase activity. Together, our data show that HCV infection induces sCD55 expression in HCV-infected cell culture-conditioned medium and inhibits C3 convertase activity. This may have implications for modulating complement-mediated immune function in the microenvironment and on HCV-harboring cells. PMID:27357152

  14. Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII.

    PubMed

    Ibrahim, Hany S; Abou-Seri, Sahar M; Tanc, Muhammet; Elaasser, Mahmoud M; Abdel-Aziz, Hatem A; Supuran, Claudiu T

    2015-10-20

    New series of benzenesulfonamide derivatives incorporating pyrazole and isatin moieties were prepared using celecoxib as lead molecule. Biological evaluation of the target compounds was performed against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely against the human isoforms hCA I, II (cytosolic), IX and XII (transmembrane, tumor-associated enzymes). Most of the tested compounds efficiently inhibited hCA I, II and IX, with KIs of 2.5-102 nM, being more effective than the reference drug acetazolamide. Compounds 11e, 11f, 16e and 16f were found to inhibit hCA XII with Ki of 3.7, 6.5, 5.4 and 7.2 nM, respectively. Compounds 11e and 16e, with 5-NO2 substitution on the isatin ring, were found to be selective inhibitors of hCA IX and hCA XII. Docking studies revealed that the NO2 group of both compounds participate in interactions with Asp132 within the hCA IX active site, and with residues Lys67 and Asp130 in hCA XII, respectively. PMID:26408817

  15. Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

    PubMed

    Gong, Yan; Bourhis, Eric; Chiu, Cecilia; Stawicki, Scott; DeAlmeida, Venita I; Liu, Bob Y; Phamluong, Khanhky; Cao, Tim C; Carano, Richard A D; Ernst, James A; Solloway, Mark; Rubinfeld, Bonnee; Hannoush, Rami N; Wu, Yan; Polakis, Paul; Costa, Mike

    2010-01-01

    β-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for

  16. Genetic deletion and pharmacological inhibition of Akt1 isoform attenuates bladder cancer cell proliferation, motility and invasion.

    PubMed

    Sabbineni, Harika; Alwhaibi, Abdulrahman; Goc, Anna; Gao, Fei; Pruitt, Alanna; Somanath, Payaningal R

    2015-10-01

    Isoform specific expression, intracellular localization and function of Akt in bladder cancer are not known. In the current study, we identified Akt1, followed by Akt2 and Akt3 as the predominant Akt isoform in human T24 and UM-UC-3 metastatic bladder cancer cells. Whereas Akt1 is localized at the membrane, cytoplasm and nucleus, Akt2 is solely cytoplasmic and Akt3 is mostly localized in the nucleus in T24 cells. ShRNA-mediated Akt1 knockdown resulted in impaired T24 cell survival, proliferation, colony formation, migration and microinvasion. Whereas pharmacological inhibition of Akt1 resulted in impaired T24 and UM-UC-3 cell motility, viability and proliferation, effect of pharmacological inhibition by Akt2 inhibitor was limited to proliferation in T24, but not UM-UC-3 cells. Our data provide important clues on the therapeutic benefits of targeting Akt1 for bladder cancer therapy. PMID:26148825

  17. Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform.

    PubMed

    Bau, Jason T; Kang, Zhili; Austin, Caroline A; Kurz, Ebba U

    2014-02-01

    Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity. PMID:24220011

  18. Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

    PubMed

    Luttgeharm, Kyle D; Cahoon, Edgar B; Markham, Jonathan E

    2016-03-01

    Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism. PMID:26635357

  19. Dynamic and Static Simulations of Fluvoxamine-Perpetrated Drug-Drug Interactions Using Multiple Cytochrome P450 Inhibition Modeling, and Determination of Perpetrator-Specific CYP Isoform Inhibition Constants and Fractional CYP Isoform Contributions to Victim Clearance.

    PubMed

    Iga, Katsumi

    2016-03-01

    Fluvoxamine-perpetrated drug-drug interactions (DDIs) of victims metabolized by multiple cytochrome P450 isoforms (CYP1A2, CYP2C19, and CYP3A4) were simulated using 2 compartment-based tube modeling, assuming a multiple inhibition-constant (Ki) model, as well as a previously reported single Ki model. Good fittings were obtained for all DDIs using consistent perpetrator-specific CYP isoform Kis and fractional CYP isoform contributions to victim clearance in concordance with literature information. Through these simulations, the following rules to predict DDI were derived. Overall enzymatic inhibitory activity calculated from static DDI data determines entirely dynamic DDIs. DDI-relevant time-dependent hepatic blood unbound perpetrator levels can be approximated to mean hepatic blood unbound perpetrator levels in any victim DDIs when a perpetrator is supplied consistently. Static and dynamic multiple CYP model-based simulations agree with one another. Fluvoxamine-perpetrated DDIs can be bridged to other perpetrator DDIs. The derived rules will allow simpler prediction of DDIs from in vivo DDI databases. Tens or hundreds of Ki gaps between in vitro and in vivo data could be reduced to within severalfold using the liver-microsome contamination model, thus suggesting that microsomes qualified with contamination would greatly improve prediction of DDIs from in vitro data. PMID:26886336

  20. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

    PubMed

    Göckeritz, Elisa; Kerwien, Susan; Baumann, Michael; Wigger, Marion; Vondey, Verena; Neumann, Lars; Landwehr, Thomas; Wendtner, Clemens M; Klein, Christian; Liu, Ningshu; Hallek, Michael; Frenzel, Lukas P; Krause, Günter

    2015-11-01

    Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation. PMID:25912635

  1. Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

    PubMed

    Ahuja, Shivani; Mukund, Susmith; Deng, Lunbin; Khakh, Kuldip; Chang, Elaine; Ho, Hoangdung; Shriver, Stephanie; Young, Clint; Lin, Sophia; Johnson, J P; Wu, Ping; Li, Jun; Coons, Mary; Tam, Christine; Brillantes, Bobby; Sampang, Honorio; Mortara, Kyle; Bowman, Krista K; Clark, Kevin R; Estevez, Alberto; Xie, Zhiwei; Verschoof, Henry; Grimwood, Michael; Dehnhardt, Christoph; Andrez, Jean-Christophe; Focken, Thilo; Sutherlin, Daniel P; Safina, Brian S; Starovasnik, Melissa A; Ortwine, Daniel F; Franke, Yvonne; Cohen, Charles J; Hackos, David H; Koth, Christopher M; Payandeh, Jian

    2015-12-18

    Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists. PMID:26680203

  2. The γ-Protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane

    PubMed Central

    Mah, Kar Men; Houston, Douglas W.; Weiner, Joshua A.

    2016-01-01

    The 22 γ-Protocadherin (γ-Pcdh) adhesion molecules encoded by the Pcdhg gene cluster play critical roles in nervous system development, including regulation of dendrite arborisation, neuronal survival, and synaptogenesis. Recently, they have been implicated in suppression of tumour cell growth by inhibition of canonical Wnt signalling, though the mechanisms through which this occurs remain unknown. Here, we show differential regulation of Wnt signalling by individual γ-Pcdhs: The C3 isoform uniquely inhibits the pathway, whilst 13 other isoforms upregulate signalling. Focusing on the C3 isoform, we show that its unique variable cytoplasmic domain (VCD) is the critical one for Wnt pathway inhibition. γ-Pcdh-C3, but not other isoforms, physically interacts with Axin1, a key component of the canonical Wnt pathway. The C3 VCD competes with Dishevelled for binding to the DIX domain of Axin1, which stabilizes Axin1 at the membrane and leads to reduced phosphorylation of Wnt co-receptor Lrp6. Finally, we present evidence that Wnt pathway activity can be modulated up (by γ-Pcdh-A1) or down (by γ-Pcdh-C3) in the cerebral cortex in vivo, using conditional transgenic alleles. Together, these data delineate opposing roles for γ-Pcdh isoforms in regulating Wnt signalling and identify Axin1 as a novel protein interactor of the widely-expressed γ-Pcdh-C3 isoform. PMID:27530555

  3. HPV E6/p53 mediated down-regulation of miR-34a inhibits Warburg effect through targeting LDHA in cervical cancer

    PubMed Central

    Zhang, Rong; Su, Jing; Xue, Song-Lin; Yang, Hui; Ju, Li-Li; Ji, Ying; Wu, Kai-Hua; Zhang, Yan-Wei; Zhang, Ye-Xin; Hu, Jian-Fang; Yu, Min-Min

    2016-01-01

    MicroRNAs (miRNA) play crucial roles in regulating cell proliferation, differentiation and developmental timing. Aberrantly expressed miRNAs have recently emerged as key regulators of metabolism. However, little is known about its role in tumor metabolism of cervical cancer. In this study, we determined the oncogenic effects of miRNAs on Warburg effect, a metabolic phenotype that allows cancer cells to utilize glucose even under aerobic conditions. A gain-of-function study was performed in 12 down-regulated miRNAs that frequently reported in cervical cancer. We found that miR-34a plays a suppressive role in Warburg effect as evidenced by decreased lactate production and glucose consumption. Knockdown of oncoprotein E6 expression of human papillomavirus in SiHa and HeLa cells by siRNAs lead to an increased protein level of p53, decreased level of miR-34a, as well as reduced Warburg effect. Subsequently, lactate dehydrogenase A (LDHA), which catalyzes the last key step in glycolysis, was identified as a direct target of miR-34a. Silencing of LDHA or introduction of miR-34a significantly attenuated colony formation ability and invasive capacity of SiHa and HeLa cells, and these effects were fully compromised by reintroduction of LDHA. In conclusion, our findings demonstrated that deregulated miR-34a/LDHA axis induced by HPV E6/p53 signaling facilitates tumor growth and invasion through regulating Warburg effect in cervical cancer, and provided new insights into the mechanism by which miR-34a contributes to the development and progression of cervical cancer.

  4. Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

    PubMed

    Peng, Sheng-Bin; Henry, James R; Kaufman, Michael D; Lu, Wei-Ping; Smith, Bryan D; Vogeti, Subha; Rutkoski, Thomas J; Wise, Scott; Chun, Lawrence; Zhang, Youyan; Van Horn, Robert D; Yin, Tinggui; Zhang, Xiaoyi; Yadav, Vipin; Chen, Shih-Hsun; Gong, Xueqian; Ma, Xiwen; Webster, Yue; Buchanan, Sean; Mochalkin, Igor; Huber, Lysiane; Kays, Lisa; Donoho, Gregory P; Walgren, Jennie; McCann, Denis; Patel, Phenil; Conti, Ilaria; Plowman, Gregory D; Starling, James J; Flynn, Daniel L

    2015-09-14

    LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation. PMID:26343583

  5. Isoform-selective inhibition of facilitative glucose transporters: elucidation of the molecular mechanism of HIV protease inhibitor binding.

    PubMed

    Hresko, Richard C; Kraft, Thomas E; Tzekov, Anatoly; Wildman, Scott A; Hruz, Paul W

    2014-06-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  6. Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3.

    PubMed

    Akhter, S; Nath, S K; Tse, C M; Williams, J; Zasloff, M; Donowitz, M

    1999-01-01

    Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 micrograms/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator. PMID:9886929

  7. Selective inhibition of tumor cell associated Vacuolar-ATPase 'a2' isoform overcomes cisplatin resistance in ovarian cancer cells.

    PubMed

    Kulshrestha, Arpita; Katara, Gajendra K; Ginter, Jordyn; Pamarthy, Sahithi; Ibrahim, Safaa A; Jaiswal, Mukesh K; Sandulescu, Corina; Periakaruppan, Ramayee; Dolan, James; Gilman-Sachs, Alice; Beaman, Kenneth D

    2016-06-01

    Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its 'a2' isoform (V-ATPase-V0a2) is a component of functional plasma-membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the 'a' subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo

  8. Inhibition of the synthesis of a cytochrome-c-oxidase subunit isoform by antisense RNA.

    PubMed

    Sandonà, D; Bisson, R

    1994-02-01

    To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictyostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity. PMID:8112318

  9. Inhibition of UDP-Glucuronosyltransferase (UGT) Isoforms by Arctiin and Arctigenin.

    PubMed

    Zhang, Hui; Zhao, Zhenying; Wang, Tao; Wang, Yijia; Cui, Xiao; Zhang, Huijuan; Fang, Zhong-Ze

    2016-07-01

    Arctiin is the major pharmacological ingredient of Fructus Arctii, and arctigenin is the metabolite of arctiin formed via the catalysis of human intestinal bacteria. The present study aims to investigate the inhibition profile of arctiin and arctigenin on important phase II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGTs), indicating the possible herb-drug interaction. In vitro screening experiment showed that 100 μM of arctiin and arctigenin inhibited the activity of UGT1A3, 1A9, 2B7, and 2B15. Homology modeling-based in silico docking of arctiin and arctigenin into the activity cavity of UGT2B15 showed that hydrogen bonds and hydrophobic interactions contributed to the strong binding free energy of arctiin (-8.14 kcal/mol) and arctigenin (-8.43 kcal/mol) with UGT2B15. Inhibition kinetics study showed that arctiin and arctigenin exerted competitive and noncompetitive inhibition toward UGT2B15, respectively. The inhibition kinetic parameters (Ki ) were calculated to be 16.0 and 76.7 μM for the inhibition of UGT2B15 by arctiin and arctigenin, respectively. Based on the plasma concentration of arctiin and arctigenin after administration of 100 mg/kg of arctiin, the [I]/Ki values were calculated to be 0.3 and 0.007 for arctiin and arctigenin, respectively. Based on the inhibition evaluation standard ([I]/Ki  < 0.1, low possibility; 0.1 < [I]/Ki  < 1, medium possibility; [I]/Ki  > 1, high possibility), arctiin might induce drug-drug interaction with medium possibility. Based on these results, clinical monitoring the utilization of Fructus Arctii is very important and necessary. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145339

  10. Selective inhibition of the inducible isoform of nitric oxide synthase prevents pulmonary transvascular flux during acute endotoxemia.

    PubMed

    Arkovitz, M S; Wispé, J R; Garcia, V F; Szabó, C

    1996-08-01

    The inducible isoform of nitric oxide synthase (iNOS) is expressed in various organs, including the lung, during systemic endotoxemia. Overproduction of nitric oxide (NO) by iNOS contributes significantly to the vascular failure and end-organ damage in endotoxemia. Using selective pharmacological inhibitors of iNOS, the purpose of this study was to define the role of iNOS in a rat model of endotoxin-induced pulmonary transvascular flux (TVF). Lung TVF was assessed by a method of Evans Blue permeability index (PI). Bacterial lipopolysaccharide (LPS) (15 mg/kg intraperitoneally [IP]) significantly increased pulmonary iNOS activity and serum levels of nitrite/nitrate (NO2/NO3). This was accompanied by a significant elevation of the PI 5 hours after injection. Selective iNOS inhibition with either S-methyl isothiourea (SMT; 5 mg/kg IP) or aminoguanidine (AG; 20 mg/kg IP), administered 2 hours after LPS injection, significantly prevented the increase in PI associated with LPS injection. Similarly, inhibition of the induction of iNOS with dexamethasone (10 mg/kg IP), given 3 hours before LPS, also inhibited the increase in PI. All three treatments significantly prevented the increase in both lung iNOS activity and serum NO2/NO3 associated with endotoxemia. In conclusion, the overproduction of NO generated by iNOS during systemic endotoxemia causes a vascular leak in the lung. Thus, it is speculated that selective inhibition of iNOS may be beneficial in preventing the development of acute respiratory failure in sepsis. PMID:8863222

  11. Comparative evaluation of 12 immature citrus fruit extracts for the inhibition of cytochrome P450 isoform activities.

    PubMed

    Fujita, Tadashi; Kawase, Atsushi; Niwa, Toshiro; Tomohiro, Norimichi; Masuda, Megumi; Matsuda, Hideaki; Iwaki, Masahiro

    2008-05-01

    In a previous study we found that 50% ethanol extracts of immature fruits of Citrus unshiu (satsuma mandarin) have anti-allergic effects against the Type I, II and IV allergic reactions. However, many adverse interactions between citrus fruit, especially grapefruit juice, and drugs have been reported due to the inhibition of cytochrome P450 (CYP) activities. The purpose of this study was to examine the competitive inhibitory effects of extracts from immature citrus fruit on CYP activity. Extracts were prepared from 12 citrus species or cultivars, and were tested against three kinds of major CYPs, CYP2C9, CYP2D6 and CYP3A4, in human liver microsomes. We also estimated the amounts of flavonoids (narirutin, hesperidin, naringin and neohesperidin) and furanocoumarins (bergapten, 6',7'-dihydroxybergamottin and bergamottin) in each extract using HPLC. Citrus paradisi (grapefruit) showed the greatest inhibition of CYP activities, while Citrus unshiu which has an antiallergic effect, showed relatively weak inhibitory effects. Extracts having relatively strong inhibitory effects for CYP3A4 tended to contain higher amounts of naringin, bergamottin and 6',7'-dihydroxybergamottin. These results, providing comparative information on the inhibitory effects of citrus extracts on CYP isoforms, suggest that citrus extracts containing high levels of narirutin and hesperidin and lower levels of furanocoumarins such as C. unshiu are favorable as antiallergic functional ingredients. PMID:18451520

  12. Inhibition of PaCaMKII-E isoform in the dorsal unpaired median neurosecretory cells of cockroach reduces nicotine- and clothianidin-induced currents.

    PubMed

    List, Olivier; Calas-List, Delphine; Taillebois, Emiliane; Juchaux, Marjorie; Heuland, Emilie; Thany, Steeve H

    2014-08-01

    Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), which transduces the signal into downstream effects. We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms, and only PaCaMKII-E isoform is specifically expressed in the dorsal unpaired median neurosecretory cells. In the present study, using antisense oligonucleotides, we demonstrated that PaCaMKII-E isoform inhibition reduced nicotine-induced currents through α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptor subtypes. Specifically, PaCaMKII-E isoform is sufficient to repress nicotinic current amplitudes as a result of its depression by antisense oligonucleotides. Similar results were found using the neonicotinoid insecticide clothianidin, which acted as a full agonist of dorsal unpaired median neuron nicotinic acetylcholine receptors. Clothianidin current amplitudes are strongly reduced under bath application of PaCaMKII-E antisense oligonucleotides but no significant results are found with α-bungarotoxin co-applied, demonstrating that CaMKII-E isoform affects nicotine currents through α-bungarotoxin-sensitive and -insensitive receptor subtypes whereas clothianidin currents are reduced via α-bungarotoxin-insensitive receptors. In addition, we found that intracellular calcium increase induced by nicotine and clothianidin were reduced by PaCaMKII-E antisense oligonucleotides, demonstrating that intracellular calcium increase induced by nicotine and clothianidin are affected by PaCaMKII-E inhibition. Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII). We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms and only PaCaMKII-E isoform was specifically expressed in the dorsal unpaired median neurosecretory cells. Here we show that specific inhibition of PaCaMKII-E isoform is

  13. Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

    PubMed

    Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

    2016-11-01

    The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP. PMID:27530298

  14. Purified canola lutein selectively inhibits specific isoforms of mammalian DNA polymerases and reduces inflammatory response.

    PubMed

    Horie, Sho; Okuda, Chiaki; Yamashita, Takatoshi; Watanabe, Kenichi; Kuramochi, Kouji; Hosokawa, Masashi; Takeuchi, Toshifumi; Kakuda, Makiko; Miyashita, Kazuo; Sugawara, Fumio; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2010-08-01

    In the screening of DNA polymerase (pol) inhibitor, we isolated lutein, a carotenoid, from the crude (unrefined) pressed oil of canola (low erucic acid rapeseed, Brassica napus L.). Commercially prepared carotenoids such as lutein (1), zeaxanthin (2), beta-cryptoxanthin (3), astaxanthin (4), canthaxanthin (5), beta-carotene (6), lycopene (7), capsanthin (8), fucoxanthin (9) and fucoxanthinol (10), were investigated for the inhibitory activities of pols. Compounds 1, 2 and 8 exhibited strong inhibition of the activities of mammalian pols beta and lambda, which are DNA repair- and/or recombination-related pols. On the other hand, all carotenoids tested had no influence on the activity of a mammalian pol alpha, which is a DNA replicative pol. Lutein (1) was the strongest pol inhibitor of mammalian pols beta and lambda in the prepared ten carotenoids tested, but did not influence of the activities of mammalian pols alpha, gamma, delta and epsilon. The tendency for pols beta and lambda inhibition by these carotenoids showed a positive correlation with the suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation. These results suggest that cold pressed unrefined canola/rapeseed oil, or other oils with high levels of lutein and other carotenoids, may be useful for their anti-inflammatory properties. PMID:20669052

  15. Microwave assisted synthesis of novel acridine-acetazolamide conjugates and investigation of their inhibition effects on human carbonic anhydrase isoforms hCA I, II, IV and VII.

    PubMed

    Ulus, Ramazan; Aday, Burak; Tanç, Muhammet; Supuran, Claudiu T; Kaya, Muharrem

    2016-08-15

    4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide was condensed with cyclic-1,3-diketones (dimedone and cyclohexane-1,3-dione) and aromatic aldehydes under microwave irradiation, leading to a series of acridine-acetazolamide conjugates. The new compounds were investigated as inhibitors of carbonic anhydrases (CA, EC 4.2.1.1), and more precisely cytosolic isoforms hCA I, II, VII and membrane-bound one hCA IV. All investigated isoforms were inhibited in low micromolar and nanomolar range by the new compounds. hCA IV and VII were inhibited with KIs in the range of 29.7-708.8nM (hCA IV), and of 1.3-90.7nM (hCA VII). For hCA I and II the KIs were in the range of 6.7-335.2nM (hCA I) and of 0.5-55.4nM (hCA II). The structure-activity relationships (SAR) for the inhibition of these isoforms with the acridine-acetazolamide conjugates reported here were delineated. PMID:27298005

  16. 7,3',4'-Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase.

    PubMed

    Lee, Dong Eun; Lee, Ki Won; Song, Nu Ry; Seo, Sang Kwon; Heo, Yong-Seok; Kang, Nam Joo; Bode, Ann M; Lee, Hyong Joo; Dong, Zigang

    2010-07-01

    Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents. PMID:20444693

  17. Sodium pump isoform specificity for the digitalis-like factor isolated from human peritoneal dialysate.

    PubMed

    Tao, Q F; Hollenberg, N K; Price, D A; Graves, S W

    1997-03-01

    We have isolated a labile, specific sodium pump inhibitor or digitalis-like factor from the peritoneal dialysate of volume-expanded renal failure patients whose levels correlated closely with volume status and blood pressure. This study characterizes the inhibitory profile of this agent compared with that of ouabain against the three alpha-isoforms of the sodium pump. We prepared microsomal Na,K-ATPase from rat tissues representing the highest proportion of one of the alpha-isoforms. Both Northern and Western blot analyses confirmed that kidney had predominantly the alpha1-isoform, skeletal muscle the alpha2-isoform, and fetal brain the alpha3-isoform. Ouabain (5 x 10(-6) mol/L) produced little inhibition of kidney Na,K-ATPase (3.4+/-2.0%) but significant inhibition of skeletal muscle (37.2+/-3.7%, P<.001) and fetal brain (38.8+/-3.5%, P<.001) activity. In contrast, the labile digitalis-like factor, causing comparable inhibition of fetal brain Na,K-ATPase activity (33.3+/-4.7%), produced markedly greater inhibition of kidney (42.5+/-5.6%, P<.001) and moderately greater inhibition of skeletal muscle pump activity (57.7+/-6.3%, P<.05). In addition, the labile digitalis-like factor produced a marked concentration-dependent inhibition of the alpha2- and alpha3-isoforms (r=.79, P=.00005). Experiments combining the labile digitalis-like factor and ouabain confirmed that digitalis-like factor, unlike ouabain, was an effective inhibitor of all three isoforms in rat, in particular alpha2. The different pattern of isoform sensitivity displayed by the labile digitalis-like factor and ouabain further differentiates the two agents and raises some interesting possibilities about the functional implications of the endogenous factor. PMID:9052901

  18. Novel, isoform-selective, cholecystokinin A receptor antagonist inhibits colon and pancreatic cancers in preclinical models through novel mechanism of action.

    PubMed

    Ponnusamy, Suriyan; Lattmann, Eric; Lattmann, Pornthip; Thiyagarajan, Thirumagal; Padinjarethalakal, Balaram N; Narayanan, Ramesh

    2016-04-01

    Colon and pancreatic cancers contribute to 90,000 deaths each year in the USA. These cancers lack targeted therapeutics due to heterogeneity of the disease and multiple causative factors. One important factor that contributes to increased colon and pancreatic cancer risk is gastrin. Gastrin mediates its actions through two G-protein coupled receptors (GPCRs): cholecystokinin receptor A (CCK-A) and CCK-B/gastrin receptor. Previous studies have indicated that colon cancer predominantly expresses CCK-A and responds to CCK-A isoform antagonists. However, many CCK-A antagonists have failed in the clinic due to poor pharmacokinetic properties or lack of efficacy. In the present study, we synthesized a library of CCK-A isoform-selective antagonists and tested them in various colon and pancreatic cancer preclinical models. The lead CCK-A isoform, selective antagonist PNB-028, bound to CCK-A at 12 nM with a 60-fold selectivity towards CCK-A over CCK-B. Furthermore, it inhibited the proliferation of CCK-A-expressing colon and pancreatic cancer cells without affecting the proliferation of non-cancerous cells. PNB-028 was also extremely effective in inhibiting the growth of MAC-16 and LoVo colon cancer and MIA PaCa pancreatic cancer xenografts in immune-compromised mice. Genome‑wide microarray and kinase-array studies indicate that PNB-028 inhibited oncogenic kinases and angiogenic factors to inhibit the growth of colon cancer xenografts. Safety pharmacology and toxicology studies have indicated that PNB-028 is extremely safe and has a wide safety margin. These studies suggest that targeting CCK-A selectively renders promise to treat colon and pancreatic cancers and that PNB-028 could become the next-generation treatment option. PMID:26820391

  19. Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p.

    PubMed

    Jamaluddin, M Fairuz B; Bailey, Ulla-Maja; Tan, Nikki Y J; Stark, Anthony P; Schulz, Benjamin L

    2011-05-01

    Asparagine-linked glycosylation is a common and vital co- and post-translocational modification of diverse secretory and membrane proteins in eukaryotes that is catalyzed by the multiprotein complex oligosaccharyltransferase (OTase). Two isoforms of OTase are present in Saccharomyces cerevisiae, defined by the presence of either of the homologous proteins Ost3p or Ost6p, which possess different protein substrate specificities at the level of individual glycosylation sites. Here we present in vitro characterization of the polypeptide binding activity of these two subunits of the yeast enzyme, and show that the peptide-binding grooves in these proteins can transiently bind stretches of polypeptide with amino acid characteristics complementary to the characteristics of the grooves. We show that Ost6p, which has a peptide-binding groove with a strongly hydrophobic base lined by neutral and basic residues, binds peptides enriched in hydrophobic and acidic amino acids. Further, by introducing basic residues in place of the wild type neutral residues lining the peptide-binding groove of Ost3p, we engineer binding of a hydrophobic and acidic peptide. Our data supports a model of Ost3/6p function in which they transiently bind stretches of nascent polypeptide substrate to inhibit protein folding, thereby increasing glycosylation efficiency at nearby asparagine residues. PMID:21384453

  20. Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

    PubMed Central

    Jamaluddin, M Fairuz B; Bailey, Ulla-Maja; Tan, Nikki YJ; Stark, Anthony P; Schulz, Benjamin L

    2011-01-01

    Asparagine-linked glycosylation is a common and vital co- and post-translocational modification of diverse secretory and membrane proteins in eukaryotes that is catalyzed by the multiprotein complex oligosaccharyltransferase (OTase). Two isoforms of OTase are present in Saccharomyces cerevisiae, defined by the presence of either of the homologous proteins Ost3p or Ost6p, which possess different protein substrate specificities at the level of individual glycosylation sites. Here we present in vitro characterization of the polypeptide binding activity of these two subunits of the yeast enzyme, and show that the peptide-binding grooves in these proteins can transiently bind stretches of polypeptide with amino acid characteristics complementary to the characteristics of the grooves. We show that Ost6p, which has a peptide-binding groove with a strongly hydrophobic base lined by neutral and basic residues, binds peptides enriched in hydrophobic and acidic amino acids. Further, by introducing basic residues in place of the wild type neutral residues lining the peptide-binding groove of Ost3p, we engineer binding of a hydrophobic and acidic peptide. Our data supports a model of Ost3/6p function in which they transiently bind stretches of nascent polypeptide substrate to inhibit protein folding, thereby increasing glycosylation efficiency at nearby asparagine residues. PMID:21384453

  1. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression.

    PubMed

    Esber, Nathalie; Le Billan, Florian; Resche-Rigon, Michèle; Loosfelt, Hugues; Lombès, Marc; Chabbert-Buffet, Nathalie

    2015-01-01

    The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required. PMID:26474308

  2. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression

    PubMed Central

    Esber, Nathalie; Le Billan, Florian; Resche-Rigon, Michèle; Loosfelt, Hugues; Lombès, Marc; Chabbert-Buffet, Nathalie

    2015-01-01

    The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required. PMID:26474308

  3. Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    PubMed Central

    Passacquale, Gabriella; Phinikaridou, Alkystis; Warboys, Christina; Cooper, Margaret; Lavin, Begona; Alfieri, Alessio; Andia, Marcelo E; Botnar, Rene M; Ferro, Albert

    2015-01-01

    Background and Purpose There are conflicting data regarding whether netrin-1 retards or accelerates atherosclerosis progression, as it can lead either to monocyte repulsion from or retention within plaques depending on its cellular source. We investigated the effect of aspirin, which is widely used in cardiovascular prophylaxis, on the synthesis of different isoforms of netrin-1 by endothelial cells under pro-inflammatory conditions, and defined the net effect of aspirin-dependent systemic modulation of netrin-1 on atherosclerosis progression. Experimental Approach Netrin-1 synthesis was studied in vitro using human endothelial cells stimulated with TNF-α, with or without aspirin treatment. In vivo experiments were conducted in ApoE−/− mice fed with a high-fat diet (HFD), receiving either aspirin or clopidogrel. Key Results TNF-α-induced NF-κB activation up-regulated the nuclear isoform of netrin-1, while simultaneously reducing secreted netrin-1. Down-regulation of the secreted isoform compromised the chemorepellent action of the endothelium against monocyte chemotaxis. Aspirin counteracted TNF-α-mediated effects on netrin-1 synthesis by endothelial cells through COX-dependent inhibition of NF-κB and concomitant histone hyperacetylation. Administration of aspirin to ApoE−/− mice on HFD increased blood and arterial wall levels of netrin-1 independently of its effects on platelets, accompanied by reduced plaque size and content of monocytes/macrophages, compared with untreated or clopidogrel-treated mice. In vivo blockade of netrin-1 enhanced monocyte plaque infiltration in aspirin-treated ApoE−/− mice. Conclusions and Implications Aspirin counteracts down-regulation of secreted netrin-1 induced by pro-inflammatory stimuli in endothelial cells. The aspirin-dependent increase of netrin-1 in ApoE−/− mice exerts anti-atherogenic effects by preventing arterial accumulation of monocytes. PMID:25824964

  4. Inhibition of Recombinant Cytochrome P450 Isoforms 2D6 and 2C9 by Diverse Drug-like Molecules

    PubMed Central

    McMasters, Daniel R.; Torres, Rhonda A.; Crathern, Susan J.; Dooney, Deborah L.; Nachbar, Robert B.; Sheridan, Robert P.; Korzekwa, Kenneth R.

    2008-01-01

    The affinities of a diverse set of 500 drug-like molecules to cytochrome P450 isoforms 2C9 and 2D6 were measured using recombinant expressed enzyme. The dose–response curve of each compound was fitted with a series of equations representing typical or various types of atypical kinetics. Atypical kinetics was identified where the Akaike Information Criterion, plus other criteria, suggested the kinetics was more complex than expected for a Michaelis–Menten model. Approximately 20% of the compounds were excluded due to poor solubility, and approximately 15% were excluded due to fluorescence interference. Of the remaining compounds, roughly half were observed to bind with an affinity of 200 μM or lower for each of the two isoforms. Atypical kinetics were observed in 18 percent of the compounds that bind to cytochrome 2C9 but less than 2 percent for 2D6. The resulting collection of competitive inhibitors and inactive compounds was analyzed for trends in binding affinity. For CYP2D6, a clear relationship between polar surface area and charge was observed, with the most potent inhibitors having a formal positive charge and a low percent polar surface area. For CYP2C9, no clear trend between activity and physicochemical properties could be seen for the group as a whole; however, certain classes of compounds have altered frequencies of activity and atypical kinetics. PMID:17559204

  5. The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1.

    PubMed

    Voets, Erik; Marsman, Judith; Demmers, Jeroen; Beijersbergen, Roderick; Wolthuis, Rob

    2015-01-01

    Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase. PMID:26423135

  6. Antibody-Mediated Inhibition of the FGFR1c Isoform Induces a Catabolic Lean State in Siberian Hamsters.

    PubMed

    Samms, Ricardo J; Lewis, Jo E; Lory, Alex; Fowler, Maxine J; Cooper, Scott; Warner, Amy; Emmerson, Paul; Adams, Andrew C; Luckett, Jeni C; Perkins, Alan C; Wilson, Dana; Barrett, Perry; Tsintzas, Kostas; Ebling, Francis J P

    2015-11-16

    Hypothalamic tanycytes are considered to function as sensors of peripheral metabolism. To facilitate this role, they express a wide range of receptors, including fibroblast growth factor receptor 1 (FGFR1). Using a monoclonal antibody (IMC-H7) that selectively antagonizes the FGFR1c isoform, we investigated possible actions of FGFR1c in a natural animal model of adiposity, the Siberian hamster. Infusion of IMC-H7 into the third ventricle suppressed appetite and increased energy expenditure. Likewise, peripheral treatment with IMC-H7 decreased appetite and body weight and increased energy expenditure and fat oxidation. A greater reduction in body weight and caloric intake was observed in response to IMC-H7 during the long-day fat state as compared to the short-day lean state. This enhanced response to IMC-H7 was also observed in calorically restricted hamsters maintained in long days, suggesting that it is the central photoperiodic state rather than the peripheral adiposity that determines the response to FGFR1c antagonism. Hypothalamic thyroid hormone availability is controlled by deiodinase enzymes (DIO2 and DIO3) expressed in tanycytes and is the key regulator of seasonal cycles of energy balance. Therefore, we determined the effect of IMC-H7 on hypothalamic expression of these deiodinase enzymes. The reductions in food intake and body weight were always associated with decreased expression of DIO2 in the hypothalamic ependymal cell layer containing tanycytes. These data provide further support for the notion the tanycytes are an important component of the mechanism by which the hypothalamus integrates central and peripheral signals to regulate energy intake and expenditure. PMID:26549257

  7. Synthesis and structure of [(η6-p-cymene)Ru(2-anthracen-9-ylmethylene-N-ethylhydrazinecarbothioamide)Cl]Cl; biological evaluation, topoisomerase II inhibition and reaction with DNA and human serum albumin

    PubMed Central

    Thessing, Jeffrey; Woods, Jason; Didion, Jacob; Gerasimchuk, Nikolay; Gonzalez-Sarrias, Antonio; Seeram, Navindra P.

    2012-01-01

    We have synthesized and evaluated the biological properties of a compound of the type [η6-p-cymene)Ru(EtATSC)Cl]Cl (1) where EtATSC = 2-anthracen-9-ylmethylene-N-ethylhydrazinecarbothioamide, a thiosemicarbazone. The complex has been characterized by elemental analysis, spectroscopically (NMR, UV-Vis, and IR) and structurally by XRD. The in vitro anticancer activity of 1 has been evaluated against two human colon cancer cell lines. The IC50 value for activity against HCT-116 was 224 ± 7 μM and 205 ± 5 μM against the Caco-2 cell line. The proficiency of 1 as an antibacterial agent was also evaluated against six bacterial strains. The minimum inhibitory concentration for Bacillus cereus was determined to be 5 μM and for Enterococcus faecalis it was 20 μM. At the maximum concentration tested the complex showed no activity against the Gram-negative strains. The complex binds strongly to human serum albumin with a binding constant of 1.37 ± 0.02 M−1 at 308 K on a single binding site. It is also a strong binder to DNA with an apparent binding constant of 2.82 × 105 M−1 at 308 K. 1 shows very good activity as a catalytic inhibitor of human topoisomerase II at concentrations as low as 20 μM. PMID:21347491

  8. Melatonin inhibits insulin secretion in rat insulinoma β-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2.

    PubMed

    Mühlbauer, Eckhard; Albrecht, Elke; Hofmann, Kathleen; Bazwinsky-Wutschke, Ivonne; Peschke, Elmar

    2011-10-01

    Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic β-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma β-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease. PMID:21585522

  9. Tumour growth inhibition and anti-metastatic activity of a mutated furin-resistant Semaphorin 3E isoform

    PubMed Central

    Casazza, Andrea; Kigel, Boaz; Maione, Federica; Capparuccia, Lorena; Kessler, Ofra; Giraudo, Enrico; Mazzone, Massimiliano; Neufeld, Gera; Tamagnone, Luca

    2012-01-01

    Secreted Semaphorin 3E (Sema3E) promotes cancer cell invasiveness and metastatic spreading. The pro-metastatic activity of Sema3E is due to its proteolytic fragment p61, capable of transactivating the oncogenic tyrosine kinase ErbB2 that associates with the Sema3E receptor PlexinD1 in cancer cells. Here, we show that a mutated, uncleavable variant of Sema3E (Uncl-Sema3E) binds to PlexinD1 like p61-Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading. Furthermore, Uncl-Sema3E competes with endogenous p61-Sema3E produced by tumour cells, thereby hampering their metastatic ability. Uncl-Sema3E also acts independently as a potent anti-angiogenic factor. It activates a PlexinD1-mediated signalling cascade in endothelial cells that leads to the inhibition of adhesion to extracellular matrix, directional migration and cell survival. The putative therapeutic potential of Uncl-Sema3E was validated in multiple orthotopic or spontaneous tumour models in vivo, where either local or systemic delivery of Uncl-Sema3E-reduced angiogenesis, growth and metastasis, even in the case of tumours refractory to treatment with a soluble vascular endothelial growth factor trap. In summary, we conclude that Uncl-Sema3E is a novel inhibitor of tumour angiogenesis and growth that concomitantly hampers metastatic spreading. PMID:22247010

  10. Chromosome 6p amplification and cancer progression

    PubMed Central

    Santos, Gda C; Zielenska, M; Prasad, M; Squire, J A

    2007-01-01

    Chromosomal imbalances represent an important mechanism in cancer progression. A clear association between DNA copy‐number aberrations and prognosis has been found in a variety of tumours. Comparative genomic hybridisation studies have detected copy‐number increases affecting chromosome 6p in several types of cancer. A systematic analysis of large tumour cohorts is required to identify genomic imbalances of 6p that correlate with a distinct clinical feature of disease progression. Recent findings suggest that a central part of the short arm of chromosome 6p harbours one or more oncogenes directly involved in tumour progression. Gains at 6p have been associated with advanced or metastatic disease, poor prognosis, venous invasion in bladder, colorectal, ovarian and hepatocellular carcinomas. Copy number gains of 6p DNA have been described in a series of patients who presented initially with follicle centre lymphoma, which subsequently transformed to diffuse large B cell lymphoma. Melanoma cytogenetics has consistently identified aberrations of chromosome 6, and a correlation with lower overall survival has been described. Most of the changes observed in tumours to date map to the 6p21–p23 region, which encompasses approximately half of the genes on all of chromosome 6 and one third of the number of CpG islands in this chromosome. Analyses of the genes that cluster to the commonly amplified regions of chromosome 6p have helped to identify a small number of molecular pathways that become deregulated during tumour progression in diverse tumour types. Such pathways offer promise for new treatments in the future. PMID:16790693

  11. Metabolism of glucose 1,6-P2--III. Partial purification and characterization of glucose 1,6-P2 synthase from pig skeletal muscle.

    PubMed

    Carreras, M; Carreras, J; Climent, F

    1988-01-01

    1. Glycerate 1,3-P2-dependent glucose, 1,6-P2 synthase has been purified 2000-fold from pig skeletal muscle, with a yield of 75%. 2. The enzyme possesses fructose 1,6-P2-dependent glucose 1,6-P2 synthase and phosphoglucomutase activities, which represent 0.1 and 60% of the main activity, respectively. 3. Both glucose 1-P and glucose 6-P can act as acceptors of the phosphoryl group from glycerate 1,3-P2. 4. The Km values are 19 microM and 67 nM for glucose 1-P and glycerate 1,3-P2, respectively. 5. The enzyme is inhibited by glycerate 2,3-P2, fructose 1,6-P2, glycerate 3-P, phosphoenolpyruvate and lithium, the inhibition pattern varying with the compound. PMID:2854765

  12. Carbonic anhydrase inhibitors: synthesis and inhibition of the human carbonic anhydrase isoforms I, II, VII, IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety.

    PubMed

    Sethi, Kalyan K; Vullo, Daniella; Verma, Saurabh M; Tanç, Muhammet; Carta, Fabrizio; Supuran, Claudiu T

    2013-10-01

    A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000nM), but were moderately effective, as hCA II inhibitors (Kis of 47-190nM) and poor hCA VII inhibitors (Kis in the range of 54-175nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234nM and hCA XII with inhibition constants in the range of 6.1-197nM with high selectivity ratio. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking study of compounds was performed to rationalize the SAR reported over here. PMID:23965175

  13. Novel action of apolipoprotein E (ApoE): ApoE isoform specifically inhibits lipid-particle-mediated cholesterol release from neurons

    PubMed Central

    Gong, Jian-Sheng; Morita, Shin-ya; Kobayashi, Mariko; Handa, Tetsurou; Fujita, Shinobu C; Yanagisawa, Katsuhiko; Michikawa, Makoto

    2007-01-01

    Background Since the majority of apolipoprotein E (apoE) existing in the cerebrospinal fluid is associated with high-density lipoprotein (HDL), one should focus on the role of the apoE-HDL complex rather than on that of free apoE in cholesterol metabolism in the central nervous system. However, the apoE-isoform-specific effect of apoE-HDL on cholesterol transport remains unclarified. Results Here we show that apoE3-HDL induced a marked cholesterol release from neurons, while apoE4-HDL induced little. To elucidate the mechanism underlying this phenomenon, we used a complex of lipid emulsion (EM) with recombinant apoE3 or apoE4 (apoE-EM) at various apoE concentrations. When a small number of apoE molecules were associated with EM, apoE3- and apoE4-EM, induced a marked cholesterol release to a level similar to that induced by EM alone. However, when apoE at given concentrations was incubated with EM, apoE3-EM induced a marked cholesterol release, while apoE4-EM induced little. Under these conditions, a greater number of apoE4 molecules were associated with EM than apoE3 molecules. When an increasing number of apoE molecules were associated with EM, both apoE3-EM and apoE4-EM induced little cholesterol release. Preincubation with β-mercaptoethanol increased the number of apoE3 molecules associated with EM similar to that of apoE4 molecules, indicating that the presence (apoE3) or absence (apoE4) of intermolecular disulfide bond formation is responsible for the association of a greater number of apoE4 molecules to EM than apoE3 molecules. Conclusion These results suggest that although apoE and a lipid particle are lipid acceptors, when apoE and a lipid particle form a complex, apoE on the particle surface inhibits the lipid particle-mediated cholesterol release from cells in an apoE-concentration-dependent manner. PMID:17504523

  14. INCREASED SENSITIVITY OF OXIDIZED LARGE ISOFORM OF RUBISCO ACTIVASE TO ADP INHIBITION IS DUE TO AN INTERACTION BETWEEN ITS CARBOXYL-EXTENSION AND NUCLEOTIDE-BINDING POCKET

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Arabidopsis, oxidation of the large (46-kDa) isoform activase to form a disulfide bond in the carboxyl-terminal extension (C-extension) significantly increases its ADP sensitivity for both ATP hydrolysis and Rubisco activation, thereby decreasing both activities at physiological ratios of ADP/ATP...

  15. PI3K pan-inhibition impairs more efficiently proliferation and survival of T-cell acute lymphoblastic leukemia cell lines when compared to isoform-selective PI3K inhibitors

    PubMed Central

    Spartà, Antonino Maria; Chiarini, Francesca; Buontempo, Francesca; Evangelisti, Camilla; Evangelisti, Cecilia; Orsini, Ester; McCubrey, James A.; Martelli, Alberto Maria

    2015-01-01

    Class I phosphatidylinositol 3-kinases (PI3Ks) are frequently activated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to the loss of PTEN function. Therefore, targeting PI3Ks is a promising innovative approach for T-ALL treatment, however at present no definitive evidence indicated which is the better therapeutic strategy between pan or selective isoform inhibition, as all the four catalytic subunits might participate in leukemogenesis. Here, we demonstrated that in both PTEN deleted and PTEN non deleted T-ALL cell lines, PI3K pan-inhibition exerted the highest cytotoxic effects when compared to both selective isoform inhibition or dual p110γ/δ inhibition. Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome. PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms. PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis. We further demonstrated that in some T-ALL cell lines, autophagy could exert a protective role against PI3K inhibition. Our findings strongly support clinical application of class I PI3K pan-inhibitors in T-ALL treatment, with the possible exception of ETP-ALL cases. PMID:25871383

  16. The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin.

    PubMed

    Wang, Yifan; Bernhardy, Andrea J; Cruz, Cristina; Krais, John J; Nacson, Joseph; Nicolas, Emmanuelle; Peri, Suraj; van der Gulden, Hanneke; van der Heijden, Ingrid; O'Brien, Shane W; Zhang, Yong; Harrell, Maribel I; Johnson, Shawn F; Candido Dos Reis, Francisco J; Pharoah, Paul D P; Karlan, Beth; Gourley, Charlie; Lambrechts, Diether; Chenevix-Trench, Georgia; Olsson, Håkan; Benitez, Javier J; Greene, Mark H; Gore, Martin; Nussbaum, Robert; Sadetzki, Siegal; Gayther, Simon A; Kjaer, Susanne K; D'Andrea, Alan D; Shapiro, Geoffrey I; Wiest, David L; Connolly, Denise C; Daly, Mary B; Swisher, Elizabeth M; Bouwman, Peter; Jonkers, Jos; Balmaña, Judith; Serra, Violeta; Johnson, Neil

    2016-05-01

    Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR. PMID:27197267

  17. 1,2-Benzisothiazole Derivatives Bearing 4-, 5-, or 6-Alkyl/arylcarboxamide Moieties Inhibit Carbonic Anhydrase Isoform IX (CAIX) and Cell Proliferation under Hypoxic Conditions.

    PubMed

    Coviello, Vito; Marchi, Beatrice; Sartini, Stefania; Quattrini, Luca; Marini, Anna Maria; Simorini, Francesca; Taliani, Sabrina; Salerno, Silvia; Orlandi, Paola; Fioravanti, Anna; Desidero, Teresa Di; Vullo, Daniela; Da Settimo, Federico; Supuran, Claudiu T; Bocci, Guido; La Motta, Concettina

    2016-07-14

    Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated. PMID:27305384

  18. PKC Isoform Expression in Modeled Microgravity

    NASA Technical Reports Server (NTRS)

    Risin, Diana; Sundaresan, Alamelu; Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

  19. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells

    PubMed Central

    Chakedis, Jeffery; French, Randall; Babicky, Michele; Jaquish, Dawn; Howard, Haleigh; Mose, Evangeline; Lam, Raymond; Holman, Patrick; Miyamoto, Jaclyn; Walterscheid, Zakk; Lowy, Andrew M.

    2015-01-01

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  20. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

    PubMed

    Chakedis, J; French, R; Babicky, M; Jaquish, D; Howard, H; Mose, E; Lam, R; Holman, P; Miyamoto, J; Walterscheid, Z; Lowy, A M

    2016-06-23

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription domain. The splice variant is detected in 73% of xenografts derived from pancreatic adenocarcinoma patients and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirm translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing cells, activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  1. Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms

    PubMed Central

    Tatsumi, Naoya; Hojo, Nozomi; Sakamoto, Hiroyuki; Inaba, Rena; Moriguchi, Nahoko; Matsuno, Keiko; Fukuda, Mari; Matsumura, Akihide; Hayashi, Seiji; Morimoto, Soyoko; Nakata, Jun; Fujiki, Fumihiro; Nishida, Sumiyuki; Nakajima, Hiroko; Tsuboi, Akihiro; Oka, Yoshihiro; Hosen, Naoki; Sugiyama, Haruo; Oji, Yusuke

    2015-01-01

    The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. PMID:26090994

  2. Spinach pyruvate kinase isoforms: partial purification and regulatory properties

    SciTech Connect

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K/sub m/ values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K/sub i/ for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K/sub a/ of 0.05 millimolar, and glutamate is an inhibitor with a K/sub i/ of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, the authors suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.

  3. Tropomyosin isoforms and reagents

    PubMed Central

    Schevzov, Galina; Whittaker, Shane P; Fath, Thomas; Lin, Jim JC

    2011-01-01

    Tropomyosins are rod-like dimers which form head-to-tail polymers along the length of actin filaments and regulate the access of actin binding proteins to the filaments.1 The diversity of tropomyosin isoforms, over 40 in mammals, and their role in an increasing number of biological processes presents a challenge both to experienced researchers and those new to this field. The increased appreciation that the role of these isoforms expands beyond that of simply stabilizing actin filaments has lead to a surge of reagents and techniques to study their function and mechanisms of action. This report is designed to provide a basic guide to the genes and proteins and the availability of reagents which allow effective study of this family of proteins. We highlight the value of combining multiple techniques to better evaluate the function of different tm isoforms and discuss the limitations of selected reagents. Brief background material is included to demystify some of the unfortunate complexity regarding this multi-gene family of proteins including the unconventional nomenclature of the isoforms and the evolutionary relationships of isoforms between species. Additionally, we present step-by-step detailed experimental protocols used in our laboratory to assist new comers to the field and experts alike. PMID:22069507

  4. The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production.

    PubMed

    Ghosh, Rajeshwary; Bank, Sarbashri; Maji, Uttam K; Bhattacharya, Rabindra; Guha, Santanu; Khan, Nighat N; Sinha, A Kumar

    2014-09-01

    Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was found to reduce both the elevated systolic and diastolic blood pressures to normal levels within 3 h due to the normalization of dermcidin level in these subjects. The plasma cortexin level at day 0, 1, 30 and 90 were 0.5 pmol/ml, 155.5 pmol/ml, 160.2 pmol/ml, 190.5 pmol/ml respectively with increased NO synthesis (r=+0.994). In vitro studies demonstrated that the incubation of the goat kidney cortex cells with aspirin stimulated (r)-cortexin synthesis due to NO synthesis. It could be suggested that the use of aspirin might control EH in man. PMID:25324696

  5. The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production

    PubMed Central

    Ghosh, Rajeshwary; Bank, Sarbashri; Maji, Uttam K.; Bhattacharya, Rabindra; Guha, Santanu; Khan, Nighat N.; Sinha, A. Kumar

    2014-01-01

    Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was found to reduce both the elevated systolic and diastolic blood pressures to normal levels within 3 h due to the normalization of dermcidin level in these subjects. The plasma cortexin level at day 0, 1, 30 and 90 were 0.5 pmol/ml, 155.5 pmol/ml, 160.2 pmol/ml, 190.5 pmol/ml respectively with increased NO synthesis (r=+0.994). In vitro studies demonstrated that the incubation of the goat kidney cortex cells with aspirin stimulated (r)-cortexin synthesis due to NO synthesis. It could be suggested that the use of aspirin might control EH in man. PMID:25324696

  6. Carbonic anhydrase inhibitors. Inhibition of human cytosolic isoforms I and II with (reduced) Schiff's bases incorporating sulfonamide, carboxylate and carboxymethyl moieties.

    PubMed

    Nasr, Gihane; Cristian, Alina; Barboiu, Mihail; Vullo, Daniella; Winum, Jean-Yves; Supuran, Claudiu T

    2014-05-15

    A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide, carboxylic acid or carboxymethyl functionalities as Zn(2+)-binding groups, with aromatic aldehydes incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The Ki values of the Schiff bases were in the range of 7.0-21,400nM against hCA II and of 52-8600nM against hCA I, respectively. The corresponding amines showed Ki values in the range of 8.6nM-5.3μM against hCA II, and of 18.7-251nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble derivatives may show pharmacologic applications. PMID:24746465

  7. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

    SciTech Connect

    Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro; Ariga, Toyohiko; Kusumi, Yoshiaki; Mitsumata, Masako; Yamada, Sachiko; Makishima, Makoto

    2008-07-15

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

  8. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  9. DNA signals at isoform promoters.

    PubMed

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  10. Molecular identification of the dominant-negative, splicing isoform of the two-pore domain K(+) channel K(2P)5.1 in lymphoid cells and enhancement of its expression by splicing inhibition.

    PubMed

    Endo, Kyoko; Kurokawa, Natsumi; Kito, Hiroaki; Nakakura, Sawa; Fujii, Masanori; Ohya, Susumu

    2015-12-01

    The two-pore domain background K(+) channel K2P5.1 is expected as a possible therapeutic target for autoimmune and inflammatory disorders and cancers because it plays an important role in maintaining the resting membrane potential and regulation of Ca(2+) signaling in T lymphocytes and cancer cells. However, the lack of selective K2P5.1 blockers has led to difficulties conducting experimental studies on this K(+) channel. We identified a novel splicing isoform of K2P5.1, K2P5.1B from the mammalian spleen, which lacked the N-terminus of full-length K2P5.1A. A co-immunoprecipitation assay using mice spleen lysates revealed an interaction between K2P5.1A and K2P5.1B in the cytoplasmic C-terminal domain. In a heterologous HEK293 expression system, K2P5.1B inhibited the trafficking of K2P5.1A to the plasma membrane. The alkaline pHe-induced hyperpolarizing response was significantly suppressed in K2P5.1B-transfected human leukemia K562 cells. Enhancement in cell proliferation by the overexpression of K2P5.1A in K562 was significantly prevented by the transfection of K2P5.1B. The spliceosome inhibitor pladienolide B significantly enhanced the relative expression of K2P5.1B in K562, resulting in decreases in the activity of K2P5.1A. K2P5.1B suppresses the function of the K2P5.1 K(+) channel in a dominant-negative manner, suggesting that the mRNA splicing mechanisms underlying the transcriptional regulation of K2P5.1B may be a new therapeutic strategy for autoimmune and inflammatory disorders and cancers. PMID:26475531

  11. CASC15-S is a tumor suppressor lncRNA at the 6p22 neuroblastoma susceptibility locus

    PubMed Central

    Russell, Mike R.; Penikis, Annalise; Oldridge, Derek A.; Alvarez-Dominguez, Juan R.; McDaniel, Lee; Diamond, Maura; Padovan, Olivia; Raman, Pichai; Li, Yimei; Wei, Jun S.; Zhang, Shile; Gnanchandran, Janahan; Seeger, Robert; Asgharzadeh, Shahab; Khan, Javed; Diskin, Sharon J.; Maris, John M.; Cole, Kristina A.

    2015-01-01

    Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single nucleotide polymorphism (SNP) associations reside within CASC15, a long non-coding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression. PMID:26100672

  12. NMR studies on polyphosphide Ce6Ni6P17

    NASA Astrophysics Data System (ADS)

    Koyama, T.; Yamada, H.; Ueda, K.; Mito, T.; Aoyama, Y.; Nakano, T.; Takeda, N.

    2016-02-01

    We report the result of 31P nuclear magnetic resonance (NMR) studies on Ce6Ni6P17. The observed NMR spectra show a Lorentzian-type and an asymmetric shapes, reflecting the local symmetry around each P site in the cubic unit cell. We have identified the observed NMR lines corresponding to three inequivalent P sites and deduced the temperature dependence of the Knight shift for each site. The Knight shifts increase with decreasing temperature down to 1.5 K, indicating a localized spin system of Ce6Ni6P17. Antiferromagnetic correlation between 4f spins is suggested from the negative sign of the Weiss-temperature.

  13. Renin and the IGFII/M6P Receptor System in Cardiac Biology

    PubMed Central

    Heger, Jacqueline; Schlüter, Klaus-Dieter

    2013-01-01

    Nonenzymatic cardiac activities of renin are well described during the last years and contribute either to cardiac-specific effects of the renin-angiotensin-aldosterone-system (RAAS) or to the pharmacological effects of RAAS inhibition. The interaction of renin with insulin-like growth factor II/mannose-6-phosphate (IGFII/M6P) receptors participates in nonclassical renin effects and contributes to cardiac remodelling caused by RAAS activation. The current findings suggest an important role for renin IGFII/M6P receptor interaction in cardiac adaptation to stress and support the idea that excessive accumulation of renin during inhibition of RAAS directly contributes to blood pressure-independent effects of these pharmacological interventions. It becomes a challenge for future studies focussing on chronic hypertension or myocardial infarction to comprise regulatory adaptations of the kidney, the main source of plasma renin and prorenin, because they directly contribute to key steps in regulation of cardiac (mal)adaptation via IGFII/M6P receptors. This receptor system is part of peptide/receptor interactions that modifies and possibly limits adverse remodelling effects caused by angiotensin II. Evaluation of interactions of renin with other pro-hypertrophic agonists is required to decide whether this receptor may become a target of pharmacological intervention. PMID:24288471

  14. Regulation of Ins(3,4,5,6)P(4) signaling by a reversible kinase/phosphatase.

    PubMed

    Ho, Melisa W Y; Yang, Xiaonian; Carew, Mark A; Zhang, Tong; Hua, Len; Kwon, Yong-Uk; Chung, Sung-Kee; Adelt, Stephan; Vogel, Günter; Riley, Andrew M; Potter, Barry V L; Shears, Stephen B

    2002-03-19

    Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases. PMID:11909533

  15. Linkage of Inflammatory Bowel Disease to Human Chromosome 6p

    PubMed Central

    Hampe, Jochen; Shaw, Sarah H.; Saiz, Robert; Leysens, Nancy; Lantermann, Annette; Mascheretti, Silvia; Lynch, Nicholas J.; MacPherson, Andrew J. S.; Bridger, Stephen; van Deventer, Sander; Stokkers, Pieter; Morin, Phil; Mirza, Mudassar M.; Forbes, Alastair; Lennard-Jones, John E.; Mathew, Christopher G.; Curran, Mark E.; Schreiber, Stefan

    1999-01-01

    Summary Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation. IBD is subdivided into Crohn disease and ulcerative colitis phenotypes. Given the immunologic dysregulation in IBD, the human-leukocyte-antigen region on chromosome 6p is of significant interest. Previous association and linkage analysis has provided conflicting evidence as to the existence of an IBD-susceptibility locus in this region. Here we report on a two-stage linkage and association analysis of both a basic population of 353 affected sibling pairs (ASPs) and an extension of this population to 428 white ASPs of northern European extraction. Twenty-eight microsatellite markers on chromosome 6 were genotyped. A peak multipoint LOD score of 4.2 was observed, at D6S461, for the IBD phenotype. A transmission/disequilibrium test (TDT) result of P=.006 was detected for D6S426 in the basic population and was confirmed in the extended cohort (P=.004; 97 vs. 56 transmissions). The subphenotypes of Crohn disease, ulcerative colitis, and mixed IBD contributed equally to this linkage, suggesting a general role for the chromosome 6 locus in IBD. Analysis of five single-nucleotide polymorphisms in the TNFA and LTA genes did not reveal evidence for association of these important candidate genes with IBD. In summary, we provide firm linkage evidence for an IBD-susceptibility locus on chromosome 6p and demonstrate that TNFA and LTA are unlikely to be susceptibility loci for IBD. PMID:10577918

  16. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    SciTech Connect

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  17. Pharmacological targeting of PI3K isoforms as a therapeutic strategy in chronic lymphocytic leukaemia

    PubMed Central

    Blunt, Matthew D.; Steele, Andrew J.

    2015-01-01

    PI3Kδ inhibitors such as idelalisib are providing improved therapeutic options for the treatment of chronic lymphocytic leukaemia (CLL). However under certain conditions, inhibition of a single PI3K isoform can be compensated by the other PI3K isoforms, therefore PI3K inhibitors which target multiple PI3K isoforms may provide greater efficacy. The development of compounds targeting multiple PI3K isoforms (α, β, δ, and γ) in CLL cells, in vitro, resulted in sustained inhibition of BCR signalling but with enhanced cytotoxicity and the potential for improve clinical responses. This review summarises the progress of PI3K inhibitor development and describes the rationale and potential for targeting multiple PI3K isoforms. PMID:26500849

  18. Cluster harvesting in the WBr6-P system.

    PubMed

    Ströbele, Markus; Eichele, Klaus; Meyer, Hans-Jürgen

    2015-02-01

    A combined thermal scanning-X-ray diffraction (XRD) approach was performed for the WBr(6)-P system to detect and analyze phases in this system, including metal-rich phases generated with increasing amounts of elemental (red) phosphorus under partial PBr(3) release. Phases were characterized by powder XRD. A black crystalline powder of W(4)(PBr)Br(10) was obtained after reduction of WBr(6) with elemental phosphorus at 450 °C. The crystal structure of the new compound was found to be isotypic with the structure of W(4)(PCl)Cl(10) on the basis of powder XRD data. The structure of W(4)(PBr)Br(10) is represented by a cyclobutadiene-like tetranuclear tungsten cluster interconnected into a layered (W(4)(μ(4)-PBr)Br(6)(i))Br(8)/(2)(a-a) arrangement via outer bromide ligands. The μ(4)-capping bromophosphinidene ligand was verified by solid-state magic-angle spinning (31)P NMR spectroscopy. PMID:25392957

  19. Differential Roles of PML Isoforms.

    PubMed

    Nisole, Sébastien; Maroui, Mohamed Ali; Mascle, Xavier H; Aubry, Muriel; Chelbi-Alix, Mounira K

    2013-01-01

    The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed. PMID:23734343

  20. Physicochemical Studies and Anticancer Potency of Ruthenium η6-p-Cymene Complexes Containing Antibacterial Quinolones

    PubMed Central

    2011-01-01

    With the aim of exploring the anticancer properties of organometallic compounds with bioactive ligands, Ru(arene) compounds of the antibacterial quinolones nalidixic acid (2) and cinoxacin (3) were synthesized, and their physicochemical properties were compared to those of chlorido(η6-p-cymene)(ofloxacinato-κ2O,O)ruthenium(II) (1). All compounds undergo a rapid ligand exchange reaction from chlorido to aqua species. 2 and 3 are significantly more stable than 1 and undergo minor conversion to an unreactive [(cym)Ru(μ-OH)3Ru(cym)]+ species (cym = η6-p-cymene). In the presence of human serum albumin 1−3 form adducts with this transport protein within 20 min of incubation. With guanosine 5′-monophosphate (5′-GMP; as a simple model for reactions with DNA) very rapid reactions yielding adducts via its N7 atom were observed, illustrating that DNA is a possible target for this compound class. A moderate capacity of inhibiting tumor cell proliferation in vitro was observed for 1 in CH1 ovarian cancer cells, whereas 2 and 3 turned out to be inactive. PMID:21552495

  1. Regulation of the formin for3p by cdc42p and bud6p.

    PubMed

    Martin, Sophie G; Rincón, Sergio A; Basu, Roshni; Pérez, Pilar; Chang, Fred

    2007-10-01

    Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Delta cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition. PMID:17699595

  2. Basal activity of GIRK5 isoforms.

    PubMed

    Salvador, Carolina; Mora, Silvia I; Ordaz, Benito; Antaramian, Anaid; Vaca, Luis; Escobar, Laura I

    2003-02-14

    G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results. PMID:12535718

  3. P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

    SciTech Connect

    Liu Yang; Dong Qianze; Zhao Yue; Dong Xinjun; Miao Yuan; Dai Shundong; Yang Zhiqiang; Zhang Di; Wang Yan; Li Qingchang; Zhao Chen; Wang Enhua

    2009-03-10

    Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and {beta}-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms.

  4. Isoform-specific targeting of ROCK proteins in immune cells

    PubMed Central

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders. PMID:27254302

  5. Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against carbonic anhydrase isoforms I, II, IX and XII.

    PubMed

    Barresi, Elisabetta; Salerno, Silvia; Marini, Anna Maria; Taliani, Sabrina; La Motta, Concettina; Simorini, Francesca; Da Settimo, Federico; Vullo, Daniela; Supuran, Claudiu T

    2016-02-15

    Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (KIs ranging between 40.8 and 92.7nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications. PMID:26796953

  6. Exo70 Isoform Switching upon Epithelial-Mesenchymal Transition Mediates Cancer Cell Invasion

    PubMed Central

    Lu, Hezhe; Liu, Jianglan; Liu, Shujing; Zeng, Jingwen; Ding, Deqiang; Carstens, Russ P.; Cong, Yusheng; Xu, Xiaowei; Guo, Wei

    2014-01-01

    Summary Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2, and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoforms expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal but not the epithelial isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion. PMID:24331928

  7. Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p.

    PubMed

    Sivaram, Mylavarapu V S; Saporita, Jennifer A; Furgason, Melonnie L M; Boettcher, Angela J; Munson, Mary

    2005-04-26

    Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation. PMID:15835919

  8. 6p22.3 amplification as a biomarker and potential therapeutic target of advanced stage bladder cancer

    PubMed Central

    Zhang, Jianmin; Underwood, Willie; Yang, Nuo; Frangou, Costa; Eng, Kevin; Head, Karen; Bollag, Roni J.; Kavuri, Sravan K.; Rojiani, Amyn M.; Li, Yingwei; Yan, Li; Hill, Annette; Woloszynska-Read, Anna; Wang, Jianmin; Liu, Song; Trump, Donald L.; Candace, Johnson S.

    2013-01-01

    Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type. PMID:24231253

  9. Cell, Isoform, and Environment Factors Shape Gradients and Modulate Chemotaxis

    PubMed Central

    Chang, S. Laura; Cavnar, Stephen P.; Takayama, Shuichi; Luker, Gary D.; Linderman, Jennifer J.

    2015-01-01

    Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-γ isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinder CXCL12-γ-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment. PMID:25909600

  10. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides

    SciTech Connect

    Silver, Kristopher S.; Soderlund, David M. . E-mail: dms6@cornell.edu

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel {alpha} subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na{sub v}1.2a, Na{sub v}1.4, Na{sub v}1.5, and Na{sub v}1.8 sodium channel {alpha} subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat {beta}1 auxiliary subunit on the sensitivity of the Na{sub v}1.2a and Na{sub v}1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel {alpha} subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na{sub v}1.4 > Na{sub v}1.2a > Na{sub v}1.5 > Na{sub v}1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na{sub v}1.8 isoform was most sensitive, the Na{sub v}1.4 isoform was completely insensitive, and the sensitivities of the Na{sub v}1.5 and Na{sub v}1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na{sub v}1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na{sub v}1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na{sub v}1.2a or Na{sub v}1.4 isoforms with the {beta}1 subunit was the modest reduction in the sensitivity of the Na{sub v}1.2a isoform to RH 3421. These results demonstrate that mammalian sodium

  11. In Vitro Inhibition of Human UDP-Glucuronosyl-Transferase (UGT) Isoforms by Astaxanthin, β-Cryptoxanthin, Canthaxanthin, Lutein, and Zeaxanthin: Prediction of in Vivo Dietary Supplement-Drug Interactions.

    PubMed

    Zheng, Yu Fen; Min, Jee Sun; Kim, Doyun; Park, Jung Bae; Choi, Sung-Wook; Lee, Eun Seong; Na, Kun; Bae, Soo Kyung

    2016-01-01

    Despite the widespread use of the five major xanthophylls astaxanthin, β-cryptoxanthin, canthaxanthin, lutein, and zeaxanthin as dietary supplements, there have been no studies regarding their inhibitory effects on hepatic UDP-glucuronosyltransferases (UGTs). Here, we evaluated the inhibitory potential of these xanthophylls on the seven major human hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15) in vitro by LC-MS/MS using specific marker reactions in human liver microsomes (except UGT2B15) or recombinant supersomes (UGT2B15). We also predicted potential dietary supplement-drug interactions for β-cryptoxanthin via UGT1A1 inhibition. We demonstrated that astaxanthin and zeaxanthin showed no apparent inhibition, while the remaining xanthophylls showed only weak inhibitory effects on the seven UGTs. β-Cryptoxanthin mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC50 values of 18.8 ± 2.07, 28.3 ± 4.40 and 34.9 ± 5.98 μM, respectively. Canthaxanthin weakly inhibited UGT1A1 and UGT1A3, with IC50 values of 38.5 ± 4.65 and 41.2 ± 3.14 μM, respectively; and lutein inhibited UGT1A1 and UGT1A4, with IC50 values of 45.5 ± 4.01 and 28.7 ± 3.79 μM, respectively. Among the tested xanthophyll-UGT pairs, β-cryptoxanthin showed the strongest competitive inhibition of UGT1A1 (Ki, 12.2 ± 0.985 μM). In addition, we predicted the risk of UGT1A1 inhibition in vivo using the reported maximum plasma concentration after oral administration of β-cryptoxanthin in humans. Our data suggests that these xanthophylls are unlikely to cause dietary supplement-drug interactions mediated by inhibition of the hepatic UGTs. These findings provide useful information for the safe clinical use of the tested xanthophylls. PMID:27529203

  12. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase. PMID:26348913

  13. The TRAPP Subunit Trs130p Interacts with the GAP Gyp6p to Mediate Ypt6p Dynamics at the Late Golgi

    PubMed Central

    Brunet, Stephanie; Saint-Dic, Djenann; Milev, Miroslav P.; Nilsson, Tommy; Sacher, Michael

    2016-01-01

    Small GTPases of the Rab superfamily participate in virtually all vesicle-mediated trafficking events. Cycling between an active GTP-bound form and an inactive GDP-bound form is accomplished in conjunction with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Rab cascades have been described in which an effector of an activated Rab is a GEF for a downstream Rab, thus ensuring activation of a pathway in an ordered fashion. Much less is known concerning crosstalk between GEFs and GAPs although regulation between these factors could also contribute to the overall physiology of a cell. Here we demonstrate that a subunit of the TRAPP II multisubunit tethering factor, a Rab GEF, participates in the recruitment of Gyp6p, a GAP for the GTPase Ypt6p, to Golgi membranes. The extreme carboxy-terminal portion of the TRAPP II subunit Trs130p is required for the interaction between TRAPP II and Gyp6p. We further demonstrate that TRAPP II mutants, but not a TRAPP III mutant, display a defect in Gyp6p interaction. A consequence of this defective interaction is the enhanced localization of Ypt6p at late Golgi membranes. Although a ypt31/32 mutant also resulted in an enhanced localization of Gyp6p at the late Golgi, the effect was not as dramatic as that seen for TRAPP II mutants, nor was Ypt31/32 detected in the same TRAPP II purification that detected Gyp6p. We propose that the interaction between TRAPP II and Gyp6p represents a parallel mechanism in addition to that mediated by Ypt31/32 for the recruitment of a GAP to the appropriate membrane, and is a novel example of crosstalk between a Rab GAP and GEF. PMID:27252941

  14. Inference of Isoforms from Short Sequence Reads

    NASA Astrophysics Data System (ADS)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  15. Vanadate induces apoptosis in epidermal JB6 P+ cells via hydrogen peroxide-mediated reactions.

    PubMed

    Ye, J; Ding, M; Leonard, S S; Robinson, V A; Millecchia, L; Zhang, X; Castranova, V; Vallyathan, V; Shi, X

    1999-12-01

    Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process. PMID:10705990

  16. [Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

    PubMed

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation. PMID:25372487

  17. Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE) for 5α-reductase isoform II inhibition using a cell-free in vitro test system

    PubMed Central

    Pais, Pilar; Villar, Agustí; Rull, Santiago

    2016-01-01

    Background The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen – 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE) has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive – some have shown significant results, and others have not – possibly the result of varying bioactivities of the SPEs used in the studies. Purpose To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE), an inhibitor of the 5α-reductase isoenzyme type II. Materials and methods The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. Results By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 μg/mL), SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%–75% inhibition of 5α-reductase type II. Conclusion SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The bioactivity of SPSE corresponds favorably to that reported for the hexane extract used in a large

  18. Inhibiting the neuronal isoform of nitric oxide synthase has similar effects on the compensatory choroidal and axial responses to myopic defocus in chicks as does the non-specific inhibitor L-NAME

    PubMed Central

    Nickla, Debora L.; Damyanova, Petya; Lytle, Grace

    2010-01-01

    In birds, the choroid plays a role in the visual regulation of eye growth, thickening in response to myopic defocus, and thinning in response to hyperopic defocus, in both cases moving the retina towards the image plane. This response is rapid, occurring within hours of the defocus stimulus. These changes are consistently associated with slower changes in the sclera, that result in the appropriate changes in axial elongation, decreasing growth in response to myopic defocus and increasing it in response to hyperopic defocus. The molecular mechanisms underlying the scleral response involve changes in the synthesis of extracellular matrix molecules, however, those underlying the changes in choroidal thickness are not known. However, evidence suggests that it may involve the gaseous signal molecule nitric oxide, as nitric oxide is a potent smooth muscle relaxant, and injections of the non-specific nitric oxide synthase inhibitor L-NAME transiently inhibits the thickening response. Interestingly, it also dis-inhibits ocular growth, in accordance with a mechanistic link between the two responses. If nitric oxide is part of the signal cascade underlying the visual regulation of eye growth, it would be important to ascertain the source of the molecule. As a first step towards doing so, we used various more specific NOS inhibitors and studied their effects on the choroidal and growth responses. Birds (7–12 days old) were fitted with +10 D lenses on one eye. On that day, single intravitreal injections (30 μl) of the following inhibitors were used: nNOS inhibitor N ω-propyl-L-arginine (n=12), iNOS inhibitor L-NIL (n=16), eNOS/iNOS inhibitor L-NIO (n=15), non-specific inhibitor L-NMMA (n=30) or physiological saline (n=18). Ocular dimensions were measured using high-frequency A-scan ultrasonography at the start of the experiment, and at 7, 24 and 48 hours after. We found that the nNOS inhibitor N ω-propyl-L-arginine had the same inhibitory effects on the choroidal response

  19. Fructose 2,6-bisphosphate (Fru-2,6-P/sub 2/) binds at the active site of rabbit liver fructose-1,6-bisphosphate

    SciTech Connect

    Scheffler, J.E.; Fromm, H.J.

    1986-05-01

    Fru-2,6-P/sub 2/ and AMP are potent inhibitors of fructose-1,6-bisphosphatase (FBPase). AMP inhibits by binding to an allosteric site. While Fru-2,6-P/sub 2/ and fructose 1,6-bisphosphate (Fru-1,6-P/sub 2/) binding is mutually exclusive, a controversy exists as to whether Fru-2,6-P/sub 2/ inhibits by binding to the active site or a regulatory site. To address this question, the aromatic region of the /sup 1/H NMR spectrum of FBPase was examined in the absence and presence of Fru-1,6-P/sub 2/, Fru-2,6-P/sub 2/, and AMP. All of the spectral perturbations produced by Fru-1,6-P/sub 2/ binding were also seen with Fru-2,6-P/sub 2/: (1) the C2-H of His 1 (pKa=7.1) shifted downfield 0.03 ppm (pH 5.3); (2) the C2-H of His 2 shifted downfield 0.03 ppm (pH 5.3) and broadened; (3) several resonances in the phe/tyr region of the spectrum decreased in intensity. In addition, both fructose bisphosphates produced a change in the exchange rate of AMP from past/intermediate in the binary enzyme AMP complex to slow exchange in the ternary complex. AMP produced similar changes in the phe/tyr region of the spectrum. Unlike the fructose bisphosphates, AMP binding produced: (1) broadening of the C2-H of His 1; (2) no effect on His 2 and; (3) an enhancement of the intensity of a resonance at 7.25 ppm (pH 5.3). The results are consistent with an active site binding mode of inhibition for Fru-2,6-P/sub 2/. AMP binding to a regulatory site was apparent from the different results obtained with this ligand.

  20. Secretion of PDGF isoforms during osteoclastogenesis and its modulation by anti-osteoclast drugs.

    PubMed

    Rahman, M Motiur; Matsuoka, Kazuhiko; Takeshita, Sunao; Ikeda, Kyoji

    2015-06-26

    In an attempt to identify secretory products of osteoclasts that mediate the coupling of bone formation to resorption, we found that along with osteoclast differentiation, PDGF-A gene expression increase occurred first, by 12 h after stimulation of bone marrow macrophages with M-CSF and RANKL, and peaked at 36 h. This was next followed by a progressive increase in PDGF-B gene expression until a peak at 60 h, when mature osteoclasts formed. Isoform-specific ELISA of the conditioned medium collected every 24 h revealed that all three of the isoforms of PDGF-AA, AB and BB were secreted, in this temporal order as differentiation proceeded. Their secretion was enhanced when osteoclasts were activated by placing them on dentin slices. The secretion of all three isoforms was decreased in cathepsin K-deficient osteoclasts compared with wild-type osteoclasts. Pharmacological inhibition of cathepsin K with odanacatib also inhibited the secretion of all three isoforms, as was also the case with alendronate treatment. The secretion of sphingosine-1-phosphate, which increased during osteoclastogenesis, was reduced from cathepsin K-deficient osteoclasts, and was inhibited by treatment with odanacatib more profoundly than with alendronate. Thus, all three isoforms of PDGF, which are secreted at distinct differentiation stages of osteoclasts, appear to have distinct roles in the cell-cell communication that takes place in the microenvironment of bone remodeling, especially from the osteoclast lineage to mesenchymal cells and vascular cells, thereby stimulating osteogenesis and angiogenesis. PMID:25951977

  1. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  2. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  3. A novel alternative splicing isoform of NF2 identified in human Schwann cells

    PubMed Central

    Su, Fang; Zhou, Zhengguang; Su, Wen; Wang, Zishu; Wu, Qiong

    2016-01-01

    Vestibular schwannoma (VS) is a benign, slow-growing cranial tumor that originates from the hypertrophy of Schwann cells. The majority of sporadic VS are unilateral, and the mechanisms underlying VS tumorigenesis are not fully understood. The human neurofibromin 2 (NF2) gene encodes the tumor suppressor protein merlin and the NF2 transcript can be alternatively spliced to form numerous isoforms. The present study investigated human Schwann cells (HSCs) at the mRNA and protein level to understand the function of the alternative splicing (AS) isoform of NF2. The total RNA of HSCs was isolated and the full-length coding sequence of NF2 was amplified. The amplified products were excised from agarose gels, purified and sequenced. NF2 at a protein level was assayed by immunoprecipitation and western blot analysis. The full-length and spliced NF2 forms were amplified by polymerase chain reaction (PCR) from the HSC complementary DNA and ligated into eukaryotic expression vector pcDNA3.1(+). The plasmids were transfected into the HSC HEI-193 cell line and cell proliferation assays were performed using Cell Counting Kit-8. PCR analysis using HSC total RNA as a template revealed the presence of a shortened NF2 transcript, which was due to splicing at the 3′-end of the NF2 mRNA. Sequence analysis confirmed that this AS isoform omitted exons 11, 12, 13, 14, 15 and 16. Immunoprecipitation and western blot analysis demonstrated that the AS isoform was highly expressed in the HSCs at 38 kDa, while the wild-type (WT) isoform, which was expected at 66 kDa, was undetectable. Transfection and cell proliferation assays revealed that the WT isoform exhibited significant growth inhibition, while the AS isoform did not suppress cell growth. In conclusion, the present study detected AS NF2 isoforms in HSC for the first time, and investigated the function of the principle AS isoform. The present study suggests that although HSCs have an undetectable level of WT isoform of the NF2 protein

  4. Candidate variants at 6p21.33 and 6p22.1 and risk of non-small cell lung cancer in a Chinese population

    PubMed Central

    Zhang, Mingfeng; Hu, Lingmin; Shen, Hao; Dong, Jing; Shu, Yongqian; Xu, Lin; Jin, Guangfu; Tian, Tian; Hu, Zhibin; Shen, Hongbing

    2010-01-01

    Chromosome 6p21.33, containing BAT3 and MSH5 genes, together with chromosome 6p22.1 were recently identified as susceptible regions for lung cancer in Caucasian populations. These findings interest us in assessing whether genetic variants in these regions also contribute to lung cancer risk in Chinese populations. We genotyped the most significant single nucleotide polymorphism (SNP) (rs9295740) reported in Caucasian populations at Chromosome 6p22.1 and one common potentially functional variant (rs2075789) located at exon 2 of MSH5 in a case-control study including 1009 histologically confirmed non-small cell lung cancer (NSCLC) cases and 1127 cancer-free controls in a Chinese population. We found that the distributions of genotypes of both SNPs between cases and controls were not significantly different (P = 0.624 for rs9295740 and P = 0.937 for rs2075789). Logistic regression analyses revealed neither of the two SNPs was significantly associated with altered risk of NSCLC in dominant or recessive genetic models. When we compared the combined variant genotypes (GA+AA) with the common homozygote GG, assuming a dominant genetic model, the adjusted ORs were 1.03 (95% CI = 0.86-1.25) for rs9295740 and 1.03 (95% CI = 0.85-1.25) for rs2075789. In addition, no significant associations were observed in subgroups stratified by age, gender, smoking status or histologic types. Our results indicate that the most significant SNP rs9295740 identified in Caucasians in 6p22.1 and the potentially functional SNP rs2075789 in 6p21.33, seem not applicable to Chinese populations as susceptible markers for lung cancer. Re-sequencing and fine-mapping this region, along with extensive functional evaluations, is required. PMID:21537448

  5. Magnetic properties of cubic compound Ce6Ni6P17 with geometric frustrations

    NASA Astrophysics Data System (ADS)

    Takeda, N.; Izumi, K.; Ono, H.; Yodono, S.; Nakano, T.

    2012-12-01

    We report the magnetic susceptibility and the low-temperature specific heat of Ce6Ni6P17 with geometric frustrations and La6Ni6P17 as a nonmagnetic counterpart. The magnetic susceptibility of Ce6Ni6P17 decreases monotonically with decreasing temperature and the specific heat shows a broad peak around 1.4K. The evaluated magnetic entropy is about a half of Rln2 at 5.0 K. This result suggests that the frustrations persist down to very low temperatures and the residual entropy originate from frustration is present.

  6. The 6p3/2ns(J = 1,2) autoionizing states of barium

    NASA Astrophysics Data System (ADS)

    Li, S. B.; Dai, C. J.; Sun, W.; Xue, P.

    2001-06-01

    Using a three-step laser excitation scheme we have selectively excited the Ba 6p3/2ns(J = 1,2) autoionizing Rydberg states with different polarization configurations. The level energies and widths of Ba 6p3/2ns(J = 1) autoionizing states with n = 9-33 and 6p3/2ns(J = 2) autoionizing states with n = 9-20 are reported, most of which were observed for the first time. Their spectroscopic properties are discussed in detail. The experimental data are in good agreement with the theoretical analysis from the multichannel quantum defect theory.

  7. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

    PubMed

    Cao, Yun; Bender, Ingrid K; Konstantinidis, Athanasios K; Shin, Soon Cheon; Jewell, Christine M; Cidlowski, John A; Schleimer, Robert P; Lu, Nick Z

    2013-02-28

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  8. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans

    PubMed Central

    Cao, Yun; Bender, Ingrid K.; Konstantinidis, Athanasios K.; Shin, Soon Cheon; Jewell, Christine M.; Cidlowski, John A.; Schleimer, Robert P.

    2013-01-01

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  9. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    SciTech Connect

    Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  10. Low-frequency germline variants across 6p22.2-6p21.33 are associated with non-obstructive azoospermia in Han Chinese men.

    PubMed

    Ni, Bixian; Lin, Yuan; Sun, Liangdan; Zhu, Meng; Li, Zheng; Wang, Hui; Yu, Jun; Guo, Xuejiang; Zuo, Xianbo; Dong, Jing; Xia, Yankai; Wen, Yang; Wu, Hao; Li, Honggang; Zhu, Yong; Ping, Ping; Chen, Xiangfeng; Dai, Juncheng; Jiang, Yue; Xu, Peng; Du, Qiang; Yao, Bing; Weng, Ning; Lu, Hui; Wang, Zhuqing; Zhu, Xiaobin; Yang, Xiaoyu; Xiong, Chenliang; Ma, Hongxia; Jin, Guangfu; Xu, Jianfeng; Wang, Xinru; Zhou, Zuomin; Liu, Jiayin; Zhang, Xuejun; Conrad, Donald F; Hu, Zhibin; Sha, Jiahao

    2015-10-01

    Genome-wide association studies (GWAS) have identified several common loci contributing to non-obstructive azoospermia (NOA). However, a substantial fraction of NOA heritability remains undefined, especially those low-frequency [defined here as having a minor allele frequency (MAF) between 0.5 and 5%] and rare (MAF below 0.5%) variants. Here, we performed a 3-stage exome-wide association study in Han Chinese men to evaluate the role of low-frequency or rare germline variants in NOA development. The discovery stage included 962 NOA cases and 1348 healthy male controls genotyped by exome chips and was followed by a 2-stage replication with an additional 2168 cases and 5248 controls. We identified three low-frequency variants located at 6p22.2 (rs2298090 in HIST1H1E encoding p.Lys152Arg: OR = 0.30, P = 2.40 × 10(-16)) and 6p21.33 (rs200847762 in FKBPL encoding p.Pro137Leu: OR = 0.11, P = 3.77 × 10(-16); rs11754464 in MSH5: OR = 1.78, P = 3.71 × 10(-7)) associated with NOA risk after Bonferroni correction. In summary, we report an instance of newly identified signals for NOA risk in genes previously undetected through GWAS on 6p22.2-6p21.33 in a Chinese population and highlight the role of low-frequency variants with a large effect in the process of spermatogenesis. PMID:26199320

  11. Non-Classical Inhibition of Carbonic Anhydrase

    PubMed Central

    Lomelino, Carrie L.; Supuran, Claudiu T.; McKenna, Robert

    2016-01-01

    Specific isoforms from the carbonic anhydrase (CA) family of zinc metalloenzymes have been associated with a variety of diseases. Isoform-specific carbonic anhydrase inhibitors (CAIs) are therefore a major focus of attention for specific disease treatments. Classical CAIs, primarily sulfonamide-based compounds and their bioisosteres, are examined as antiglaucoma, antiepileptic, antiobesity, antineuropathic pain and anticancer compounds. However, many sulfonamide compounds inhibit all CA isoforms nonspecifically, diluting drug effectiveness and causing undesired side effects due to off-target inhibition. In addition, a small but significant percentage of the general population cannot be treated with sulfonamide-based compounds due to a sulfa allergy. Therefore, CAIs must be developed that are not only isoform specific, but also non-classical, i.e. not based on sulfonamides, sulfamates, or sulfamides. This review covers the classes of non-classical CAIs and the recent advances in the development of isoform-specific inhibitors based on phenols, polyamines, coumarins and their derivatives. PMID:27438828

  12. Non-Classical Inhibition of Carbonic Anhydrase.

    PubMed

    Lomelino, Carrie L; Supuran, Claudiu T; McKenna, Robert

    2016-01-01

    Specific isoforms from the carbonic anhydrase (CA) family of zinc metalloenzymes have been associated with a variety of diseases. Isoform-specific carbonic anhydrase inhibitors (CAIs) are therefore a major focus of attention for specific disease treatments. Classical CAIs, primarily sulfonamide-based compounds and their bioisosteres, are examined as antiglaucoma, antiepileptic, antiobesity, antineuropathic pain and anticancer compounds. However, many sulfonamide compounds inhibit all CA isoforms nonspecifically, diluting drug effectiveness and causing undesired side effects due to off-target inhibition. In addition, a small but significant percentage of the general population cannot be treated with sulfonamide-based compounds due to a sulfa allergy. Therefore, CAIs must be developed that are not only isoform specific, but also non-classical, i.e. not based on sulfonamides, sulfamates, or sulfamides. This review covers the classes of non-classical CAIs and the recent advances in the development of isoform-specific inhibitors based on phenols, polyamines, coumarins and their derivatives. PMID:27438828

  13. Tryptophan Inhibits Biofilm Formation by Pseudomonas aeruginosa

    PubMed Central

    Brandenburg, Kenneth S.; Rodriguez, Karien J.; McAnulty, Jonathan F.; Murphy, Christopher J.; Abbott, Nicholas L.; Schurr, Michael J.

    2013-01-01

    Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the d and l isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of d and l isoforms producing the greatest inhibitory effect. Addition of d-/l-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa. PMID:23318791

  14. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  15. Spontaneous Hepatocellular Carcinoma after the Combined Deletion of Akt Isoforms.

    PubMed

    Wang, Qi; Yu, Wan-Ni; Chen, Xinyu; Peng, Xiao-Ding; Jeon, Sang-Min; Birnbaum, Morris J; Guzman, Grace; Hay, Nissim

    2016-04-11

    Akt is frequently hyperactivated in human cancers and is targeted for cancer therapy. However, the physiological consequences of systemic Akt isoform inhibition were not fully explored. We showed that while combined Akt1 and Akt3 deletion in adult mice is tolerated, combined Akt1 and Akt2 deletion induced rapid mortality. Akt2(-/-) mice survived hepatic Akt1 deletion but all developed spontaneous hepatocellular carcinoma (HCC), which is associated with FoxO-dependent liver injury and inflammation. The gene expression signature of HCC-bearing livers is similar to aggressive human HCC. Consistently, neither Akt1(-/-) nor Akt2(-/-) mice are resistant to diethylnitrosamine-induced hepatocarcinogenesis, and Akt2(-/-) mice display a high incidence of lung metastasis. Thus, in contrast to other cancers, hepatic Akt inhibition induces liver injury that could promote HCC. PMID:26996309

  16. hMENA(11a), a hMENA isoform sending survival signals.

    PubMed

    Trono, Paola; Di Modugno, Francesca; Nisticò, Paola

    2016-03-01

    Human MENA(11a) (hMENA(11a)), an epithelial-associated isoform of the actin binding protein enabled homolog (ENAH, also known as mammalian ENA [MENA]), is upregulated and phosphorylated following the activation of human epidermal growth factor receptor (HER) 1, HER2, and HER3. Here, we reveal a novel role of this isoform in sustaining cell survival and propose hMENA(11a) as a marker of HER3 activation and resistance to phosphatidylinositol-3-kinase inhibition therapies. PMID:27308605

  17. Terminal 6p deletion syndrome mimicking CHARGE syndrome: A case report

    PubMed Central

    Freire, Gabrielle; Russell, Laura; Oskoui, Maryam

    2013-01-01

    The clinical features associated with terminal 6p deletion syndrome include anterior eye chamber defects, hearing loss, congenital heart anomalies and characteristic facies along with developmental delays. These features overlap with a number of other conditions including CHARGE syndrome. This acronym stands for non-random association of anomalies including coloboma of the eye, heart anomalies, choanal atresia, retardation of growth and development, genital hypoplasia and ear anomalies/deafness now known to be caused by CHD7 mutations. We describe a boy initially diagnosed with CHARGE syndrome who was subsequently found to have a terminal 6p deletion. Screening for 6p deletions in individuals presenting with atypical CHARGE syndrome may be warranted, with direct consequences for genetic counseling.

  18. Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

    PubMed Central

    Pinard, Melissa A.

    2015-01-01

    The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3−. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors. PMID:25811028

  19. Versican V1 Isoform Induces Neuronal Differentiation and Promotes Neurite Outgrowth

    PubMed Central

    Wu, Yaojiong; Sheng, Wang; Chen, Liwen; Dong, Haiheng; Lee, Vivian; Lu, Fred; Wong, C. Shun; Lu, Wei-Yang; Yang, Burton B.

    2004-01-01

    The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, β1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair. PMID:14978219

  20. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  1. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies.

    PubMed

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-10-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  2. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    PubMed Central

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M.

    2016-01-01

    ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. PMID:26563652

  3. Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation

    PubMed Central

    Stastna, Jana; Pan, Xiaoyu; Wang, Haicui; Kollmannsperger, Alina; Kutscheidt, Stefan; Lohmann, Volker; Grosse, Robert; Fackler, Oliver T

    2012-01-01

    Plasma membrane (PM) blebs are dynamic actin-rich cell protrusions that occur, e.g., during cytokinesis, amoeboid cell motility and cell attachment. Using a targeted siRNA screen against 21 actin nucleation factors, we identify a novel and essential role of the human diaphanous formin DIAPH3 in PM blebbing during cell adhesion. Suppression of DIAPH3 inhibited blebbing to promote rapid cell spreading involving β1-integrin. Multiple isoforms of DIAPH3 were detected on the mRNA and protein level of which isoforms 3 and 7 were the largest and most abundant isoforms that however did not induce formation of actin-rich protrusions. Rather, PM blebbing specifically involved the low abundance isoform 1 of DIAPH3 and activation of isoform 7 by deletion of the diaphanous-autoregulatory domain caused the formation of filopodia. Dimerization and actin assembly activity were essential for induction of specific cell protrusions by DIAPH3 isoforms 1 and 7. Our data suggest that the N-terminal region comprising the GTPase-binding domain determined the subcellular localization of the formin as well as its protrusion activity between blebs and filopodia. We propose that isoform-selective actin assembly by DIAPH3 exerts specific and differentially regulated functions during cell adhesion and motility. PMID:22184005

  4. Physical Map of Human 6p21.2–6p21.3: Region Flanking the Centromeric End of the Major Histocompatibility Complex

    PubMed Central

    Tripodis, Nicos; Mason, Ruth; Humphray, Sean J.; Davies, Angela F.; Herberg, Jethro A.; Trowsdale, John; Nizetic, Dean; Senger, Gabriele; Ragoussis, Jiannis

    1998-01-01

    We have physically mapped and cloned a 2.5-Mb chromosomal segment flanking the centromeric end of the major histocompatibility complex (MHC). We characterized in detail 27 YACs, 144 cosmids, 51 PACs, and 5 BACs, which will facilitate the complete genomic sequencing of this region of chromosome 6. The contig contains the genes encoding CSBP, p21, HSU09564 serine kinase, ZNF76, TCP-11, RPS10, HMGI(Y), BAK, and the human homolog of Tctex-7 (HSET). The GLO1 gene was mapped further centromeric in the 6p21.2–6p21.1 region toward TCTE-1. The gene order of the GLO1–HMGI(Y) segment in respect to the centromere is similar to the gene order in the mouse t-chromosome distal inversion, indicating that there is conservation in gene content but not gene order between humans and mice in this region. The close linkage of the BAK and CSBP genes to the MHC is of interest because of their possible involvement in autoimmune disease. PMID:9647638

  5. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

    SciTech Connect

    Gilbreth, Ryan N.; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B.; Li, Nan-Sheng; Piccirilli, Joseph A.; Chen, Yuan; Koide, Shohei

    2011-07-25

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a 'monobody,' a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K{sub d} values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  6. Three-step laser excitation of the 6p3/2ns, nd, ng autoionizing Rydberg levels via the 6p5f 1/2[5/2]2 level of lead

    NASA Astrophysics Data System (ADS)

    Ahad, A.; Nadeem, A.; Bhatti, S. A.; Baig, M. A.

    2005-03-01

    Odd parity autoionizing Rydberg levels of atomic lead in the energy region above the 6p1/2 ionization threshold have been investigated using three-step laser excitation in conjunction with an atomic beam apparatus. The 6p3/2ns (J = 1, 2), 6p3/2nd (J = 1, 2, 3) and 6p3/2ng (J = 2, 3) levels have been observed from the 6p5f 1/2[5/2]2 intermediate level. Energy values and FWHM of forty levels belonging to the 6p3/2ns, 6p3/2nd and 6p3/2ng configurations are presented. Six levels based on the 6p3/2ng (5, 13 ≤n ≤15) configurations and three levels attached to the 6p3/28d configuration are reported for the first time. The present study of the low-lying autoionizing levels attached to the 6p3/25g (J = 2, 3) configuration completes the series adjacent to the 6p1/2 limit.

  7. Osteopontin (OPN/SPP1) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

    PubMed Central

    Lin, Jules; Myers, Amy L.; Wang, Zhuwen; Nancarrow, Derek J.; Ferrer-Torres, Daysha; Handlogten, Amy; Leverenz, Kimmy; Bao, Julia; Thomas, Dafydd G.; Wang, Thomas D.; Orringer, Mark B.; Reddy, Rishindra M.; Chang, Andrew C.; Beer, David G.; Lin, Lin

    2015-01-01

    Esophageal adenocarcinoma (EAC) is often diagnosed at an advanced stage, thus understanding the molecular basis for EAC invasion and metastasis is critical. Here we report that SPP1/OPN was highly overexpressed in primary EACs and intracellularly localized to tumor cells. We further demonstrate that all known OPN isoforms (OPNa, b, c, 4 and 5) were frequently co-overexpressed in primary EACs. Distinct pro-invasion and dissemination phenotypes of isoform-specific OPNb and OPNc stable transfectants were observed. Expression of OPNb significantly enhanced cell migration and adhesion to laminin. In contrast, OPNc cells showed significantly decreased cell migration yet increased cell detachment. Enhanced invasion, both in vitro and in vivo, was observed for OPNb- but not OPNc-expressing cells. Inhibition of RGD integrins, one family of OPN receptors, attenuated OPNb cell migration, abrogated OPNb cell adhesion and significantly reduced OPNb cell clonogenic survival but did not affect OPNc phenotypes, indicating that OPNb but not OPNc acts through integrin-dependent signaling. Differential expression of vimentin, E-cadherin and β-catenin in OPN stable cells may account for the variation in cell adhesion and detachment between these isoforms. We conclude that while all OPN isoforms are frequently co-overexpressed in primary EACs, isoforms OPNb and OPNc enhance invasion and dissemination through collective yet distinct mechanisms. PMID:26068949

  8. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    PubMed Central

    Volpi, Nila; Pecorelli, Alessandra; Lorenzoni, Paola; Di Lazzaro, Francesco; Belmonte, Giuseppe; Aglianò, Margherita; Giannini, Fabio; Grasso, Giovanni

    2013-01-01

    Objective. To investigate expression of vascular endothelial growth factor (VEGF) antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM) and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis) and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides. PMID:23840094

  9. Myosin motor isoforms direct specification of actomyosin function by tropomyosins

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Murray, George G.; Lord, Matthew

    2015-01-01

    Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in non-muscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this regulation we examined the role of two mammalian tropomyosins (Tpm3.1cy/Tm5NM1 and Tpm4.2cy/Tm4) recently implicated in cancer cell proliferation and metastasis. Like Cdc8p, the Tpm3.1cy and Tpm4.2cy isoforms significantly enhance Myo2p and Myo52p motor activity, converting non-processive Myo52p molecules into processive motors that can walk along actin tracks as single molecules. In contrast to the positive regulation of Myo2p and Myo52p, Cdc8p and the mammalian tropomyosins potently inhibited skeletal muscle myosin-II, while having negligible effects on the highly processive mammalian myosin-Va. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1cy supported normal contractile ring function in fission yeast. Our work reveals that actomyosin regulation by tropomyosin is dependent on the myosin isoform, highlighting a general role for specific isoforms of tropomyosin in sorting myosin motor outputs. PMID:25712463

  10. Locomotion in Lymphocytes is Altered by Differential PKC Isoform Expression

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    1999-01-01

    Lymphocyte locomotion is critical for proper elicitation of the immune response. Locomotion of immune cells via the interstitium is essential for optimal immune function during wound healing, inflammation and infection. There are conditions which alter lymphocyte locomotion and one of them is spaceflight. Lymphocyte locomotion is severely inhibited in true spaceflight (true microgravity) and in rotating wall vessel culture (modeled microgravity). When lymphocytes are activated prior to culture in modeled microgravity, locomotion is not inhibited and the levels are comparable to those of static cultured lymphocytes. When a phorbol ester (PMA) is used in modeled microgravity, lymphocyte locomotion is restored by 87%. This occurs regardless if PMA is added after culture in the rotating wall vessel or during culture. Inhibition of DNA synthesis also does not alter restoration of lymphocyte locomotion by PMA. PMA is a direct activator of (protein kinase C) PKC . When a calcium ionophore, ionomycin is used it does not possess any restorative properties towards locomotion either alone or collectively with PMA. Since PMA brings about restoration without help from calcium ionophores (ionomycin), it is infer-red that calcium independent PKC isoforms are involved. Changes were perceived in the protein levels of PKC 6 where levels of the protein were downregulated at 24,72 and 96 hours in untreated rotated cultures (modeled microgravity) compared to untreated static (1g) cultures. At 48 hours there is an increase in the levels of PKC & in the same experimental set up. Studies on transcriptional and translational patterns of calcium independent isoforms of PKC such as 8 and E are presented in this study.

  11. The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein*

    PubMed Central

    Martínez-Sernández, Victoria; Mezo, Mercedes; González-Warleta, Marta; Perteguer, María J.; Muiño, Laura; Guitián, Esteban; Gárate, Teresa; Ubeira, Florencio M.

    2014-01-01

    Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10−6 m−1. We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes. PMID:24280214

  12. N-β-glycosyl sulfamides are selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII.

    PubMed

    Rodríguez, Oscar M; Maresca, Alfonso; Témpera, Carlos A; Bravo, Rodolfo D; Colinas, Pedro A; Supuran, Claudiu T

    2011-08-01

    The transmembrane isoforms of carbonic anhydrase (CA IX and XII) have been shown to be linked to carcinogenesis and their inhibition to arrest primary tumor and metastases growth. In this Letter, we present a series of peracetylated and deprotected N-β-glycosyl sulfamides that were tested for the inhibition of 4 carbonic anhydrase isoforms: the cytosolic hCA I and hCA II and transmembrane tumor-associated IX and XII. Compounds 1-4 and 6-8 selectively target cancer-associated CAs (IX and XII) with K(I)s in the low nanomolar range. PMID:21723123

  13. Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with DeltaNp73.

    PubMed

    Petitjean, A; Ruptier, C; Tribollet, V; Hautefeuille, A; Chardon, F; Cavard, C; Puisieux, A; Hainaut, P; Caron de Fromentel, C

    2008-02-01

    TP63, a member of the TP53 gene family, encodes two groups of three isoforms (alpha, beta and gamma). The TAp63 isoforms act as transcription factors. The DeltaNp63 isoforms lack the main transcription activation domain and act as dominant-negative inhibitors of transactivation (TA) isoforms. To clarify the role of these isoforms and to better understand their functional overlap with p53, we ectopically expressed each p63 isoform in the p53-null hepatocellular carcinoma cell line Hep3B. All TA isoforms, as well as DeltaNp63alpha, had a half-life of <1 h when transiently expressed and were degraded by the proteasome pathway. The most stable form was DeltaNp63gamma, with a half-life of >8 h. As expected, TA isoforms differed in their transcriptional activities toward genes regulated by p53, TAp63gamma being the most active form. In contrast, DeltaNp63 isoforms were transcriptionally inactive on genes studied and inhibited TA isoforms in a dose-dependent manner. When stably expressed in polyclonal cell populations, TAp63beta and gamma isoforms were undetectable. However, when treated with doxorubicin (DOX), p63 proteins rapidly accumulated in the cells. This stabilization was associated with an increase in phosphorylation. Strikingly, in DOX-treated polyclonal populations, increase in TAp63 levels was accompanied by overexpression of DeltaNp73. This observation suggests complex regulatory cross talks between the different isoforms of the p53 family. In conclusion, p63 exhibits several transcriptional and stress-response properties similar to those of p53, suggesting that p63 activities should be taken into consideration in approaches to improve cancer therapies based on genotoxic agents. PMID:18048390

  14. Isoform specificity of cardiac glycosides binding to human Na+,K+-ATPase α1β1, α2β1 and α3β1

    PubMed Central

    Hauck, Christian; Potter, Tatjana; Bartz, Michaela; Wittwer, Thorsten; Wahlers, Thorsten; Mehlhorn, Uwe; Scheiner-Bobis, Georgios; McDonough, Alicia A.; Bloch, Wilhelm; Schwinger, Robert H.G.; Müller-Ehmsen, Jochen

    2016-01-01

    Cardiac glycosides inhibit the Na+,K+-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na+,K+-ATPase are expressed: α1β1, α2β1 and α3β1. It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na+,K+-ATPase isoforms α1β1, α2β1 and α3β1 were expressed in yeast which has no endogenous Na+,K+-ATPase. Isoform specific affinities of digoxin, digitoxin, β-acetyldigoxin, methyldigoxin and ouabain were assessed in [3H]-ouabain binding assays in the absence or presence of K+ (each n=5). The subcellular localizations of the Na+,K+-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K+, methyldigoxin (α1>α3>α2) and ouabain (α1=α3>α2) showed distinct isoform specific affinities, while for digoxin, digitoxin and β-acetyldigoxin no differences were found. In the presence of K+, also digoxin (α2=α3>α1) and β-acetyldigoxin (α1>α3) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only α1β1 and α2β1 are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na+,K+-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance. PMID:19751721

  15. GH1-family 6-P-β-glucosidases from human microbiome lactic acid bacteria

    PubMed Central

    Michalska, Karolina; Tan, Kemin; Li, Hui; Hatzos-Skintges, Catherine; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej

    2013-01-01

    In lactic acid bacteria and other bacteria, carbohydrate uptake is mostly governed by phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). PTS-dependent translocation through the cell membrane is coupled with phosphorylation of the incoming sugar. After translocation through the bacterial membrane, the β-glycosidic bond in 6′-­P-­β-glucoside is cleaved, releasing 6-P-β-glucose and the respective aglycon. This reaction is catalyzed by 6-P-β-glucosidases, which belong to two glycoside hydrolase (GH) families: GH1 and GH4. Here, the high-resolution crystal structures of GH1 6-P-β-glucosidases from Lactobacillus plantarum (LpPbg1) and Streptococcus mutans (SmBgl) and their complexes with ligands are reported. Both enzymes show hydrolytic activity towards 6′-P-β-glucosides. The LpPbg1 structure has been determined in an apo form as well as in a complex with phosphate and a glucose molecule corresponding to the aglycon molecule. The S. mutans homolog contains a sulfate ion in the phosphate-dedicated subcavity. SmBgl was also crystallized in the presence of the reaction product 6-P-β-glucose. For a mutated variant of the S. mutans enzyme (E375Q), the structure of a 6′-P-salicin complex has also been determined. The presence of natural ligands enabled the definition of the structural elements that are responsible for substrate recognition during catalysis. PMID:23519420

  16. Apker Award Talk: Atomic Beam Measurement of the Indium 6p1 / 2 Scalar Polarizability

    NASA Astrophysics Data System (ADS)

    Augenbraun, Benjamin

    2016-05-01

    We report on the first measurement of the scalar polarizability of the indium 6p1 / 2 -excited state using two-step laser spectroscopy in an atomic beam. This is one in a series of precise atomic structure measurements by the Majumder lab at Williams College, which serve as stringent tests of abinitio calculation methods for three-valence-electron systems. We stabilize a laser to the indium 5p1 / 2 --> 6s1 / 2 410 nm transition and scan a second laser across the 6s1 / 2 --> 6p1 / 2 1343 nm transition. The two laser beams are overlapped and interact transversely with a collimated atomic beam of indium. Two-tone FM spectroscopy allows us to observe the small (< 1 part in 103) IR absorption, and characteristic sideband features in the RF-demodulated lineshape provide built-in frequency calibration. Application of DC electric fields up to 20 kV/cm give rise to Stark shifts of order 100 MHz. Because our group has previously measured the difference in polarizabilities within the 410 nm transition, we can determine the 6p1 / 2 polarizability with no loss of precision. Preliminary results are in excellent agreement with recent theoretical calculations and can be used to infer accurate values for the indium 6 p - 5 d matrix elements.

  17. Dichlorido(η6-p-cymene)(eth­oxy­diphenyl­phosphane)ruthenium(II)

    PubMed Central

    Knapp, Spring M. M.; Zakharov, Lev N.; Tyler, David R.

    2012-01-01

    The title compound, [RuCl2(C10H14)(C14H15OP)], is an RuII complex in which an η6-p-cymene ligand, two chloride anions and the P atom of an ethoxydiphenylphosphane ligand form a piano-stool coordination environment about the central RuII atom. PMID:23468692

  18. Aberrant Liver Insulin Receptor Isoform A Expression Normalises with Remission of Type 2 Diabetes after Gastric Bypass Surgery

    PubMed Central

    Besic, Vinko; Shi, Hongjun; Stubbs, Richard S.; Hayes, Mark T.

    2015-01-01

    Type 2 diabetes mellitus (T2DM) results from a combination of progressive insulin resistance and loss of pancreatic beta cell function and/or mass. Insulin signalling occurs through the insulin receptor, (INSR) which is alternatively spliced into two isoforms: INSRA (-exon 11) and INSRB (+exon 11). Because the INSR isoforms have different functional characteristics, their relative expression ratio has been implicated in the pathogenesis of insulin resistance and T2DM. We studied levels of INSR isoform mRNA in liver samples taken from 46 individuals with or without T2DM at Roux-en-Y (RYGB) surgery, and on average 17 (± 5.6) months later in 16 of the same individuals (8 diabetic and non-diabetic patients). INSRA or INSRB was also overexpressed in HepG2 cells to ascertain their effect on AKT phosphorylation and PCK1 expression as markers of insulin-mediated metabolic signalling. We found the INSRB:A isoform ratio was reduced in individuals with T2DM in comparison to those with normal glucose tolerance and normalised with remission of diabetes. The INSRB:A ratio increased due to a reduction in the alternatively spliced INSRA isoform following remission of diabetes. Overexpressing INSRA isoform in HepG2 hepatoma cells reduced inhibition of PCK1 transcription and did not increase AKT phosphorylation in response to insulin load compared to the effect of overexpressing the B isoform. Data presented here revitalizes the role of the INSR isoforms in the pathogenesis of T2DM, and suggests that an abrogated INSRB:A ratio that favours the INSRA isoform may negatively impact insulin-mediated metabolic signalling. PMID:25742416

  19. Role of Rho kinase isoforms in murine allergic airway responses.

    PubMed

    Zhu, M; Liu, P-Y; Kasahara, D I; Williams, A S; Verbout, N G; Halayko, A J; Fedulov, A; Shoji, T; Williams, E S; Noma, K; Shore, S A; Liao, J K

    2011-10-01

    Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses. PMID:21565918

  20. BDNF isoforms: a round trip ticket between neurogenesis and serotonin?

    PubMed

    Foltran, Rocío Beatriz; Diaz, Silvina Laura

    2016-07-01

    The brain-derived neurotrophic factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the pro-BDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin, and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. BDNF is synthetized as a pre-proneurotrophin. After removal of the pre-region, proBDNF can be cleaved by intracellular or extracellular proteases. Mature BDNF can bind TrkB receptors, promoting their homodimerization and intracellular phosphorylation. Phosphorylated-TrkB can activate three different signaling pathways. Whereas G-protein-coupled receptors can transactivate TrkB receptors, truncated forms can inhibit mBDNF signaling. Pro-BDNF binds p75(NTR) by its mature domain, whereas the pro-region binds co-receptors. PMID:27167299

  1. CGX1037 is a novel PKC isoform delta selective inhibitor in platelets

    PubMed Central

    BHAVANASI, DHEERAJ; KOSTYAK, JOHN C.; SWINDLE, JOHN; KILPATRICK, LAURIE E.; KUNAPULI, SATYA P.

    2014-01-01

    Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions. Novel PKC isoform δ has been shown to play an important role in some pathological processes. Lack of specific inhibitors for PKCδ has restricted analysis of its role in various cells. The current study was carried out to evaluate a novel small molecule PKCδ inhibitor, CGX1037 in platelets. Platelet aggregation, dense granule secretion and western blotting experiments were performed to evaluate CGX1037. In human platelets, CGX1037 inhibited PAR4-mediated phosphorylation on PKD2, a PKCδ-specific substrate. Pretreatment of human or murine platelets with CGX1037 inhibited PAR4-mediated dense granule secretion whereas it potentiated GPVI-mediated dense granule secretion similar to the responses observed in murine platelets lacking PKCδ Furthermore, pre-treatment of platelets from PKCδ−/− mice with CGX1037 had no significant additive effect on platelet responses suggesting the specificity of CGX1037. Hence, we show that CGX1037 is a selective small molecule inhibitor of PKCδ in platelets. PMID:24433221

  2. The anti-angiogenic isoforms of VEGF in health and disease.

    PubMed

    Qiu, Yan; Hoareau-Aveilla, Coralie; Oltean, Sebastian; Harper, Steven J; Bates, David O

    2009-12-01

    Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF(165)b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGF(xxx)b isoforms to the pro-angiogenic VEGF(xxx) isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGF(xxx)b isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology. PMID:19909248

  3. The anti-angiogenic isoforms of VEGF in health and disease

    PubMed Central

    Qiu, Yan; Hoareau-Aveilla, Coralie; Oltean, Sebastian; Harper, Steven J.; Bates, David O.

    2010-01-01

    Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology. PMID:19909248

  4. Synthesis and Pharmacological Evaluation of 4-Iminothiazolidinones for Inhibition of PI3 Kinase

    PubMed Central

    Pinson, Jo-Anne; Schmidt-Kittler, Oleg; Frazzetto, Mark; Zheng, Zhaohua; Jennings, Ian G.; Kinzler, Kenneth W.; Vogelstein, Bert; Chalmers, David K.; Thompson, Philip E.

    2012-01-01

    The thiazolidinedione, compound 1, has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound 1 could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity. PMID:23997244

  5. Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

    PubMed Central

    Tseng, Yeu-Yang; Lin, Fong-Yuan; Cheng, Sun-Fang; Chulakasian, Songkhla; Chou, Chia-Chi; Liu, Ya-Fen; Chang, Wei-Shan; Wong, Min-Liang

    2015-01-01

    ABSTRACT Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. PMID:25694596

  6. Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

    PubMed Central

    Malathi, K; Xiao, Y; Mitchell, A P

    1997-01-01

    Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex. PMID:9372955

  7. The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles.

    PubMed

    Sivaram, Mylavarapu V S; Furgason, Melonnie L M; Brewer, Daniel N; Munson, Mary

    2006-06-01

    The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite <10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins. PMID:16699513

  8. Apolipoprotein E isoform-dependent microglia migration

    PubMed Central

    Cudaback, Eiron; Li, Xianwu; Montine, Kathleen S.; Montine, Thomas J.; Keene, C. Dirk

    2011-01-01

    Complement component C5a and ATP are potent effectors of microglial movement and are increased in diverse neurodegenerative diseases and at sites of injury. Apolipoprotein E (apoE) influences microglial function, and different human apoE isoforms confer variable risk for development of neurodegenerative disorders, especially Alzheimer's disease. The purpose of this investigation was to test the hypothesis that mouse apoE and human apoE isoforms influence microglial migration. Using primary wild-type and apoE-deficient microglia, we show that C5a- and ATP-stimulated chemotaxis are largely apoE-dependent processes with different molecular bases. Although the C5a-dependent chemotaxis of wild-type microglia was completely blocked by receptor-associated protein (RAP), suggesting apoE receptor involvement, ATP-stimulated migration was unaffected by RAP but was associated with differential ERK phosphorylation. Studies using primary microglia derived from targeted replacement mice “humanized” for the coding exons (protein isoform) of human ε2 (apoE2), ε3 (apoE3), or ε4 (apoE4) allele of APOE revealed that primary mouse microglia expressing apoE4 or apoE2 exhibited significantly reduced C5a- and ATP-stimulated migration compared with microglia expressing human apoE3. This study, for the first time, demonstrates apoE dependence and apoE isoform-specific modulation of microglial migration in response to distinct chemotactic stimuli commonly associated with neurodegenerative disease.—Cudaback, E., Li, X., Montine, K. S., Montine, T. J., Keene, C. D. Apolipoprotein E isoform-dependent microglia migration. PMID:21385991

  9. Sec6p anchors the assembled exocyst complex at sites of secretion.

    PubMed

    Songer, Jennifer A; Munson, Mary

    2009-02-01

    The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two "patches" of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion. PMID:19073882

  10. Nonsyndromic cleft lip and palate: Evidence of linkage to a microsatellite marker on 6p23

    SciTech Connect

    Carinci, F.; Pezzetti, F.; Scapoli, L.; Padula, E.; Baciliero, U.; Curioni, C.; Tognon, M.

    1995-01-01

    Nonsydromic cleft lip with or without secondary clefting of the palate (CL+/{minus}P) is one of the most common birth defects. A previous linkage study concerning CL+/{minus}P and cleft palate (CP) families indicated chromosome 6p, near F13A locus, as a possible region for the presence of a clefting gene. More recently, another linkage study performed on a sample of 12 families with nonsyndromic CL+/{minus}P seemed to exclude this association. To test the hypothesis on the possible presence of a major gene on chromosome 6p, we carried out a study on a large sample (21) of CL+/{minus}P families from northeastern Italy. In conclusion, our investigation can be summarized as follows: (i) CL+/{minus}P disease appears to be heterogeneous; (ii) {approximately}66% of the pedigrees showed an autosomal dominant inheritance with incomplete penetrance; and (iii) CL+/{minus}P locus maps on 6p23 very close to or at the microsatellite marker D6S89. To verify whether the D6S89 is the closest marker to the CL+/{minus}P locus, additional examinations with new markers are underway. 19 refs., 1 fig., 1 tab.

  11. Congenital adrenal hyperplasia, ovarian failure and Ehlers-Danlos syndrome due to a 6p deletion.

    PubMed

    Moysés-Oliveira, Mariana; Mancini, Tatiane I; Takeno, Sylvia S; Rodrigues, Andressa D S; Bachega, Tania A S S; Bertola, Debora; Melaragno, Maria Isabel

    2014-01-01

    Cryptic deletions in balanced de novo translocations represent a frequent cause of abnormal phenotypes, including Mendelian diseases. In this study, we describe a patient with multiple congenital abnormalities, such as late-onset congenital adrenal hyperplasia (CAH), primary ovarian failure and Ehlers-Danlos syndrome (EDS), who carries a de novo t(6;14)(p21;q32) translocation. Genomic array analysis identified a cryptic 1.1-Mb heterozygous deletion, adjacent to the breakpoint on chromosome 6, extending from 6p21.33 to 6p21.32 and affecting 85 genes, including CYP21A2,TNXB and MSH5. Multiplex ligation-dependent probe amplification analysis of the 6p21.3 region was performed in the patient and her family and revealed a 30-kb deletion in the patient's normal chromosome 6, inherited from her mother, resulting in homozygous loss of genes CYP21A1P and C4B. CYP21A2 sequencing showed that its promoter region was not affected by the 30-kb deletion, suggesting that the deletion of other regulatory sequences in the normal chromosome 6 caused a loss of function of the CYP21A2 gene. EDS and primary ovarian failure phenotypes could be explained by the loss of genes TNXB and MSH5, a finding that may contribute to the characterization of disease-causing genes. The detection of this de novo microdeletion drastically reduced the estimated recurrence risk for CAH in the family. PMID:24970489

  12. A novel mechanistic spectrum underlies glaucoma-associated chromosome 6p25 copy number variation

    PubMed Central

    Chanda, Bhaskar; Asai-Coakwell, Mika; Ye, Ming; Mungall, Andrew J.; Barrow, Margaret; Dobyns, William B.; Behesti, Hourinaz; Sowden, Jane C.; Carter, Nigel P.; Walter, Michael A.; Lehmann, Ordan J.

    2008-01-01

    The factors that mediate chromosomal rearrangement remain incompletely defined. Among regions prone to structural variant formation, chromosome 6p25 is one of the few in which disease-associated segmental duplications and segmental deletions have been identified, primarily through gene dosage attributable ocular phenotypes. Using array comparative genome hybridization, we studied ten 6p25 duplication and deletion pedigrees and amplified junction fragments from each. Analysis of the breakpoint architecture revealed that all the rearrangements were non-recurrent, and in contrast to most previous examples the majority of the segmental duplications and deletions utilized coupled homologous and non-homologous recombination mechanisms. One junction fragment exhibited an unprecedented 367 bp insert derived from tandemly arranged breakpoint elements. While this accorded with a recently described replication-based mechanism, it differed from the previous example in being unassociated with template switching, and occurring in a segmental deletion. These results extend the mechanisms involved in structural variant formation, provide strong evidence that a spectrum of recombination, DNA repair and replication underlie 6p25 rearrangements, and have implications for genesis of copy number variations in other genomic regions. These findings highlight the benefits of undertaking the extensive studies necessary to characterize structural variants at the base pair level. PMID:18694899

  13. Synthesis and biological evaluation of novel FK228 analogues as potential isoform selective HDAC inhibitors.

    PubMed

    Narita, Koichi; Matsuhara, Keisuke; Itoh, Jun; Akiyama, Yui; Dan, Singo; Yamori, Takao; Ito, Akihiro; Yoshida, Minoru; Katoh, Tadashi

    2016-10-01

    Novel C4- and C7-modified FK228 analogues were efficiently synthesized in a highly convergent and unified manner. This synthesis features the amide condensation of glycine-d-cysteine-containing segments with d-valine-containing segments for the direct assembly of the corresponding seco-acids, which are key precursors of macrolactones. The HDAC inhibition assay and cell-growth inhibition analysis of the synthesized analogues revealed novel aspects of their structure-activity relationship. This study demonstrated that simple modification at the C4 and C7 side chains in FK228 is effective for improving both HDAC inhibitory activity and isoform selectivity; moreover, potent and highly isoform-selective class I HDAC1 inhibitors were identified. PMID:27318982

  14. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

    PubMed Central

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  15. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    PubMed

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  16. Anti-angiogenic VEGFA164B isoform mRNA is more abundant in E2-inactive, atretic follicles while expression of angiogenic VEGFA isoforms is greater in granulosa cells from developing bovine follicles prior to the LH surge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vascular endothelial growth factor A (VEGFA) is expressed by granulosa cells of the follicle and if its actions are blocked, ovulation and antral follicle development is inhibited. However, the role of anti-angiogenic VEGFA isoforms in bovine dominant follicle development, especially prior to and a...

  17. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    SciTech Connect

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  18. RELATIONSHIP BETWEEN BRAIN AND OVARY AROMATASE ACTIVITY AND ISOFORM-SPECIFIC AROMATASE MRNA EXPRESSION IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

    EPA Science Inventory

    There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead min...

  19. Tau Interconverts Between Diffusive and Stable Populations on the Microtubule Surface in an Isoform and Lattice Specific Manner

    PubMed Central

    McVicker, Derrick P.; Hoeprich, Gregory J.; Thompson, Andrew R.; Berger, Christopher L.

    2014-01-01

    It has been demonstrated that Tau exists on the microtubule lattice in both diffusing and static populations, but how this may relate to Tau function is currently unclear. Tau isoforms are developmentally regulated and have been shown to have disparate effects on microtubule polymerization, the ability to bind microtubules, and the ability to inhibit kinesin. It has also been shown that Tau is sensitive to microtubule stabilizing agents and the ability to affect the persistence length of microtubules and to inhibit kinesin can be altered by stabilizing microtubules with various nucleotide analogs. Given these observations, it is likely the behavior of Tau is dictated by both the isoform of Tau and by structural changes in the microtubule lattice. In the present study, we use single molecule imaging to examine the behavior of the three-repeat short (3RS) isoform and the four-repeat long (4RL) isoform on different microtubule tracks stabilized with either paclitaxel or guanylyl-(α, β)-methylene-diphosphate (GMPCPP). On paclitaxel-stabilized microtubules, we find 3RS-Tau favors the static conformation and forms complexes consisting of 2–3 molecules, while 4RL-Tau predominantly exists as a single molecule equally distributed between the static and diffusing populations. However, on GMPCPP-stabilized microtubules both isoforms favor the diffusing conformation and do not form static complexes composed of more than one Tau molecule. We find both isoforms of Tau interconvert between static and diffusing populations on the microtubule surface, and the equilibrium between these two states depends on both the isoform of Tau and the structure of the underlying microtubule lattice. PMID:24520046

  20. VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

    PubMed Central

    2010-01-01

    Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into

  1. Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum--over expression and modification of G6P dehydrogenase.

    PubMed

    Becker, Judith; Klopprogge, Corinna; Herold, Andrea; Zelder, Oskar; Bolten, Christoph J; Wittmann, Christoph

    2007-10-31

    In the present work, metabolic flux engineering of Corynebacterium glutamicum was carried out to increase lysine production. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. Over expression of the zwf gene, encoding G6P dehydrogenase, in the feedback-deregulated lysine-producing strain C. glutamicum ATCC 13032 lysC(fbr) resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose. The additional introduction of the A243T mutation into the zwf gene and the over expression of fructose 1,6-bisphosphatase resulted in a further successive improvement of lysine production. Hereby the point mutation resulted in higher affinity of G6P dehydrogenase towards NADP and reduced sensitivity against inhibition by ATP, PEP and FBP. Overall, the lysine yield increased up to 70% through the combination of the different genetic modifications. Through strain engineering formation of trehalose was reduced by up to 70% due to reduced availability of its precursor G6P. Metabolic flux analysis revealed a 15% increase of PPP flux in response to over expression of the zwf gene. Overall a strong apparent NADPH excess resulted. Redox balancing indicated that this excess is completely oxidized by malic enzyme. PMID:17624457

  2. Comparative methylome analysis in solid tumors reveals aberrant methylation at chromosome 6p in nasopharyngeal carcinoma

    PubMed Central

    Dai, Wei; Cheung, Arthur Kwok Leung; Ko, Josephine Mun Yee; Cheng, Yue; Zheng, Hong; Ngan, Roger Kai Cheong; Ng, Wai Tong; Lee, Anne Wing Mui; Yau, Chun Chung; Lee, Victor Ho Fu; Lung, Maria Li

    2015-01-01

    Altered patterns of DNA methylation are key features of cancer. Nasopharyngeal carcinoma (NPC) has the highest incidence in Southern China. Aberrant methylation at the promoter region of tumor suppressors is frequently reported in NPC; however, genome-wide methylation changes have not been comprehensively investigated. Therefore, we systematically analyzed methylome data in 25 primary NPC tumors and nontumor counterparts using a high-throughput approach with the Illumina HumanMethylation450 BeadChip. Comparatively, we examined the methylome data of 11 types of solid tumors collected by The Cancer Genome Atlas (TCGA). In NPC, the hypermethylation pattern was more dominant than hypomethylation and the majority of de novo methylated loci were within or close to CpG islands in tumors. The comparative methylome analysis reveals hypermethylation at chromosome 6p21.3 frequently occurred in NPC (false discovery rate; FDR=1.33 × 10−9), but was less obvious in other types of solid tumors except for prostate and Epstein–Barr virus (EBV)-positive gastric cancer (FDR<10−3). Bisulfite pyrosequencing results further confirmed the aberrant methylation at 6p in an additional patient cohort. Evident enrichment of the repressive mark H3K27me3 and active mark H3K4me3 derived from human embryonic stem cells were found at these regions, indicating both DNA methylation and histone modification function together, leading to epigenetic deregulation in NPC. Our study highlights the importance of epigenetic deregulation in NPC. Polycomb Complex 2 (PRC2), responsible for H3K27 trimethylation, is a promising therapeutic target. A key genomic region on 6p with aberrant methylation was identified. This region contains several important genes having potential use as biomarkers for NPC detection. PMID:25924914

  3. Strategies for the Discovery of Target-Specific or Isoform-Selective Modulators.

    PubMed

    Zhan, Peng; Itoh, Yukihiro; Suzuki, Takayoshi; Liu, Xinyong

    2015-10-01

    Currently, the creation of class- and isoform-selective modulators of biologically important targets is a particularly challenging problem because different isoforms within a protein family often show striking similarity in spatial quaternary structure, especially at the catalytic sites or binding pockets. Therefore, an understanding of both the precise three-dimensional structure of the target protein and the mechanisms of action of modulators is important for developing more effective and selective agents. In this Perspective, we discuss currently available rational design strategies for obtaining class- and isoform-selective inhibitors and we illustrate these strategies with the aid of specific examples from the recent literature. The strategies covered include: (1) target-derived (-dependent) de novo drug discovery methodologies, and (2) follow-on derivatization approaches from initially identified active molecules (hit-to-lead and lead-to-candidate efforts). We also comment on prospects for further development and integration of strategies to achieve target-specific or isoform-selective inhibition. PMID:26086931

  4. Isoform-selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases.

    PubMed

    Court, Michael H

    2005-01-01

    The majority of UDP-glucuronosyltransferases (UGT), like other drug-metabolizing enzymes, display broad and often overlapping substrate specificities, complicating evaluation of the function of individual UGT isoforms within human tissues. Despite this, there have been recent advances in identifying UGT-selective probes--UGT substrates that are primarily glucuronidated by a single isoform. Such probes can be used to (1) facilitate identification of UGT isoforms mediating a particular glucuronidation activity in human liver through activity correlation analysis; (2) evaluate the role of particular UGTs in drug-drug interactions through either enzyme induction or inhibition; and (3) elucidate the functional significance of genetic polymorphisms associated with the gene encoding the UGT of interest. UGT-selective probes currently being used in our laboratory for the evaluation of glucuronidation activities in human liver tissues include estradiol (3OH-glucuronidation; UGT1A1), trifluoperazine (UGT1A4) serotonin (UGT1A6), propofol (UGT1A9), 3'-azidothymidine (UGT2B7), and S-oxazepam (UGT2B15). In vitro incubation protocols and the HPLC analysis methods used to determine each of these activities are described in detail. Future work is needed to elucidate more highly selective probes than those in current usage, as well as probes for the extrahepatic UGT isoforms. PMID:16399346

  5. An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.

    PubMed

    Kikuchi, Ryosuke; Nakamura, Kazuto; MacLauchlan, Susan; Ngo, Doan Thi-Minh; Shimizu, Ippei; Fuster, Jose Javier; Katanasaka, Yasufumi; Yoshida, Sumiko; Qiu, Yan; Yamaguchi, Terry P; Matsushita, Tadashi; Murohara, Toyoaki; Gokce, Noyan; Bates, David O; Hamburg, Naomi M; Walsh, Kenneth

    2014-12-01

    Peripheral artery disease (PAD) generates tissue ischemia through arterial occlusions and insufficient collateral vessel formation. Vascular insufficiency in PAD occurs despite higher circulating levels of vascular endothelial growth factor A (VEGF-A), a key regulator of angiogenesis. Here we show that clinical PAD is associated with elevated levels of an antiangiogenic VEGF-A splice isoform (VEGF-A165b) and a corresponding reduction in levels of the proangiogenic VEGF-A165a splice isoform. In mice, VEGF-A165b expression was upregulated by conditions associated with impaired limb revascularization, including leptin deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells. In a mouse model of PAD, delivery of VEGF-A165b inhibited revascularization of ischemic hind limbs, whereas treatment with an isoform-specific neutralizing antibody reversed impaired revascularization caused by metabolic dysfunction or perturbations in the Wnt5a-Sfrp5 regulatory system. These results indicate that inflammation-driven expression of the antiangiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease. PMID:25362254

  6. Metallicity of Ca2Cu6P5 with single and double copper-pnictide layers

    DOE PAGESBeta

    Li, Li; Parker, David; Chi, Miaofang; Tsoi, Georgiy M.; Vohra, Yogesh K.; Sefat, Athena S.

    2016-02-16

    Here, we report thermodynamic and transport properties, and also theoretical calculations, for Cu-based compound Ca2Cu6P5 and compare with CaCu2-dP2. Both materials have layers of edge-sharing copper pnictide tetrahedral CuP4, similar to Fe–As and Fe–Se layers (with FeAs4, FeSe4) in the iron-based superconductors. Despite the presence of this similar transition-metal pnictide layer, we find that both Ca2Cu6P5 and CaCu2-δP2 have temperature-independent magnetic susceptibility and show metallic behavior with no evidence of either magnetic ordering or superconductivity down to 1.8 K CaCu2-δP2 is slightly off-stoichiometric, with δ = 0.14. Theoretical calculations suggest that unlike Fe 3d-based magnetic materials with a large densitymore » of states (DOS) at the Fermi surface, Cu have comparatively low DOS, with the majority of the 3d spectral weight located well below Fermi level. The room-temperature resistivity value of Ca2Cu6P5 is only 9 μΩ-cm, due to a substantial plasma frequency and an inferred electron-phonon coupling λ of 0.073 (significantly smaller than that of metallic Cu). Also, microscopy result shows that Cu–Cu distance along the c-axis within the double layers can be very short (2.5 Å), even shorter than metallic elemental copper bond (2.56 Å). The value of dρ/dT for CaCu2-δP2 at 300 K is approximately three times larger than in Ca2Cu6P5, which suggests the likelihood of stronger electron-phonon coupling. This study shows that the details of Cu-P layers and bonding are important for their transport characteristics. In addition, it emphasizes the remarkable character of the DOS of ‘122’ iron-based materials, despite much structural similarities.« less

  7. Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

    PubMed

    Yao, Lin-Lin; Shen, Mei; Lu, Zekuan; Ikebe, Mitsuo; Li, Xiang-dong

    2016-04-01

    Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5. PMID:26912658

  8. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    PubMed

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway. PMID:17588539

  9. Structural Basis of Dscam Isoform Specificity

    SciTech Connect

    Meijers,R.; Puettmann-Holgado, R.; Skiniotis, G.; Liu, J.; Walz, T.; Wang, J.; Schmucker, D.

    2007-01-01

    The Dscam gene gives rise to thousands of diverse cell surface receptors1 thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

  10. The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis

    PubMed Central

    Ibrahim, Sulaiman S.; Riveron, Jacob M.; Stott, Robert; Irving, Helen; Wondji, Charles S.

    2016-01-01

    Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools. PMID:26548743

  11. The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis.

    PubMed

    Ibrahim, Sulaiman S; Riveron, Jacob M; Stott, Robert; Irving, Helen; Wondji, Charles S

    2016-01-01

    Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools. PMID:26548743

  12. Role of nuclear progesterone receptor isoforms in uterine pathophysiology

    PubMed Central

    Patel, Bansari; Elguero, Sonia; Thakore, Suruchi; Dahoud, Wissam; Bedaiwy, Mohamed; Mesiano, Sam

    2015-01-01

    BACKGROUND Progesterone is a key hormonal regulator of the female reproductive system. It plays a major role to prepare the uterus for implantation and in the establishment and maintenance of pregnancy. Actions of progesterone on the uterine tissues (endometrium, myometrium and cervix) are mediated by the combined effects of two progesterone receptor (PR) isoforms, designated PR-A and PR-B. Both receptors function primarily as ligand-activated transcription factors. Progesterone action on the uterine tissues is qualitatively and quantitatively determined by the relative levels and transcriptional activities of PR-A and PR-B. The transcriptional activity of the PR isoforms is affected by specific transcriptional coregulators and by PR post-translational modifications that affect gene promoter targeting. In this context, appropriate temporal and cell-specific expression and function of PR-A and PR-B are critical for normal uterine function. METHODS Relevant studies describing the role of PRs in uterine physiology and pathology (endometriosis, uterine leiomyoma, endometrial cancer, cervical cancer and recurrent pregnancy loss) were comprehensively searched using PubMed, Cochrane Library, Web of Science, and Google Scholar and critically reviewed. RESULTS Progesterone, acting through PR-A and PR-B, regulates the development and function of the endometrium and induces changes in cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs promotes myometrial relaxation and cervical closure. Withdrawal of PR-mediated progesterone signaling triggers menstruation and parturition. PR-mediated progesterone signaling is anti-mitogenic in endometrial epithelial cells, and as such, mitigates the tropic effects of estrogen on eutopic normal endometrium, and on ectopic implants in endometriosis. Similarly, ligand-activated PRs function as tumor suppressors in endometrial cancer cells through inhibition of key

  13. Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins

    PubMed Central

    Gorynia, Sabine; Lorenz, Todd C.; Costaguta, Giancarlo; Daboussi, Lydia; Cascio, Duilio; Payne, Gregory S.

    2012-01-01

    Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6∆ cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein–like family. PMID:22993212

  14. Functions of sensor 1 and sensor 2 regions of Saccharomyces cerevisiae Cdc6p in vivo and in vitro.

    PubMed

    Takahashi, Naoko; Tsutsumi, Shinji; Tsuchiya, Tomofusa; Stillman, Bruce; Mizushima, Tohru

    2002-05-01

    Cdc6p is a key regulator of the cell cycle in eukaryotes and is a member of the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins. In this family of proteins, the sensor 1 and sensor 2 regions are important for their function and ATPase activity. Here, site-directed mutagenesis has been used to examine the role of these regions of Saccharomyces cerevisiae Cdc6p in controlling the cell cycle progression and initiation of DNA replication. Two important amino acid residues (Asn(263) in sensor 1 and Arg(332) in sensor 2) were identified as key residues for Cdc6p function in vivo. Cells expressing mutant Cdc6p (N263A or R332E) grew slowly and accumulated in the S phase. In cells expressing mutant Cdc6p, loading of the minichromosome maintenance (MCM) complex of proteins was decreased, suggesting that the slow progression of S phase in these cells was due to inefficient MCM loading on chromatin. Purified wild type Cdc6p but not mutant Cdc6p (N263A and R332E) caused the structural modification of origin recognition complex proteins. These results are consistent with the idea that Cdc6p uses its ATPase activity to change the conformation of origin recognition complex, and then together they recruit the MCM complex. PMID:11827963

  15. Structural Basis and Biological Consequences for JNK2/3 Isoform Selective Aminopyrazoles

    PubMed Central

    Park, HaJeung; Iqbal, Sarah; Hernandez, Pamela; Mora, Rudy; Zheng, Ke; Feng, Yangbo; LoGrasso, Philip

    2015-01-01

    Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death. PMID:25623238

  16. A gene for autosomal dominant congenital nystagmus localizes to 6p12

    SciTech Connect

    Kerrison, J.B.; Arnould, V.J.; Koenekoop, R.K.

    1996-05-01

    Congenital nystagmus is an idiopathic disorder characterized by bilateral ocular oscillations usually manifest during infancy. Vision is typically decreased due to slippage of images across the fovea. As such, visual acuity correlates with nystagmus intensity, which is the amplitude and frequency of eye movements at a given position of gaze. X-linked, autosomal dominant, and autosomal recessive pedigrees have been described, but no mapping studies have been published. We recently described a large pedigree with autosomal dominant congenital nystagmus. A genome-wide search resulted in six markers on 6p linked by two-point analysis at {theta} = 0 (D6S459, D6S452, D6S465, FTHP1, D6S257, D6S430). Haplotype analysis localizes the gene for autosomal dominant congenital motor mystagmus to an 18-cM region between D6S271 and D6S455. 16 refs., 1 fig., 1 tab.

  17. Concordance between Parental Origin of Chromosome 13q Loss and 6p Duplication in Sporadic Retinoblastoma

    PubMed Central

    Naumova, A.; Hansen, M.; Strong, L.; Jones, P. A.; Hadjistilianou, D.; Mastrangelo, D.; Griegel, S.; Rajewsky, M. F.; Shields, J.; Donoso, L.; Wang, M.; Sapienza, C.

    1994-01-01

    Two hypotheses are capable of explaining nonrandom loss of one parent's alleles at tumor suppressor loci in sporadic cases of several pediatric cancers, including retinoblastoma—namely, preferential germ-line mutation or chromosome imprinting. We have examined 74 cases of sporadic retinoblastoma for tumors in which at least two genetic events—loss of heterozygosity for chromosome 13q markers and formation of an isochromosome 6p—have occurred. Sixteen cases were found to contain both events. In 13 of 16 such tumors, the chromosomes 13q that were lost and chromosomes 6p that were duplicated are derived from the same parent. These data may be explained within the framework of the genome imprinting model but are not predicted by preferential germ-line mutation. ImagesFigure 2Figure 3 PMID:8304344

  18. Fine genetic mapping of a gene for autosomal recessive retinitis pigmentosa on chromosome 6p21

    SciTech Connect

    Shugart, Yin Y.; Banerjee, P.; Knowles, J.A.

    1995-08-01

    The inherited retinal degenerations known as retinitis pigmentosa (RP) can be caused by mutations at many different loci and can be inherited as an autosomal recessive, autosomal dominant, or X-linked recessive trait. Two forms of autosomal recessive (arRP) have been reported to cosegregate with mutations in the rhodopsin gene and the beta-subunit of rod phosphodiesterase on chromosome 4p. Genetic linkage has been reported on chromosomes 6p and 1q. In a large Dominican family, we reported an arRp gene near the region of the peripherin/RDS gene. Four recombinations were detected between the disease locus and an intragenic marker derived from peripherin/RDS. 26 refs., 2 figs., 1 tab.

  19. Juvenile myoclonic epilepsy in chromosome 6p12-p11: Locus heterogeneity and recombinations

    SciTech Connect

    Liu, A.W.; Delgado-Escueta, A.V.; Serratosa, J.M.

    1996-06-14

    We recently analyzed under homogeneity a large pedigree from Belize with classic juvenile myoclonic epilepsy (JME). After a genome-wide search with 146 microsatellites, we obtained significant linkage between chromosome 6p markers, D6S257 and D6S272, and both convulsive and EEG traits of JME. Recombinations in two affected members defined a 40 cM JME region flanked by D6S313 and D6S258. In the present communication, we explored if the same chromosome 6p11 microsatellites also have a role in JME mixed with pyknoleptic absences. We allowed for heterogeneity during linkage analyses. We tested for heterogeneity by the admixture test and looked for more recombinations. D6S272, D6S466, D6S294, and D6S257 were significantly linked (Z{sub max} > 3.5) to the clinical and EEG traits of 22 families, assuming autosomal dominant inheritance with 70% penetrance. Pairwise Z{sub max} were 4.230 for D6S294 ({theta}{sub m=f} at 0.133) and 4.442 for D6S466 ({theta}{sub m=f} at 0.111). Admixture test (H{sub 2} vs. H{sub 1}) was significant (P = 0.0234 for D6S294 and 0.0128 for D6S272) supporting the hypotheses of linkage with heterogeneity. Estimated proportion of linked families, {alpha}, was 0.50 (95% confidence interval 0.05-0.99) for D6S294 and D6S272. Multipoint analyses and recombinations in three new families narrowed the JME locus to a 7 cM interval flanked by D6S272 and D6S257. 44 refs., 3 figs., 4 tabs.

  20. Genetic Rearrangements of Six Wheat–Agropyron cristatum 6P Addition Lines Revealed by Molecular Markers

    PubMed Central

    Su, Junji; Zhang, Jinpeng; Song, Liqiang; Gao, Ainong; Yang, Xinming; Li, Xiuquan; Liu, Weihua; Li, Lihui

    2014-01-01

    Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP) not only is cultivated as pasture fodder but also could provide many desirable genes for wheat improvement. It is critical to obtain common wheat–A. cristatum alien disomic addition lines to locate the desired genes on the P genome chromosomes. Comparative analysis of the homoeologous relationships between the P genome chromosome and wheat genome chromosomes is a key step in transferring different desirable genes into common wheat and producing the desired alien translocation line while compensating for the loss of wheat chromatin. In this study, six common wheat–A. cristatum disomic addition lines were produced and analyzed by phenotypic examination, genomic in situ hybridization (GISH), SSR markers from the ABD genomes and STS markers from the P genome. Comparative maps, six in total, were generated and demonstrated that all six addition lines belonged to homoeologous group 6. However, chromosome 6P had undergone obvious rearrangements in different addition lines compared with the wheat chromosome, indicating that to obtain a genetic compensating alien translocation line, one should recombine alien chromosomal regions with homoeologous wheat chromosomes. Indeed, these addition lines were classified into four types based on the comparative mapping: 6PI, 6PII, 6PIII, and 6PIV. The different types of chromosome 6P possessed different desirable genes. For example, the 6PI type, containing three addition lines, carried genes conferring high numbers of kernels per spike and resistance to powdery mildew, important traits for wheat improvement. These results may prove valuable for promoting the development of conventional chromosome engineering techniques toward molecular chromosome engineering. PMID:24595330

  1. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    SciTech Connect

    Ivanov, Sergey V.; Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I.

    2009-05-08

    Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

  2. Na(+),K(+)-ATPase isoform selectivity for digitalis-like compounds is determined by two amino acids in the first extracellular loop.

    PubMed

    Weigand, Karl M; Laursen, Mette; Swarts, Herman G P; Engwerda, Anthonius H J; Prüfert, Christian; Sandrock, Julia; Nissen, Poul; Fedosova, Natalya U; Russel, Frans G M; Koenderink, Jan B

    2014-12-15

    Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na(+),K(+)-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na(+),K(+)-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed α1 isoform and the more restricted α2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the α2 isoform, we were able to link residues Met(119) and Ser(124) to differences in affinity between the α1 and α2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups. PMID:25361285

  3. Design and Synthesis of Simplified Largazole Analogues as Isoform-Selective Human Lysine Deacetylase Inhibitors.

    PubMed

    Reddy, Damodara N; Ballante, Flavio; Chuang, Timothy; Pirolli, Adele; Marrocco, Biagina; Marshall, Garland R

    2016-02-25

    Selective inhibition of KDAC isoforms while maintaining potency remains a challenge. Using the largazole macrocyclic depsipeptide structure as a starting point for developing new KDACIs with increased selectivity, a combination of four different simplified largazole analogue (SLA) scaffolds with diverse zinc-binding groups (for a total of 60 compounds) were designed, synthesized, and evaluated against class I KDACs 1, 3, and 8, and class II KDAC6. Experimental evidence as well as molecular docking poses converged to establish the cyclic tetrapeptides (CTPs) as the primary determinant of both potency and selectivity by influencing the correct alignment of the zinc-binding group in the KDAC active site, providing a further basis for developing new KDACIs of higher isoform selectivity and potency. PMID:26681404

  4. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  5. Decoding RAS isoform and codon-specific signalling

    PubMed Central

    Newlaczyl, Anna U.; Hood, Fiona E.; Coulson, Judy M.; Prior, Ian A.

    2014-01-01

    RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms. PMID:25109951

  6. Microglial Signaling in Chronic Pain with a Special Focus on Caspase 6, p38 MAP Kinase, and Sex Dependence.

    PubMed

    Berta, T; Qadri, Y J; Chen, G; Ji, R R

    2016-09-01

    Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6-a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia-is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some

  7. Expression profile of Wilms Tumor 1 (WT1) isoforms in undifferentiated and all-trans retinoic acid differentiated neuroblastoma cells

    PubMed Central

    Maugeri, Grazia; D'Amico, Agata Grazia; Rasà, Daniela Maria; Reitano, Rita; Saccone, Salvatore; Federico, Concetta; Parenti, Rosalba; Magro, Gaetano; D'Agata, Velia

    2016-01-01

    Wilms tumor 1 gene (WT1) is a tumor suppressor gene originally identified in nephroblastoma. It is also expressed in neuroblastoma which represents the most aggressive extracranial pediatric tumor. Many evidences have shown that neuroblastoma may undergo maturation, by transforming itself in a more differentiated tumors such as ganglioneuroblastoma and ganglioneuroma, or progressing into a highly aggressive metastatic malignancy. To date, 13 WT1 mRNA alternative splice variants have been identified. However, most of the studies have focused their attention only on isoform of ∼49 kDa. In the present study, it has been investigated the expression pattern of WT1 isoforms in an in vitro model of neuroblastoma consisting in undifferentiated or all-trans retinoic acid (RA) differentiated cells. These latter representing the less malignant phenotype of this tumor. Results have demonstrated that WT1.1-WT1.5, WT1.6-WT1.9, WT1.10 WT1.11-WT1.12 and WT1.13 isoforms are expressed in both groups of cells, but their levels are significantly increased after RA treatment. These data have also been confirmed by immunofluorescence analysis. Moreover, the inhibition of PI3K/Akt and MAPK/ERK, that represent two signalling pathway specifically involved in NB differentiation, induces an overexpression of WT1 isoforms. These data suggest that WT1 isoforms might be involved in differentiation of neuroblastic into mature ganglion cells. PMID:27014421

  8. Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear.

    PubMed

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J; Striessnig, Jörg; Singewald, Nicolas

    2008-05-01

    Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice. PMID:18441296

  9. Separation of plasmid DNA isoforms using centrifugal ultrafiltration.

    PubMed

    Borujeni, Ehsan Espah; Zydney, Andrew L

    2012-07-01

    Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms. PMID:22780319

  10. Enantiomers of Naringenin as Pleiotropic, Stereoselective Inhibitors of Cytochrome P450 Isoforms

    PubMed Central

    Lu, Wenjie Jessie; Ferlito, Valentina; Xu, Cong; Flockhart, David A; Caccamese, Salvatore

    2011-01-01

    Interactions between naringenin and the cytochrome P450 (CYP) system have been of interest since the first demonstration that grapefruit juice reduced CYP3A activity. The effects of naringenin on other CYP isoforms have been less investigated. In addition, it is well known that interactions with enzymes are often stereospecific, but due to the lack of readily available, chirally pure naringenin enantiomers, the enantioselectivity of its effects has not been characterized. We isolated pure naringenin enantiomers by chiral HPLC and tested the ability of (R)-, (S)-and rac-naringenin to inhibit several important drug-metabolizing CYP isoforms using recombinant enzymes and pooled human liver microsomes. Naringenin was able to inhibit CYP19, CYP2C9 and CYP2C19 with IC50 values below 5 μM. No appreciable inhibition of CYP2B6 or CYP2D6 was observed at concentrations up to 10 μM. While (S)-naringenin was 2-fold more potent as an inhibitor of CYP19 and CYP2C19 than (R)-naringenin, (R)-naringenin was 2-fold more potent for CYP2C9 and CYP3A. Chiral flavanones like naringenin are difficult to separate into their enantiomeric forms, but enantioselective effects may be observed that ultimately impact clinical effects. Inhibition of specific drug metabolizing enzymes by naringenin observed in vitro may be exploited to understand pharmacokinetic changes seen in vivo. PMID:21953762

  11. Luminescence spectroscopy of matrix-isolated z 6P state atomic manganese.

    PubMed

    Collier, Martin A; McCaffrey, John G

    2005-05-01

    The relaxation of electronically excited atomic manganese isolated in solid rare gas matrices is observed from recorded emission spectra, to be strongly site specific. z 6P state excitation of Mn atoms isolated in the red absorption site in Ar and Kr produces narrow a 4D and a 6D state emissions while blue-site excitation produces z 6P state fluorescence and broadened a 4D and a 6D emissions. MnXe exhibits only a single thermally stable site whose emission at 620 nm is similar to the broad a 6D bands produced with blue-site excitation in Ar and Kr. Thus in Ar(Kr), excitation of the red site at 393 (400) nm produces narrow line emissions at 427.5 (427.8) and 590 (585.7) nm. From their spectral positions, linewidths, and long decay times, these emission bands are assigned to the a 4D72 and a 6D92 states, respectively. Excitation of the blue site at 380 (385.5) nm produces broad emission at 413 (416) nm which, because of its nanosecond radiative lifetime, is assigned to resonance z 6P --> a 6S fluorescence. Emission bands at 438 (440) and 625 (626.8) nm, also produced with blue-site excitation, are broader than their red-site equivalents at 427.5 and 590 nm (427.8 and 585.7 nm in Kr) but from their millisecond and microsecond decay times are assigned to the a 4D and a 6D states. The line features observed in high resolution scans of the red-site emission at 427.5 and 427.8 nm in MnAr and MnKr, respectively, have been analyzed with the W(p) optical line shape function and identified as resolved phonon structure originating from very weak (S=0.4) electron-phonon coupling. The presence of considerable hot-phonon emission (even in 12 K spectra) and the existence of crystal field splittings of 35 and 45 cm(-1) on the excited a 4D72 level in Ar and Kr matrices have been identified in W(p) line shape fits. The measured matrix lifetimes for the narrow red-site a 6D state emissions (0.29 and 0.65 ms) in Ar and Kr are much shorter than the calculated (3 s) gas phase value. With

  12. Extracellular and Luminal pH Regulation by Vacuolar H+-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes*

    PubMed Central

    Smith, Gina A.; Howell, Gareth J.; Phillips, Clair; Muench, Stephen P.; Ponnambalam, Sreenivasan; Harrison, Michael A.

    2016-01-01

    Plasma membrane vacuolar H+-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a1 and a3 the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a1 was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a3 was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a1 and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a1 and a3 isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype. PMID:26912656

  13. Evidence for a gene influencing reading disability on chromosome 6p in two populations

    SciTech Connect

    Smith, S.D.; Brower, A.M.; Kimberling, W.J.

    1994-09-01

    A genetic contribution to specific reading disability has been demonstrated by twin studies, and segregation analysis has supported a dominant gene effect. In an effort to localize genes influencing reading disability, we have ascertained two independent populations of families: kindreds which were selected to have a three generation history of reading diability in an autosomal dominant pattern; and families with dizygotic twins, at least one of which has been diagnosed with reading disability. All available family members were given a battery of tests to measure reading and spelling ability and intelligence, and qualitative and quantitative phenotypes for reading disability were derived. Blood samples were obtained for genotyping on all consenting family members, in concordance with IRB requirements. Linkage analysis was performed by the sib pair method utilizing the S.A.G.E. (1992) package and by a differential regression technique developed by DeFries and Fulker (1985). In both populations and by both linkage techniques, several markers in the HLA region of chromosome 6p showed results suggestive of linkage, with the effect most pronounced in the twin families. Significance levels were enhanced when only the more severely affected subjects were analyzed with the differential regression technique.

  14. Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase

    PubMed Central

    Brehm, Maria A.; Schenk, Tobias M. H.; Zhou, Xuefei; Fanick, Werner; Lin, Hongying; Windhorst, Sabine; Nalaskowski, Marcus M.; Kobras, Mario; Shears, Stephen B.; Mayr, Georg W.

    2007-01-01

    InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)–IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K. PMID:17705785

  15. Phosphodiesterase Isoform Regulation of Cell Proliferation and Fluid Secretion in Autosomal Dominant Polycystic Kidney Disease.

    PubMed

    Pinto, Cibele S; Raman, Archana; Reif, Gail A; Magenheimer, Brenda S; White, Corey; Calvet, James P; Wallace, Darren P

    2016-04-01

    cAMP stimulates cell proliferation and Cl(-)-dependent fluid secretion, promoting the progressive enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). Intracellular cAMP levels are determined by the balance of cAMP synthesis by adenylyl cyclases and degradation by phosphodiesterases (PDEs). Therefore, PDE isoform expression and activity strongly influence global and compartmentalized cAMP levels. We report here that PDE3 and PDE4 expression levels are lower in human ADPKD tissue and cells compared with those of normal human kidneys (NHKs), whereas PDE1 levels are not significantly different. Inhibition of PDE4 caused a greater increase in basal and vasopressin (AVP)-stimulated cAMP levels and Cl(-) secretion by ADPKD cells than inhibition of PDE1, and inhibition of PDE4 induced cyst-like dilations in cultured mouse Pkd1(-/-) embryonic kidneys. In contrast, inhibition of PDE1 caused greater stimulation of extracellular signal-regulated kinase (ERK) and proliferation of ADPKD cells than inhibition of PDE4, and inhibition of PDE1 enhanced AVP-induced ERK activation. Notably, inhibition of PDE1, the only family of Ca(2+)-regulated PDEs, also induced a mitogenic response to AVP in NHK cells, similar to the effect of restricting intracellular Ca(2+). PDE1 coimmunoprecipitated with B-Raf and A-kinase anchoring protein 79, and AVP increased this interaction in ADPKD but not NHK cells. These data suggest that whereas PDE4 is the major PDE isoform involved in the regulation of global intracellular cAMP and Cl(-) secretion, PDE1 specifically affects the cAMP signal to the B-Raf/MEK/ERK pathway and regulates AVP-induced proliferation of ADPKD cells. PMID:26289612

  16. Opposing roles of the two isoforms of ErbB3 binding protein 1 in human cancer cells.

    PubMed

    Ko, Hyo Rim; Chang, Yun Sil; Park, Won Soon; Ahn, Jee-Yin

    2016-09-15

    The different functions of the two isoforms of ErbB3 binding protein 1 (Ebp1), p48 and p42, have recently become the focus of interest as they reveal contradictory roles in cell growth promoting ability. The conformational change that crystal structure of p42 was shown to lack α helices at the amino-terminus present in p48 represents the differential binding partners and protein modifications of two Ebp1 isoforms. N-terminal specific phosphorylation by CDK2 and deregulation of the p53 tumor suppressor through specific interaction with HDM2 and Akt activation is postulated to contribute to p48-mediated tumorigenesis. The short isoform p42 Ebp1, which is actual binding partner of ErbB3 has been implicated as a tumor suppressor with many binding partners such as Rb, HDAC2, Sin3A and the p85 subunit of PI3K with HSP70/CHIP, inhibiting its own antiproliferative activity or inhibiting PI3K activity. The aim of the current review is to provide a summary on distinctive cellular functions of two Ebp1 proteins and their molecular partners that might be responsible for the unique functions of each isoform of Ebp1. PMID:27130196

  17. Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS.

    PubMed

    Zhang, Guigen; Chan, Baca; Samarina, Naira; Abere, Bizunesh; Weidner-Glunde, Magdalena; Buch, Anna; Pich, Andreas; Brinkmann, Melanie M; Schulz, Thomas F

    2016-02-23

    The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle. PMID:26811480

  18. Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS

    PubMed Central

    Zhang, Guigen; Chan, Baca; Samarina, Naira; Abere, Bizunesh; Weidner-Glunde, Magdalena; Buch, Anna; Pich, Andreas; Brinkmann, Melanie M.; Schulz, Thomas F.

    2016-01-01

    The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING–dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle. PMID:26811480

  19. M6P/IGF2R loss of heterozygosity in head and neck cancer associated with poor patient prognosis

    PubMed Central

    Jamieson, Timothy A; Brizel, David M; Killian, J Keith; Oka, Yoshihiko; Jang, Hong-Seok; Fu, Xiaolong; Clough, Robert W; Vollmer, Robin T; Anscher, Mitchell S; Jirtle, Randy L

    2003-01-01

    Background The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes for a multifunctional receptor involved in lysosomal enzyme trafficking, fetal organogenesis, cytotoxic T cell-induced apoptosis and tumor suppression. The purpose of this investigation was to determine if the M6P/IGF2R tumor suppressor gene is mutated in human head and neck cancer, and if allelic loss is associated with poor patient prognosis. Methods M6P/IGF2R loss of heterozygosity in locally advanced squamous cell carcinoma of the head and neck was assessed with six different gene-specific nucleotide polymorphisms. The patients studied were enrolled in a phase 3 trial of twice daily radiotherapy with or without concurrent chemotherapy; median follow-up for surviving patients is 76 months. Results M6P/IGF2R was polymorphic in 64% (56/87) of patients, and 54% (30/56) of the tumors in these informative patients had loss of heterozygosity. M6P/IGF2R loss of heterozygosity was associated with a significantly reduced 5 year relapse-free survival (23% vs. 69%, p = 0.02), locoregional control (34% vs. 75%, p = 0.03) and cause specific survival (29% vs. 75%, p = 0.02) in the patients treated with radiotherapy alone. Concomitant chemotherapy resulted in a better outcome when compared to radiotherapy alone only in those patients whose tumors had M6P/IGF2R loss of heterozygosity. Conclusions This study provides the first evidence that M6P/IGF2R loss of heterozygosity predicts for poor therapeutic outcome in patients treated with radiotherapy alone. Our findings also indicate that head and neck cancer patients with M6P/IGF2R allelic loss benefit most from concurrent chemotherapy. PMID:12589712

  20. Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p.

    PubMed

    Mohd Yusuf, Siti N H; Bailey, Ulla-Maja; Tan, Nikki Y; Jamaluddin, Muhammad Fairuz; Schulz, Benjamin L

    2013-03-15

    Oligosaccharyltransferase (OTase) glycosylates selected asparagine residues in secreted and membrane proteins in eukaryotes, and asparagine (N)-glycosylation affects the folding, stability and function of diverse glycoproteins. The range of acceptor protein substrates that are efficiently glycosylated depends on the action of several accessory subunits of OTase, including in yeast the homologous proteins Ost3p and Ost6p. A model of Ost3p and Ost6p function has been proposed in which their thioredoxin-like active site cysteines form transient mixed disulfide bonds with cysteines in substrate proteins to enhance the glycosylation of nearby asparagine residues. We tested aspects of this model with a series of in vitro assays. We developed a whole protein mixed disulfide interaction assay that showed that Ost6p could form mixed disulfide bonds with selected cysteines in pre-reduced yeast Gas1p, a model glycoprotein substrate of Ost3p and Ost6p. A complementary peptide affinity chromatography assay for mixed disulfide bond formation showed that Ost3p could also form mixed disulfide bonds with cysteines in selected reduced tryptic peptides from Gas1p. Together, these assays showed that the thioredoxin-like active sites of Ost3p and Ost6p could form transient mixed disulfide bonds with cysteines in a model substrate glycoprotein, consistent with the function of Ost3p and Ost6p in modulating N-glycosylation substrate selection by OTase in vivo. PMID:23416356

  1. Identification and Characterization of Lipoxygenase Isoforms in Senescing Carnation Petals

    PubMed Central

    Rouet-Mayer, Marie-Aude; Bureau, Jean-Marc; Laurière, Christiane

    1992-01-01

    A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents. ImagesFigure 1 PMID:16668773

  2. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

    PubMed

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A; Di Pretoro, Simona; Pires, Susana S; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A; Hossbach, Markus; MacLaren, Robert E; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W; Wood, Matthew J A; Foster, Russell G; Peirson, Stuart N

    2015-09-21

    Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  3. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  4. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    PubMed Central

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  5. IL-33 isoforms: their future as vaccine adjuvants?

    PubMed Central

    Villarreal, Daniel O; Weiner, David B

    2015-01-01

    The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy. PMID:25656504

  6. Differential and Conditional Activation of PKC-Isoforms Dictates Cardiac Adaptation during Physiological to Pathological Hypertrophy

    PubMed Central

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation. PMID:25116170

  7. The CD44s splice isoform is a central mediator for invadopodia activity.

    PubMed

    Zhao, Pu; Xu, Yilin; Wei, Yong; Qiu, Qiong; Chew, Teng-Leong; Kang, Yibin; Cheng, Chonghui

    2016-04-01

    The ability for tumor cells to spread and metastasize to distant organs requires proteolytic degradation of extracellular matrix (ECM). This activity is mediated by invadopodia, actin-rich membrane protrusions that are enriched for proteases. However, the mechanisms underlying invadopodia activity are not fully understood. Here, we report that a specific CD44 splice isoform, CD44s, is an integral component in invadopodia. We show that CD44s, but not another splice isoform CD44v, is localized in invadopodia. Small hairpin (sh)RNA-mediated depletion of CD44s abolishes invadopodia activity, prevents matrix degradation and decreases tumor cell invasiveness. Our results suggest that CD44s promotes cortactin phosphorylation and recruits MT1-MMP (also known as MMP14) to sites of matrix degradation, which are important activities for invadopodia function. Importantly, we show that depletion of CD44s inhibits breast cancer cell metastasis to the lung in animals. These findings suggest a crucial mechanism underlying the role of the CD44s splice isoform in breast cancer metastasis. PMID:26869223

  8. Semisynthetic and Natural Garcinoic Acid Isoforms as New mPGES-1 Inhibitors.

    PubMed

    Alsabil, Khaled; Suor-Cherer, Sorphon; Koeberle, Andreas; Viault, Guillaume; Lavaud, Alexis; Temml, Veronika; Waltenberger, Birgit; Schuster, Daniela; Litaudon, Marc; Lorkowski, Stefan; de Vaumas, René; Helesbeux, Jean-Jacques; Guilet, David; Stuppner, Hermann; Werz, Oliver; Seraphin, Denis; Richomme, Pascal

    2016-07-01

    Over the last twenty years, tocotrienol analogues raised great interest because of their higher level and larger domain of biological activities when compared with tocopherols. Amongst the most promising therapeutic application, anti-inflammatory potency has been evaluated through the inhibition of various mediators of inflammation. Here, we worked on the isolation of two natural isoforms of garcinoic acid (i.e., δ and γ) from two different sources, respectively, Garcinia kola seeds and Garcinia amplexicaulis bark. We also developed semisynthetic strategies to access the other two non-natural α- and β-garcinoic acid isoforms. In the next stage of our work, microsomal prostaglandin E2 synthase was defined as a target to evaluate the anti-inflammatory potential of the four garcinoic acid isomers. Both dimethylated isoforms, β- and γ-garcinoic acid, exhibited the lowest IC50, 2.8 µM and 2.0 µM, respectively. These results showed that the affinity of tocotrienol analogues to microsomal prostaglandin E2 synthase-1 most probably contributes to the anti-inflammatory potential of this class of derivatives. PMID:27286327

  9. Circadian Rhythmicity of Active GSK3 Isoforms Modulates Molecular Clock Gene Rhythms in the Suprachiasmatic Nucleus

    PubMed Central

    Besing, R.C.; Paul, J.R.; Hablitz, L.M.; Rogers, C.O.; Johnson, R.L.; Young, M.E.; Gamble, K.L.

    2015-01-01

    The suprachiasmatic nucleus (SCN) drives and synchronizes daily rhythms at the cellular level via transcriptional-translational feedback loops comprised of clock genes such as Bmal1 and Period (Per). Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, phosphorylates at least five core clock proteins and shows diurnal variation in phosphorylation state (inactivation) of the GSK3β isoform. Whether phosphorylation of the other primary isoform (GSK3α) varies across the subjective day-night cycle is unknown. The purpose of this study was to determine if the endogenous rhythm of GSK3 (α and β) phosphorylation is critical for rhythmic BMAL1 expression and normal amplitude and periodicity of the molecular clock in the SCN. Significant circadian rhythmicity of phosphorylated GSK3 (α and β) was observed in the SCN from wild-type mice housed in constant darkness for two weeks. Importantly, chronic activation of both GSK3 isoforms impaired rhythmicity of the GSK3 target BMAL1. Furthermore, chronic pharmacological inhibition of GSK3 with 20 μM CHIR-99021 enhanced the amplitude and shortened the period of PER2::luciferase rhythms in organotypic SCN slice cultures. These results support the model that GSK3 activity status is regulated by the circadian clock and that GSK3 feeds back to regulate the molecular clock amplitude in the SCN. PMID:25724980

  10. Contribution of human hepatic cytochrome p450 isoforms to the metabolism of psychotropic drugs.

    PubMed

    Niwa, Toshiro; Shiraga, Toshifumi; Ishii, Ikuko; Kagayama, Akira; Takagi, Akira

    2005-09-01

    The metabolic activities of six psychotropic drugs, diazepam, clotiazepam, tofisopam, etizolam, tandospirone, and imipramine, were determined for 14 isoforms of recombinant human hepatic cytochrome P450s (CYPs) and human liver microsomes by measuring the disappearance rate of parent compounds. In vitro kinetic studies revealed that Vmax/Km values in human liver microsomes were the highest for tofisopam, followed by tandospirone>clotiazepam>imipramine, diazepam, and etizolam. Among the recombinant CYPs, CYP3A4 exhibited the highest metabolic activities of all compounds except for clotiazepam and imipramine. The metabolism of clotiazepam was catalyzed by CYP2B6, CYP3A4, CYP2C18, and CYP2C19, and imipramine was metabolized by CYP2D6 most efficiently. In addition, the metabolic activities of diazepam, clotiazepam, and etizolam in human liver microsomes were inhibited by 2.5 microM ketoconazole, a CYP3A4 inhibitor, by 97.5%, 65.1%, and 83.5%, respectively, and the imipramine metabolism was not detected after the addition of 1 or 10 microM quinidine, a CYP2D6 inhibitor. These results suggest that the psychotropic drugs investigated are metabolized predominantly by CYP3A4, except that CYP2D6 catalyzes the metabolism of imipramine. In addition, this approach based on the disappearance rate appears to be useful for the identification of the responsible CYP isoform(s) of older drugs, for which metabolic profiles have not been reported. PMID:16141545

  11. Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG.

    PubMed

    Kovalevskaya, G; Genbacev, O; Fisher, S J; Caceres, E; O'Connor, J F

    2002-08-30

    We have previously demonstrated that a hyperglycosylated isoform of chorionic gonadotropin (hCG) (B152 hCG) is detected in the blood and urine in early pregnancy and is subsequently rapidly replaced by the hCG isoform (B109 hCG) characteristic of later pregnancy. In the current study we have extended our work on the origin of these isoforms. We have used a combination of in situ and in vitro approaches. Localization studies in placental tissues showed that monoclonal antibody B109 stained very specifically syncytiotrophoblast (STBs) from first and second trimester tissues. At term, STBs exhibited no B109 staining at all. Immunostaining with B152 antibody, that recognize the hyperglycosylated isoform of hCG, revealed only punctate staining of STBs in most villi of first trimester tissue. Both antibodies B109 and B152 failed to stain cytotrophoblasts (CTBs). To assess the functional relevance of these observations we analyzed conditioned media from purified CTBs using two immunometric assays, one of which (B152-B207*) has primary specificity for the hyperglycosylated, choriocarcinoma-like hCG and the other (B109-B108*) having primary specificity for the later pregnancy hCG isoform. Regardless of gestational age, isolated CTBs secreted predominantly B152 hCG isoform in contrast to placental villi (predominantly STBs), which released primarily the B109 hCG isoform. Isolated CTBs, however, failed to immunostain with both B109 and B152 antibodies. To resolve this contradiction, we cultured CTBs in the presence of brefeldin A, a drug known to block secretion by inhibiting protein translocation from the endoplasmic reticulum to the Golgi vesicles. Brefeldin A treated CTBs stained strongly with B109 and did not stain or stained weakly with B152 antibody. We assume that treatment with brefeldin A impaired glycosylation of beta subunit and consequently inhibited the production of hyperglycosylated form of hCG recognized by B152. In summary, our in vitro experiments indicate

  12. 14-3-3 isoforms bind directly exon B of the 5′-UTR of human surfactant protein A2 mRNA

    PubMed Central

    Noutsios, Georgios T.; Ghattas, Paul; Bennett, Stephanie

    2015-01-01

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5′-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722–L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5′-UTR mRNA. PMID:26001776

  13. Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact.

    PubMed

    Kiel, J A; Hilbrands, R E; van der Klei, I J; Rasmussen, S W; Salomons, F A; van der Heide, M; Faber, K N; Cregg, J M; Veenhuis, M

    1999-08-01

    We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Deltapex1 or Deltapex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Deltapex4, Deltapex8 or Deltapex14 cells. Both proteins are loosely membrane-bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane-bound, but only partially co-localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity. PMID:10455230

  14. Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

    PubMed

    Gao, Cui; Shi, Rong; Wang, Tianming; Tan, Hongsheng; Xu, Hongxi; Ma, Yueming

    2015-01-01

    1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions. PMID:25714435

  15. Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves.

    PubMed Central

    Herbert, D; Price, L J; Alban, C; Dehaye, L; Job, D; Cole, D J; Pallett, K E; Harwood, J L

    1996-01-01

    The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and P(i) products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K' from the Hill equation) for ACCase1 were 0.054 microM for quizalofop and 21.8 microM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the (napp) values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K' values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 microM (ACCase2) and 50

  16. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    PubMed Central

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  17. Synthesis, structure, and superconductivity in the new-structure-type compound: SrPt6P2.

    PubMed

    Lv, Bing; Jawdat, BenMaan I; Wu, Zheng; Sorolla, Maurice; Gooch, Melissa; Zhao, Kui; Deng, Liangzi; Xue, Yu-Yi; Lorenz, Bernd; Guloy, Arnold M; Chu, Ching-Wu

    2015-02-01

    A metal-rich ternary phosphide, SrPt(6)P(2), with a unique structure type was synthesized at high temperatures. Its crystal structure was determined by single-crystal X-ray diffraction [cubic space group Pa3̅; Z = 4; a = 8.474(2) Å, and V = 608.51(2) Å(3)]. The structure features a unique three-dimensional anionic (Pt(6)P(2))(2-) network of vertex-shared Pt(6)P trigonal prisms. The Sr atoms occupy a 12-coordinate (Pt) cage site and form a cubic close-packed (face-centered-cubic) arrangement, and the P atoms formally occupy tetrahedral interstices. The metallic compound becomes superconducting at 0.6 K, as evidenced by magnetic and resistivity measurements. PMID:25525885

  18. 6p22.3 deletion: report of a patient with autism, severe intellectual disability and electroencephalographic anomalies

    PubMed Central

    2013-01-01

    Background The interstitial 6p deletions, involving the 6p22-p24 chromosomal region, are rare events characterized by variable phenotypes and no clear genotype-phenotype correlation has been established so far. Results High resolution array-CGH identified 1 Mb de novo interstitial deletion in 6p22.3 chromosomal region in a patient affected by severe Intellectual Disability (ID), Autism Spectrum Disorders (ASDs), and electroencephalographic anomalies. This deletion includes ATXN1, DTNBP1, JARID2 and MYLIP genes, known to play an important role in the brain, and the GMPR gene whose function in the nervous system is unknown. Conclusions We support the suggestion that ATXN1, DTNBP1, JARID2 and MYLIP are candidate genes for the pathophysiology of ASDs and ID, and we propose that deletion of DTNBP1 and/or JARID2 contributes to the hypotonia phenotype. PMID:23324214

  19. Further evidence for the involvement of human chromosome 6p24 in the aetiology of orofacial clefting.

    PubMed Central

    Davies, A F; Imaizumi, K; Mirza, G; Stephens, R S; Kuroki, Y; Matsuno, M; Ragoussis, J

    1998-01-01

    Chromosomal translocations affecting the 6p24 region have been associated with orofacial clefting. Here we present a female patient with cleft palate, severe growth retardation, developmental delay, frontal bossing, hypertelorism, antimongoloid slant, bilateral ptosis, flat nasal bridge, hypoplastic nasal alae, protruding upper lip, microretrognathia, bilateral, low set, and posteriorly rotated ears, bilateral microtia, narrow ear canals, short neck, and a karyotype of 46,XX,t(6;9)(p24;p23). The translocation chromosomes were analysed in detail by FISH and the 6p24 breakpoint was mapped within 50-500 kb of other breakpoints associated with orofacial clefting, in agreement with the assignment of such a locus in 6p24. The chromosome 9 translocation breakpoint was identified to be between D9S156 and D9S157 in 9p23-p22, a region implicated in the 9p deletion syndrome. Images PMID:9783713

  20. RELATIONSHIP BETWEEN BRAIN AND OVARY AROMATASE ACTIVITY AND ISOFORM-SPECIFIC AROMATASE MRNA EXPRESSION IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS) - JOURNAL ARTICLE

    EPA Science Inventory

    There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead minn...

  1. [Radiative transport and collisional transfer of excitation energy in Cs(6P) atoms mixed with N2].

    PubMed

    Meng, Fan-Xin; Qin, Chen; Dai, Kang; Shen, Yi-Fan

    2008-05-01

    Applying the CW laser absorption and fluorescence method, the cross sections for the fine structure mixing and quenching of the Cs(6P) state, induced by collision with N2 molecules, were measured. Cesium atoms were optically excited to the 6P3/2 state. The excited atom density and spatial distribution were mapped by monitoring the absorption of a counterpropagating single mode laser beam, tuned to the 6P1 --> 8S(1/2) transitions, which could be translated parallel to the pump beam. The transmission factors, which describe the average probability that photons emitted within the fluorescence detection region can pass through the optically thick vapor without being absorbed, were calculated for 6P --> 6S(1/2) transitions. The N2 caused line broadening and therefore increased the effective pumping rate and radiative rates. The effective radiative rates were calculated for the 6P(J) --> 6S transitions. The fluorescence intensity I895 of the sensitized 6P(1/2) --> 6S(1/2) emission was measured as a function of N2 density in the range 2 x 10(16) < N < 1.4 x 10(17) cm(-3) at a constant temperature T = 337 K, which produced cesium density N0 = 1.25 x 10(12) cm(-3). The transparency of the cell was obtained by the absorption of light beam passing the cell. The transparency is not a simple function of N2 density. It was found that the quantity N/I895 (I895 being corrected for the cell transparency) exhibited a parabolic dependence on N, confirming that the quenching of the 6P(J) states is due to collision with N2 molecules instead of Cs ground state atoms. The coefficients of the second-order polynomial fitted through the measured data yielded the cross sections sigma3/2 --> 1/2 = (0.42 +/- 0.17) x 10(-16) cm2 and sigmaD = (1.31 +/- 0.52) x 10(-16) cm2 for the 6P(J) fine-structure mixing and quenching, respectively, due to collision with N2 molecules. The quenching rate coefficient is about 3 times larger than the rate coefficient for the fine-structure mixing. Our values for

  2. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer

    PubMed Central

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-01-01

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed. PMID:26573433

  3. Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis.

    PubMed

    Baltes-Breitwisch, Michelle M; Artac, Robin A; Bott, Rebecca C; McFee, Renee M; Kerl, Jill G; Clopton, Debra T; Cupp, Andrea S

    2010-08-01

    Vascular endothelial growth factor A (VEGFA) plays a role in both angiogenesis and seminiferous cord formation, and alternative splicing of the Vegfa gene produces both proangiogenic isoforms and antiangiogenic isoforms (B-isoforms). The objectives of this study were to evaluate the expression of pro- and antiangiogenic isoforms during testis development and to determine the role of VEGFA isoforms in testis morphogenesis. Quantitative RT-PCR determined that Vegfa_165b mRNA was most abundant between embryonic days 13.5 and 16 (E13.5 and 16; P<0.05). Compared with ovarian mRNA levels, Vegfa_120 was more abundant at E13-14 (P<0.05), Vegfa_164 was less abundant at E13 (P<0.05), and Vegfa_165b tended to be less abundant at E13 (P<0.09) in testes. Immunohistochemical staining localized antiangiogenic isoforms to subsets of germ cells at E14-16, and western blot analysis revealed similar protein levels for VEGFA_165B, VEGFA_189B, and VEGFA_206B at this time point. Treatment of E13 organ culture testes with VEGFA_120, VEGFA_164, and an antibody to antiangiogenic isoforms (anti-VEGFAxxxB) resulted in less organized and defined seminiferous cords compared with paired controls. In addition, 50 ng/ml VEGFA_120 and VEGFA_164 treatments increased vascular density in cultured testes by 60 and 48% respectively, and treatment with VEGFAxxxB antibody increased vascular density by 76% in testes (0.5 ng/ml) and 81% in ovaries (5 ng/ml) compared with controls (P<0.05). In conclusion, both pro- and antiangiogenic VEGFA isoforms are involved in the development of vasculature and seminiferous cords in rat testes, and differential expression of these isoforms may be important for normal gonadal development. PMID:20457593

  4. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290

  5. Isoform-specific cleavage of 14-3-3 proteins in apoptotic JURL-MK1 cells.

    PubMed

    Kuzelová, Katerina; Grebenová, Dana; Pluskalová, Michaela; Kavan, Daniel; Halada, Petr; Hrkal, Zbynek

    2009-03-01

    The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways. PMID:19173300

  6. Interrelation between optical, refractometric, and lattice parameters of Cu6P(S1- x Sex)5I crystals

    NASA Astrophysics Data System (ADS)

    Studenyak, I. P.; Suslikov, L. M.; Kovacs, Gy. Sh.; Kranjčec, M.; Tovt, V. V.

    2001-04-01

    The influence of the compositional disordering on the energy of the fundamental absorption edge and the refractive index dispersion of the crystalline solid solutions Cu6P(S1- x Sex)5I is studied. It is shown that the concentration dependences of the optical pseudogap width, the refractive index, and the lattice parameter in these crystalline solid solutions are interrelated.

  7. A Network of Splice Isoforms for the Mouse

    PubMed Central

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S.; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  8. Frac-seq reveals isoform-specific recruitment to polyribosomes

    PubMed Central

    Sterne-Weiler, Timothy; Martinez-Nunez, Rocio Teresa; Howard, Jonathan M.; Cvitovik, Ivan; Katzman, Sol; Tariq, Muhammad A.; Pourmand, Nader; Sanford, Jeremy R.

    2013-01-01

    Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5′UTR as well as Alu-elements and microRNA target sites in the 3′UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms. PMID:23783272

  9. A Network of Splice Isoforms for the Mouse.

    PubMed

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  10. p53 Isoforms: Key Regulators of the Cell Fate Decision.

    PubMed

    Joruiz, Sebastien M; Bourdon, Jean-Christophe

    2016-01-01

    It is poorly understood how a single protein, p53, can be responsive to so many stress signals and orchestrates very diverse cell responses to maintain/restore cell/tissue functions. The uncovering that TP53 gene physiologically expresses, in a tissue-dependent manner, several p53 splice variants (isoforms) provides an explanation to its pleiotropic biological activities. Here, we summarize a decade of research on p53 isoforms. The clinical studies and the diverse cellular and animal models of p53 isoforms (zebrafish, Drosophila, and mouse) lead us to realize that a p53-mediated cell response is, in fact, the sum of the intrinsic activities of the coexpressed p53 isoforms and that unbalancing expression of different p53 isoforms leads to cancer, premature aging, (neuro)degenerative diseases, inflammation, embryo malformations, or defects in tissue regeneration. Cracking the p53 isoforms' code is, thus, a necessary step to improve cancer treatment. It also opens new exciting perspectives in tissue regeneration. PMID:26801896

  11. Isoform dependent regulation of human HCN channels by cholesterol

    PubMed Central

    Fürst, Oliver; D’Avanzo, Nazzareno

    2015-01-01

    Cholesterol has been shown to regulate numerous ion channels. HCN channels represent the molecular correlate of If or Ih in sinoatrial node (SAN) and neuronal cells. Previous studies have implicated a role for cholesterol in the regulation of rabbit HCN4 channels with effects on pacing in the rabbit SAN. Using electrophysiological and biochemical approaches, we examined the effect of cholesterol modulation on human HCN1, HCN2 and HCN4 isoforms. Patch-clamp experiments uncovered isoform specific differences in the effect of cholesterol on gating kinetics upon depletion by MβCD or mevastatin or enrichment using MβCD/cholesterol. Most dramatically cholesterol had isoform specific effects on mode-shifting, which has been suggested to play a key role in stabilizing firing rate and preventing arrhythmic firing in SAN cells and neurons. Mode-shifting in HCN1 channels was insensitive to cholesterol manipulation, while HCN2 and HCN4 were strongly affected. Trafficking of each isoform to the plasma membrane was also affected by cholesterol modulation differentially between isoforms, however, each isoform remained localized in lipid raft domains after cholesterol depletion. These effects may contribute to the side effects of cholesterol reducing therapies including disrupted heart rhythm and neuropathic pain, as well as the susceptibility of sinus dysfunction in patients with elevated cholesterol. PMID:26404789

  12. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    PubMed Central

    Fagotti, A; Gabbiani, G; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa. PMID:9518500

  13. The balance between two isoforms of LEF-1 regulates colon carcinoma growth

    PubMed Central

    2012-01-01

    Background Colon cancer is one of the most aggressive human malignancies, with a very poor prognosis. Although it has been suggested that different isoforms of the lymphoid enhancer factor (LEF-1) have opposing biological activities, the biological outcome of aberrant LEF-1 activation in colon cancer is still unclear. The aim of this study was to evaluate the effect of the different LEF-1 phenotypes on the growth of colon carcinoma cell lines. A deeper understanding of these processes might improve the targeted therapies for colon cancer by regulating the expression of LEF-1. Methods The role of different isoforms of LEF-1 on the growth of human colon carcinoma cell lines (SW480 and HT-29) was studied using various in vitro and in vivo assays. In vitro proliferation, migration, adhesion and apoptosis of the cells stably transfected of different isoforms of LEF-1 were monitored by MTT assay, carboxyfluorescein diacetate–succinimidyl ester staining, annexin V staining, ECM adhesion assay and transwell assay, respectively. In nude mice, the formation of neovasculature in the tumors formed by our constructed cells was measured by immunohistochemistry. All the data were analyzed using a t test, and data were treated as significant when p < 0.05. Results Overexpression of truncated LEF-1 (LEF-1-ΔL) in the colon cell lines, SW480 and HT29, inhibited their growth significantly in vitro and in vivo, but the full-length LEF-1 (LEF-1-FL) promoted the proliferation of HT29. Inactivation of Wnt signaling by LEF-1-ΔL reduced the expression of CXCR4 in colon cell lines, which may lead to a decrease in activities such as migration, adhesion and survival. In nude mice, the formation of neovasculature as well as an increase in tumor volume were inhibited by the short isoform of LEF-1. LEF-1-FL, however, caused an increase in all these parameters compared with controls. Conclusions These findings suggest that LEF-1 might play an important role in colon carcinogenesis by

  14. A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms.

    PubMed

    Carradori, Simone; Secci, Daniela; De Monte, Celeste; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Codispoti, Rossella; De Cosmi, Federica; Guglielmi, Paolo; Supuran, Claudiu T

    2016-03-01

    Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with KIs ranging between 19 and 2482nM, whereas they were poorly active against hCA II (KIs >10μM) and hCA I (KIs ranging between 318nM and 50μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors. PMID:26810710

  15. Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments

    PubMed Central

    Baquero-Pérez, Belinda; Whitehouse, Adrian

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents. PMID:26587836

  16. Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

    PubMed

    Hamdollah Zadeh, Maryam A; Amin, Elianna M; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E; Ye, Xi; Heesom, Katherine J; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B; Williams, Ann C; Kerr, David J; Salmon, Andrew H J; Oltean, Sebastian; Midgley, Rachel S; Ladomery, Michael R; Harper, Steven J; Varey, Alexander H R; Bates, David O

    2015-01-01

    The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. PMID:25224594

  17. Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance

    PubMed Central

    Hamdollah Zadeh, Maryam A.; Amin, Elianna M.; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E.; Ye, Xi; Heesom, Katherine J.; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B.; Williams, Ann C.; Kerr, David J.; Salmon, Andrew H.J.; Oltean, Sebastian; Midgley, Rachel S.; Ladomery, Michael R.; Harper, Steven J.; Varey, Alexander H.R.; Bates, David O.

    2015-01-01

    The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. PMID:25224594

  18. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  19. HMGI(Y) activation by chromosome 6p21 rearrangements in multilineage mesenchymal cells from pulmonary hamartoma.

    PubMed Central

    Xiao, S.; Lux, M. L.; Reeves, R.; Hudson, T. J.; Fletcher, J. A.

    1997-01-01

    The chromosome band 6p21 high-mobility group gene, HMGI(Y), encodes DNA-binding proteins with both chromatin structural and gene regulatory roles. Although HMGI(Y) expression is associated with neoplastic transformation, no oncogenic HMGI(Y) mutations have been identified. We report pulmonary chondroid hamartoma chromosome 6p21 aberrations targeting HMGI(Y). Several pulmonary chondroid hamartomas had chromosome rearrangements mapping within 100 kb of HMGI(Y), and one pulmonary chondroid hamartoma contained an intragenic fusion juxtaposing HMGI(Y) A.T book DNA-binding domains with a laminin alpha 4 chain epidermal-growth-factor-like/ zinc-finger-like motif. These findings demonstrate an oncogenic role for HMGI(Y) and for laminin chain transcriptional regulatory motifs. Images Figure 1 Figure 4 Figure 5 PMID:9060828

  20. Reanalysis of the photoassociation spectrum of 133Cs2 (6P3/2) 1g state

    NASA Astrophysics Data System (ADS)

    Ma, Jie; Li, Yu-Qing; Wu, Ji-Zhou; Fan, Qun-Chao; Feng, Hao; Sun, Wei-Guo; Xiao, Lian-Tuan; Jia, Suo-Tang

    2013-08-01

    Reanalysis of the photoassociation spectrum of the weakly binding (6S1/2 + 6P3/2) 1g133Cs2 levels, reported in the previous study [J. Mol. Spectro. 255 (2009) 106], is performed by using a Lu—Fano graph coupled to the improved LeRoy—Bernstein formula including two additional modified terms. A more accurate coefficient (c3) is obtained for the leading long-range potential (-c3/R3) of a diatomic molecule.

  1. A {Nb6 P2 W12 }-Based Hexameric Manganese Cluster with Single-Molecule Magnet Properties.

    PubMed

    Zhang, Dongdi; Cao, Fan; Ma, Pengtao; Zhang, Chao; Song, You; Liang, Zhijie; Hu, Xiaojing; Wang, Jingping; Niu, Jingyang

    2015-12-01

    By deliberately using a metastable polyanion [(NbO2 )6 P2 W12 O56 ](12-) (1), which was formed in situ, we have discovered the unprecedented hexameric cluster {Mn15 (Nb6 P2 W12 O62 )6 } (2), in which the six polyanions [Nb6 P2 W12 O61 ](10-) are alternately connected by four intriguing trinuclear {Mn(III) 3 } moieties and four {Mn(II) } linkers. This discovery is the first in which the phosphoniobotungstate has been made accessible by using transition-metal ions; furthermore, polyanion 2 represents the largest niobotungstate cluster reported to date. Analysis by means of electrospray ionization mass spectrometry (ESI-MS) provides insight into the self-assembly process, and the peaks observed relate to the different charge states of the parent cluster, thus confirming the stability of 2. In addition, magnetic-susceptibility measurements reveal that each {Mn(III) 3 } subunit is a separate single-molecule magnet (SMM). This discovery results from the exploration of the reverse effect of metastable polyanion 1 possessing high reactivity, thereby turning a disadvantage into an advantage. This finding could define a new synthetic strategy for the design and synthesis of magnetic polyoxometalate (POM) clusters. PMID:26493685

  2. Whole genome characterization of a G6P[5] rotavirus A strain isolated from a stray cat in Japan.

    PubMed

    Kaneko, Miho; Mochizuki, Masami; Nakagomi, Osamu; Nakagomi, Toyoko

    2016-05-30

    The whole genome of an unusual G6P[5] rotavirus A strain named FRV537, which was isolated from a stray cat in Japan, was characterized to determine its species of origin. The genotype constellation of FRV537 was G6-P[5]-I2-R2-C2-M2-A13-N2- T6-E2-H3. No known feline rotavirus has this genotype constellation; the Japanese equine strain OH-4 is the only known strain that does. While FRV537 shares the same genotype with some feline rotaviruses in all genes except those encoding VP4 and NSP1, none of these genes are closely related to those of known feline rotaviruses. By contrast, G6P[5] is almost exclusively present in bovine rotaviruses. The VP7 and VP4 genes of FRV537 formed a lineage with typical bovine rotaviruses with high bootstrap values. As to the internal capsid and nonstructural gene constellation, three bovine rotavirus strains had a constellation identical to that of FRV537. Moreover, each of the genotypes of FRV537 was found to be a common bovine genotype. In addition to the high nucleotide sequence identities between FRV537 and bovine rotaviruses in each genome segment (≥95%), phylogenetic analysis revealed a close relationship to bovine/artiodactyl rotaviruses. Thus, the molecular and phylogenetic evidence suggests that FRV537 isolated from a stray cat was of bovine rotavirus origin. PMID:27139026

  3. Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus suber L).

    PubMed

    Farnet, A M; Criquet, S; Pocachard, E; Gil, G; Ferre, E

    2002-01-01

    A new isoform of laccase from Marasmius quercophilus is described in this study. The strain of this white-rot fungus was isolated for the first time on a cork oak litter. This isoform exhibited certain common properties of laccases (a molecular weight of 65 Kda, an optimum pH of 6.2 with syringaldazine). But this laccase has also particularly novel features: the best activity measured was observed at high temperatures (80 C) and this isoform was not inhibited with EDTA. Furthermore, this induced laccase was able to transform most of the aromatic compounds tested without the addition of mediators to the reaction mixture, and the transformation of certain chlorophenols (2-chlorophenol and 2,4-dichlorophenol) by a laccase isoform from M. quercophilus is reported here for the first time. We also demonstrate the importance of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator since it allowed veratryl alcohol and p-hydroxybenzoic acid transformation. Moreover, new products of transformation were observed using the combination of ABTS with this isoform of laccase. PMID:21156546

  4. SURVIV for survival analysis of mRNA isoform variation.

    PubMed

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  5. p53 isoform profiling in glioblastoma and injured brain

    PubMed Central

    Takahashi, Rie; Giannini, Caterina; Sarkaria, Jann N.; Schroeder, Mark; Rogers, Joseph; Mastroeni, Diego; Scrable, Heidi

    2014-01-01

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10–70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, which have been shown to modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain, and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry, and RT-PCR. At the protein level, we found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared to tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions. PMID:22824800

  6. Heterogeneity of presynaptic proteins: do not forget isoforms

    PubMed Central

    Bragina, Luca; Fattorini, Giorgia; Giovedì, Silvia; Bosco, Federica; Benfenati, Fabio; Conti, Fiorenzo

    2013-01-01

    Analysis of presynaptic protein expression in glutamatergic and GABAergic central synapses performed in several laboratories and with different techniques is unveiling a complex scenario, largely because each presynaptic protein exists in several isoforms. The interpretation of these findings is generally based on the notion that each synapse and each synaptic vesicle contains one of the isoforms of each family of presynaptic proteins. We verified whether this interpretation is tenable by performing triple labeling and immunoisolation studies with the aim of detecting two isoforms of a given presynaptic protein in glutamatergic or GABAergic axon terminals and/or synaptic vesicles (SVs). Here, we show that: (1) the possibility that not all families of presynaptic proteins are expressed in all terminals must be taken into serious account; (2) the expression of a given protein isoform in a terminal does not exclude the expression of other isoforms of the same protein in the same terminal and in the same vesicle. These conclusions open new and interesting problems; their experimental analysis might improve our understanding of the physiology and pathophysiology of central synapses. PMID:23382710

  7. SURVIV for survival analysis of mRNA isoform variation

    PubMed Central

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  8. Differential regulation of renal phospholipase C isoforms by catecholamines.

    PubMed

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines. PMID:7814630

  9. Specific isoforms of protein kinase C are essential for prevention of folate-resistant neural tube defects by inositol.

    PubMed

    Cogram, Patricia; Hynes, Andrew; Dunlevy, Louisa P E; Greene, Nicholas D E; Copp, Andrew J

    2004-01-01

    A proportion of neural tube defects (NTDs) can be prevented by maternal folic acid supplementation, although some cases are unresponsive. The curly tail mutant mouse provides a model of folate-resistant NTDs, in which defects can be prevented by inositol therapy in early pregnancy. Hence, inositol represents a possible novel adjunct therapy to prevent human NTDs. The present study investigated the molecular mechanism by which inositol prevents mouse NTDs. Activation of protein kinase C (PKC) is known to be essential, and we examined neurulation-stage embryos for PKC expression and applied PKC inhibitors to curly tail embryos developing in culture. Although all known PKC isoforms were detected in the closing neural tube, use of chemical PKC inhibitors identified a particular requirement for 'conventional' PKC isoforms. Peptide inhibitors offer selective inhibition of individual PKCs, and we demonstrated isoform-specific inhibition of PKC in embryonic cell cultures. Application of peptide inhibitors to neurulation-stage embryos revealed an absolute dependence on the activity of PKCbetaI and gamma for prevention of NTDs by inositol, and partial dependence on PKCzeta, whereas other PKCs (alpha, betaII delta, and epsilon) were dispensable. To investigate the cellular action of inositol and PKCs in NTD prevention, we examined cell proliferation in curly tail embryos. Defective proliferation of hindgut cells is a key component of the pathogenic sequence leading to NTDs in curly tail. Hindgut cell proliferation was stimulated specifically by inositol, an effect that required activation of PKCbetaI. Our findings reveal an essential role of specific PKC isoforms in mediating the prevention of mouse NTDs by inositol. PMID:14613966

  10. Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin.

    PubMed Central

    Rose, Christiane; Voisin, Stéphanie; Gros, Claude; Schwartz, Jean-Charles; Ouimet, Tanja

    2002-01-01

    Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity. PMID:11964170