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Sample records for a-subunit isoforms enzyme

  1. N-linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H+ -ATPase (V-ATPase) biosynthesis.

    PubMed

    Esmail, Sally; Kartner, Norbert; Yao, Yeqi; Kim, Joo Wan; Reithmeier, Reinhart A F; Manolson, Morris F

    2018-01-01

    The a subunit of the V 0 membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope-tagged human a1-a3 subunits, with or without mutations that convert Asn to Gln at putative N-glycosylation sites. Expression and N-glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N-glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild-type a subunits after enzymatic deglycosylation. Cycloheximide-chase experiments showed that unglycosylated subunits were turned over at a higher rate than N-glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co-immunoprecipitation studies showed that they were unable to associate with the V-ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N-glycosylation is crucial in all four human V-ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V-ATPase complexes. © 2017 Wiley Periodicals, Inc.

  2. [Characterization of a malic enzyme isoform V from Mucor circinelloides].

    PubMed

    Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

    2016-02-04

    We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

  3. NADP-malic enzyme isoforms in maize leaves.

    PubMed

    Maurino, V G; Drincovich, M F; Andreo, C S

    1996-02-01

    Two isoforms of NADP-malic enzyme have been characterized in maize leaves. The 72 kDa-form of the protein, present mainly in etiolated maize leaves, has lower specific activity than the 62 kDa-form, which is implicated in C4 metabolism and predominates in green leaves. The larger form of the enzyme has higher Km values for NADP and malate and lower PH optimum. The antibodies raised against the 62 kDa-form of the protein react with the 72 kDa-form. Steady state levels of NADP-malic enzyme, as measured by the amount of protein and activity, increase several-fold when dark-grown maize seedlings are illuminated. This increase in protein is about 13-fold for the 62 kDa-form of the enzyme, while the 72 kDa-form remains practically constant after a transient increase. Northern blot analysis using a specific probe against the 62 kDa-form of the enzyme, reveals the increase of a 2.2 kb mRNA during greening. Southern hybridization analysis with genomic DNA suggests the presence of more than one gene encoding NADP-malic enzyme in maize. In this paper we provide biochemical and inmunological evidence suggesting that both isoforms are closely related and that the 72 kDa-form is also present in low levels in mature green leaves.

  4. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  5. The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

    PubMed Central

    Hosokawa, Hiroyuki; Dip, Phat Vinh; Merkulova, Maria; Bakulina, Anastasia; Zhuang, Zhenjie; Khatri, Ashok; Jian, Xiaoying; Keating, Shawn M.; Bueler, Stephanie A.; Rubinstein, John L.; Randazzo, Paul A.; Ausiello, Dennis A.; Grüber, Gerhard; Marshansky, Vladimir

    2013-01-01

    Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H+-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe5, Met10, and Gln14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor. PMID:23288846

  6. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

    PubMed Central

    Schep, Daniel G.; Rubinstein, John L.

    2016-01-01

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  7. Plastidial NADP-malic enzymes from grasses: unraveling the way to the C4 specific isoforms.

    PubMed

    Saigo, Mariana; Alvarez, Clarisa E; Andreo, Carlos S; Drincovich, María F

    2013-02-01

    Malic enzyme is present in many plant cell compartments such as plastids, cytosol and mitochondria. Particularly relevant is the plastidial isoform that participates in the C(4) cycle providing CO(2) to RuBisCO in C(4) species. This type of photosynthesis is more frequent among grasses where anatomical preconditioning would have facilitated the evolution of the C(4) syndrome. In maize (C(4) grass), the photosynthetic NADP dependent Malic enzyme (ZmC(4)-NADP-ME, l-malate:NADP oxidoreductase, E.C. 1.1.1.40) and the closest related non-photosynthetic isoform (ZmnonC(4)-NADP-ME, l-malate:NADP oxidoreductase, E.C. 1.1.1.40) are both plastidial but differ in expression pattern, kinetics and structure. Features like high catalytic efficiency, inhibition by high malate concentration at pH 7.0, redox modulation and tetramerization are characteristic of the photosynthetic NADP-ME. In this work, the proteins encoded by sorghum (C(4) grass) and rice (C(3) grass) NADP-ME genes, orthologues of the plastidial NADP-MEs from maize, were recombinantly expressed, purified and characterized. In a global comparison, we could identify a small group of residues which may explain the special features of C(4) enzymes. Overall, the present work presents biochemical and molecular data that helps to elucidate the changes that took place in the evolution of C(4) NADP-ME in grasses. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  8. Identification of Specific Inhibitors of Trypanosoma cruzi Malic Enzyme Isoforms by Target-Based HTS.

    PubMed

    Ranzani, Americo T; Nowicki, Cristina; Wilkinson, Shane R; Cordeiro, Artur T

    2017-04-01

    Trypanosoma cruzi is the causative agent of Chagas disease. The lack of an efficient and safe treatment supports the research into novel metabolic targets, with the malic enzyme (ME) representing one such potential candidate. T. cruzi expresses a cytosolic (TcMEc) and a mitochondrial (TcMEm) ME isoform, with these activities functioning to generate NADPH, a key source of reducing equivalents that drives a range of anabolic and protective processes. To identify specific inhibitors that target TcMEs, two independent high-throughput screening strategies using a diversity library containing 30,000 compounds were employed. IC 50 values of 262 molecules were determined for both TcMEs, as well as for three human ME isoforms, with the inhibitors clustered into six groups according to their chemical similarity. The most potent hits belonged to a sulfonamide group that specifically target TcMEc. Moreover, several selected inhibitors of both TcMEs showed a trypanocidal effect against the replicative forms of T. cruzi. The chemical diversity observed among those compounds that inhibit TcMEs activity emphasizes the druggability of these enzymes, with a sulfonamide-based subset of compounds readily able to block TcMEc function at a low nanomolar range.

  9. Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae.

    PubMed

    Badia, Mariana Beatriz; Mans, Robert; Lis, Alicia V; Tronconi, Marcos Ariel; Arias, Cintia Lucía; Maurino, Verónica Graciela; Andreo, Carlos Santiago; Drincovich, María Fabiana; van Maris, Antonius J A; Gerrard Wheeler, Mariel Claudia

    2017-02-01

    NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO 2 , and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc - ) Saccharomyces cerevisiae strains. The heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins), and NADP-ME3 restored the growth of Pyc - S. cerevisiae on glucose, and this capacity was dependent on the availability of CO 2 . On the other hand, NADP-ME4, NAD-ME1, and NAD-ME2 could not rescue the Pyc - strains from C 4 auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C 4 compounds and redox shuttling in plant cells. © 2017 Federation of European Biochemical Societies.

  10. Membrane bound COMT isoform is an interfacial enzyme: general mechanism and new drug design paradigm.

    PubMed

    Magarkar, Aniket; Parkkila, Petteri; Viitala, Tapani; Lajunen, Tatu; Mobarak, Edouard; Licari, Giuseppe; Cramariuc, Oana; Vauthey, Eric; Róg, Tomasz; Bunker, Alex

    2018-02-15

    The enzyme catechol-O-methyltransferase (COMT) has water soluble (S-COMT) and membrane associated (MB-COMT), bitopic, isoforms. Of these MB-COMT is a drug target in relation to the treatment of Parkinson's disease. Using a combination of computational and experimental protocols, we have determined the substrate selection mechanism specific to MB-COMT. We show: (1) substrates with preferred affinity for MB-COMT over S-COMT orient in the membrane in a fashion conducive to catalysis from the membrane surface and (2) binding of COMT to its cofactor ADOMET induces conformational change that drives the catalytic surface of the protein to the membrane surface, where the substrates and Mg 2+ ions, required for catalysis, are found. Bioinformatics analysis reveals evidence of this mechanism in other proteins, including several existing drug targets. The development of new COMT inhibitors with preferential affinity for MB-COMT over S-COMT is now possible and insight of broader relevance, into the function of bitopic enzymes, is provided.

  11. Annotation and analysis of malic enzyme genes encoding for multiple isoforms in the fungus Mucor circinelloides CBS 277.49.

    PubMed

    Vongsangnak, Wanwipa; Zhang, Yingtong; Chen, Wei; Ratledge, Colin; Song, Yuanda

    2012-05-01

    Based on the newly-released genomic data of Mucor circinelloides CBS 277.49, we have annotated five genes encoding for malic enzyme: all code for proteins that contain conserved domains/motifs for malic acid binding, NAD(+) binding and NAD(P)(+) binding. Phylogenetic analysis for malic enzyme genes showed that genes ID 78524 and 11639 share ~80% amino acid identity and are grouped in cluster 1; genes ID 182779, 186772 and 116127 share ~66% amino acid identity are grouped in cluster 2. Genes ID 78524, 11639 and 166127 produce proteins that are localized in the mitochondrion, while the products from genes 182779 and 186772 are localized in the cytosol. Based on the comparative analysis published previously by Song et al. (Microbiology 147:1507-1515, 2001), we propose that malic enzyme genes ID 78524, 166127, 182779, 186772, 11639, respectively, represent protein isoforms I, II, III/IV, V, and VI.

  12. Inhibition of class IA PI3K enzymes in non-small cell lung cancer cells uncovers functional compensation among isoforms.

    PubMed

    Stamatkin, Christopher; Ratermann, Kelley L; Overley, Colleen W; Black, Esther P

    2015-01-01

    Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies while normal cell proliferation requires pathway functionality. Although inhibitors of the PI3K pathway are in clinical trials or approved for therapy, an understanding of the functional activities of pathway members in specific malignancies is needed. In lung cancers, the PI3K pathway is often aberrantly activated by mutation of genes encoding EGFR, KRAS, and PIK3CA proteins. We sought to understand whether class IA PI3K enzymes represent rational therapeutic targets in cells of non-squamous lung cancers by exploring pharmacological and genetic inhibitors of PI3K enzymes in a non-small cell lung cancer (NSCLC) cell line system. We found that class IA PI3K enzymes were expressed in all cell lines tested, but treatment of NSCLC lines with isoform-selective inhibitors (A66, TGX-221, CAL-101 and IC488743) had little effect on cell proliferation or prolonged inhibition of AKT activity. Inhibitory pharmacokinetic and pharmacodynamic responses were observed using these agents at non-isoform selective concentrations and with the pan-class I (ZSTK474) agent. Response to pharmacological inhibition suggested that PI3K isoforms may functionally compensate for one another thus limiting efficacy of single agent treatment. However, combination of ZSTK474 and an EGFR inhibitor (erlotinib) in NSCLC resistant to each single agent reduced cellular proliferation. These studies uncovered unanticipated cellular responses to PI3K isoform inhibition in NSCLC that does not correlate with PI3K mutations, suggesting that patients bearing tumors with wildtype EGFR and KRAS are unlikely to benefit from inhibitors of single isoforms but may respond to pan-isoform inhibition.

  13. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

    PubMed Central

    Hüttemann, Maik; Lee, Icksoo; Gao, Xiufeng; Pecina, Petr; Pecinova, Alena; Liu, Jenney; Aras, Siddhesh; Sommer, Natascha; Sanderson, Thomas H.; Tost, Monica; Neff, Frauke; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Naton, Beatrix; Rathkolb, Birgit; Rozman, Jan; Favor, Jack; Hans, Wolfgang; Prehn, Cornelia; Puk, Oliver; Schrewe, Anja; Sun, Minxuan; Höfler, Heinz; Adamski, Jerzy; Bekeredjian, Raffi; Graw, Jochen; Adler, Thure; Busch, Dirk H.; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Weissmann, Norbert; Doan, Jeffrey W.; Bassett, David J. P.; Grossman, Lawrence I.

    2012-01-01

    Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (−50 and −29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (−12%), reduced total oxygen consumption rate (−8%), improved glucose tolerance, and reduced grip force (−14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction

  14. Disposition of Flavonoids via Enteric Recycling: Enzyme Stability Affects Characterization of Prunetin Glucuronidation across Species, Organs, and UGT Isoforms

    PubMed Central

    Joseph, Tiby B.; Wang, Stephen W.J.; Liu, Xing; Kulkarni, Kaustubh H.; Wang, Jingrong; Xu, Haiyan; Hu, Ming

    2008-01-01

    We characterized the in vitro glucuronidation of prunetin, a prodrug of genistein that is a highly active cancer prevention agent. Metabolism studies were conducted using expressed human UGT isoforms and microsomes/S9 fractions prepared from intestine and liver of rodents and humans. The results indicated that human intestinal microsomes were more efficient than liver microsomes in glucuronidating prunetin, but rates of metabolism were dependent on time of incubation at 37°C. Human liver and intestinal microsomes mainly produced metabolite 1 (prunetin-5-O-glucuronide) and metabolite 2 (prunetin-4’-O-glucuronide), respectively. Using 12 human UGT isoforms, we showed that UGT1A7, UGT1A8 and 1A9 were mainly responsible for the formation of metabolite 1 whereas UGT1A1, UGT1A8 and UGT1A10 were mainly responsible for the formation of metabolite 2. This isoform specific metabolism was consistent with earlier results obtained using human liver and intestinal microsomes, as the former (liver) is UGT1A9-rich whereas the latter is UGT1A10-rich. Surprisingly, we found that thermo stability of the microsomes were isoform- and organ-dependent. For example, human liver microsomal UGT activities were much more heat (37°C) stable than intestinal microsomal UGT activities, consistent with the finding that human UGT1A9 is much more thermo stable than human UGT1A10 and UGT1A8. The organ-specific thermo stability profiles were also evident in rat microsomes and mouse S9 fractions, even though human intestinal glucuronidation of prunetin differ significantly from its rodent intestinal glucuronidation. In conclusion, prunetin glucuronidation is species-, organ- and UGT-isoform dependent, all of which may be impacted by thermo stability of specific UGT isoforms involved in the metabolism. PMID:18052087

  15. Disposition of flavonoids via enteric recycling: enzyme stability affects characterization of prunetin glucuronidation across species, organs, and UGT isoforms.

    PubMed

    Joseph, Tiby B; Wang, Stephen W J; Liu, Xing; Kulkarni, Kaustubh H; Wang, Jingrong; Xu, Haiyan; Hu, Ming

    2007-01-01

    We characterized the in vitro glucuronidation of prunetin, a prodrug of genistein that is a highly active cancer prevention agent. Metabolism studies were conducted using expressed human UGT isoforms and microsomes/S9 fractions prepared from intestine and liver of rodents and humans. The results indicated that human intestinal microsomes were more efficient than liver microsomes in glucuronidating prunetin, but rates of metabolism were dependent on time of incubation at 37 degrees C. Human liver and intestinal microsomes mainly produced metabolite 1 (prunetin-5- O-glucuronide) and metabolite 2 (prunetin-4'- O-glucuronide), respectively. Using 12 human UGT isoforms, we showed that UGT1A7, UGT1A8, and UGT1A9 were mainly responsible for the formation of metabolite 1, whereas UGT1A1, UGT1A8, and UGT1A10 were mainly responsible for the formation of metabolite 2. This isoform-specific metabolism was consistent with earlier results obtained using human liver and intestinal microsomes, as the former (liver) is UGT1A9-rich whereas the latter is UGT1A10-rich. Surprisingly, we found that the thermostability of the microsomes was isoform- and organ-dependent. For example, human liver microsomal UGT activities were much more heat-stable (37 degrees C) than intestinal microsomal UGT activities, consistent with the finding that human UGT1A9 is much more thermostable than human UGT1A10 and UGT1A8. The organ-specific thermostability profiles were also evident in rat microsomes and mouse S9 fractions, even though human intestinal glucuronidation of prunetin differs significantly from rodent intestinal glucuronidation. In conclusion, prunetin glucuronidation is species-, organ-, and UGT-isoform-dependent, all of which may be impacted by the thermostability of specific UGT isoforms involved in the metabolism.

  16. YtsJ Has the Major Physiological Role of the Four Paralogous Malic Enzyme Isoforms in Bacillus subtilis†

    PubMed Central

    Lerondel, Guillaume; Doan, Thierry; Zamboni, Nicola; Sauer, Uwe; Aymerich, Stéphane

    2006-01-01

    The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD+ than with NADP+. YtsJ activity, in contrast, was 70-fold higher with NADP+ than with NAD+, with Km values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion. PMID:16788182

  17. Molecular dynamics investigations of regioselectivity of anionic/aromatic substrates by a family of enzymes: a case study of diclofenac binding in CYP2C isoforms.

    PubMed

    Cui, Ying-Lu; Xu, Fang; Wu, Rongling

    2016-06-29

    The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided.

  18. Characterization of the Canine Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 (cbr1) and the Functional Isoform cbr1 V218

    PubMed Central

    Ferguson, Daniel C.; Cheng, Qiuying

    2015-01-01

    The anthracyclines doxorubicin and daunorubicin are used in the treatment of various human and canine cancers, but anthracycline-related cardiotoxicity limits their clinical utility. The formation of anthracycline C-13 alcohol metabolites (e.g., doxorubicinol and daunorubicinol) contributes to the development of anthracycline-related cardiotoxicity. The enzymes responsible for the synthesis of anthracycline C-13 alcohol metabolites in canines remain to be elucidated. We hypothesized that canine carbonyl reductase 1 (cbr1), the homolog of the prominent anthracycline reductase human CBR1, would have anthracycline reductase activity. Recombinant canine cbr1 (molecular weight: 32.8 kDa) was purified from Escherichia coli. The enzyme kinetics of “wild-type” canine cbr1 (cbr1 D218) and a variant isoform (cbr1 V218) were characterized with the substrates daunorubicin and menadione, as well as the flavonoid inhibitor rutin. Canine cbr1 catalyzes the reduction of daunorubicin to daunorubicinol, with cbr1 D218 and cbr1 V218 displaying different kinetic parameters (cbr1 D218 Km: 188 ± 144 μM versus cbr1 V218 Km: 527 ± 136 μM, P < 0.05, and cbr1 D218 Vmax: 6446 ± 3615 nmol/min per milligram versus cbr1 V218 Vmax: 15539 ± 2623 nmol/min per milligram, P < 0.01). Canine cbr1 also metabolized menadione (cbr1 D218 Km: 104 ± 50 μM, Vmax: 2034 ± 307 nmol/min per milligram). Rutin acted as a competitive inhibitor for the reduction of daunorubicin (cbr1 D218 Ki: 1.84 ± 1.02 μM, cbr1 V218 Ki: 1.38 ± 0.47 μM). These studies show that canine cbr1 metabolizes daunorubicin and provide the necessary foundation to characterize the role of cbr1 in the variable pharmacodynamics of anthracyclines in canine cancer patients. PMID:25918240

  19. Characterization of the Canine Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 (cbr1) and the Functional Isoform cbr1 V218.

    PubMed

    Ferguson, Daniel C; Cheng, Qiuying; Blanco, Javier G

    2015-07-01

    The anthracyclines doxorubicin and daunorubicin are used in the treatment of various human and canine cancers, but anthracycline-related cardiotoxicity limits their clinical utility. The formation of anthracycline C-13 alcohol metabolites (e.g., doxorubicinol and daunorubicinol) contributes to the development of anthracycline-related cardiotoxicity. The enzymes responsible for the synthesis of anthracycline C-13 alcohol metabolites in canines remain to be elucidated. We hypothesized that canine carbonyl reductase 1 (cbr1), the homolog of the prominent anthracycline reductase human CBR1, would have anthracycline reductase activity. Recombinant canine cbr1 (molecular weight: 32.8 kDa) was purified from Escherichia coli. The enzyme kinetics of "wild-type" canine cbr1 (cbr1 D218) and a variant isoform (cbr1 V218) were characterized with the substrates daunorubicin and menadione, as well as the flavonoid inhibitor rutin. Canine cbr1 catalyzes the reduction of daunorubicin to daunorubicinol, with cbr1 D218 and cbr1 V218 displaying different kinetic parameters (cbr1 D218 Km: 188 ± 144 μM versus cbr1 V218 Km: 527 ± 136 μM, P < 0.05, and cbr1 D218 Vmax: 6446 ± 3615 nmol/min per milligram versus cbr1 V218 Vmax: 15539 ± 2623 nmol/min per milligram, P < 0.01). Canine cbr1 also metabolized menadione (cbr1 D218 Km: 104 ± 50 μM, Vmax: 2034 ± 307 nmol/min per milligram). Rutin acted as a competitive inhibitor for the reduction of daunorubicin (cbr1 D218 Ki: 1.84 ± 1.02 μM, cbr1 V218 Ki: 1.38 ± 0.47 μM). These studies show that canine cbr1 metabolizes daunorubicin and provide the necessary foundation to characterize the role of cbr1 in the variable pharmacodynamics of anthracyclines in canine cancer patients. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  1. A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX

    DOE PAGES

    Pinard, Melissa A.; Aggarwal, Mayank; Mahon, Brian P.; ...

    2015-09-23

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO 2to HCO 3 $-$, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-basedmore » drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, anR cryst of 18.0% and anR free of 21.2%. Finally, the binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.« less

  2. Inhibition of α-class cytosolic human carbonic anhydrases I, II, IX and XII, and β-class fungal enzymes by carboxylic acids and their derivatives: new isoform-I selective nanomolar inhibitors.

    PubMed

    Sechi, Mario; Innocenti, Alessio; Pala, Nicolino; Rogolino, Dominga; Carcelli, Mauro; Scozzafava, Andrea; Supuran, Claudiu T

    2012-09-15

    The members of a focused series of carboxylic acids and of their derivatives (esters, amides and metal complexes) have been investigated as inhibitors of the main cytosolic/transmembrane carbonic anhydrase isoforms, CA I, II, IX and XII, belonging to the mammalian α-class of CAs. These enzymes are present in red blood cells in submillimolar concentration, and typical sulfonamide CA inhibitors do not selectively inhibit any of them. Among such isozymes, the isoform-I is an 'orphan' target that mediates hemorrhagic retinal and cerebral vascular permeability, involved in retinal and cerebral disease. In the present study, we identified the first selective CA I nanomolar inhibitors, that displayed activity against other isozymes in micromolar/millimolar concentration range. Selective CA II over CA I inhibition has also been observed with some diketo acids/metal complexes. Few diketo acid derivatives showed inhibition activities against the fungal β-class enzymes from Candida albicans and Cryptococcus neoformans in low micromolar concentration range. Prediction of drug-like properties for the most interesting compounds suggests a favorable bioavailability. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars

    USDA-ARS?s Scientific Manuscript database

    Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta and pre-implantation blastocyst. All catalyze estrogen synthesis, but the “gonadal” type enzyme is unique in also synthesizing a nonaromat...

  4. Defoliation Induces Fructan 1-Exohydrolase II in Witloof Chicory Roots. Cloning and Purification of Two Isoforms, Fructan 1-Exohydrolase IIa and Fructan 1-Exohydrolase IIb. Mass Fingerprint of the Fructan 1-Exohydrolase II Enzymes1

    PubMed Central

    Van den Ende, Wim; Michiels, An; Van Wonterghem, Dominik; Clerens, Stefan P.; De Roover, Joke; Van Laere, André J.

    2001-01-01

    The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated “Belgian endive” leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5′- and 3′-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation). PMID:11457968

  5. Defoliation induces fructan 1-exohydrolase II in Witloof chicory roots. Cloning and purification of two isoforms, fructan 1-exohydrolase IIa and fructan 1-exohydrolase IIb. Mass fingerprint of the fructan 1-exohydrolase II enzymes.

    PubMed

    Van den Ende, W; Michiels, A; Van Wonterghem, D; Clerens, S P; De Roover, J; Van Laere, A J

    2001-07-01

    The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5'- and 3'-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

  6. Repression of a Novel Isoform of Disproportionating Enzyme (stDPE2) in Potato Leads to Inhibition of Starch Degradation in Leaves But Not Tubers Stored at Low Temperature1

    PubMed Central

    Lloyd, James R.; Blennow, Andreas; Burhenne, Kim; Kossmann, Jens

    2004-01-01

    A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 4°C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anion-exchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen. PMID:15034166

  7. Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA

    SciTech Connect

    Kazi, Julhash U.; Soh, Jae-Won

    2007-12-14

    Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the pathways of signal transduction, growth control and tumorigenesis. Variations in the intracellular localization of the individual isoforms are thought to be an important mechanism for the isoform-specific regulation of enzyme activity and substrate specificity. To provide a dynamic method of analyzing the localization of the specific isoforms of PKC in living cells, we generated fluorescent fusion proteins of the various PKC isoforms by using the green fluorescent protein (GFP) as a fluorescent marker at the carboxyl termini of these enzymes. The intracellular localization of the specificmore » PKC isoforms was then examined by fluorescence microscopy after transient transfection of the respective PKC-GFP expression vector into NIH3T3 cells and subsequent TPA stimulation. We found that the specific isoforms of PKC display distinct localization patterns in untreated NIH3T3 cells. For example, PKC{alpha} is localized mainly in the cytoplasm while PKC{epsilon} is localized mainly in the Golgi apparatus. We also observed that PKC{alpha}, {beta}1, {beta}2, {gamma}, {delta}, {epsilon}, and {eta} translocate to the plasma membrane within 10 min of the start of TPA treatment, while the cellular localizations of PKC{zeta} and {iota} were not affected by TPA. Using a protein kinase inhibitor, we also showed that the kinase activity was not important for the translocation of PKC. These results suggest that specific PKC isoforms exert spatially distinct biological effects by virtue of their directed translocation to different intracellular sites.« less

  8. Only One Isoform of Drosophila melanogaster CTP Synthase Forms the Cytoophidium

    PubMed Central

    Azzam, Ghows; Liu, Ji-Long

    2013-01-01

    CTP synthase is an essential enzyme that plays a key role in energy metabolism. Several independent studies have demonstrated that CTP synthase can form an evolutionarily conserved subcellular structure termed cytoophidium. In budding yeast, there are two isoforms of CTP synthase and both isoforms localize in cytoophidium. However, little is known about the distribution of CTP synthase isoforms in Drosophila melanogaster. Here, we report that three transcripts generated at the CTP synthase gene locus exhibit different expression profiles, and three isoforms encoded by this gene locus show a distinct subcellular distribution. While isoform A localizes in the nucleus, isoform B distributes diffusely in the cytoplasm, and only isoform C forms the cytoophidium. In the two isoform C-specific mutants, cytoophidia disappear in the germline cells. Although isoform A does not localize to the cytoophidium, a mutation disrupting mostly isoform A expression results in the disassembly of cytoophidia. Overexpression of isoform C can induce the growth of the cytoophidium in a cell-autonomous manner. Ectopic expression of the cytoophidium-forming isoform does not cause any defect in the embryos. In addition, we identify that a small segment at the amino terminus of isoform C is necessary but not sufficient for cytoophidium formation. Finally, we demonstrate that an excess of the synthetase domain of CTP synthase disrupts cytoophidium formation. Thus, the study of multiple isoforms of CTP synthase in Drosophila provides a good opportunity to dissect the biogenesis and function of the cytoophidum in a genetically tractable organism. PMID:23459760

  9. VDAC isoforms in mammals.

    PubMed

    Messina, Angela; Reina, Simona; Guarino, Francesca; De Pinto, Vito

    2012-06-01

    VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

  11. Constitutive nuclear localization of an alternatively spliced sirtuin-2 isoform.

    PubMed

    Rack, Johannes G M; VanLinden, Magali R; Lutter, Timo; Aasland, Rein; Ziegler, Mathias

    2014-04-17

    Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G2/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform. © 2013.

  12. UDP-glucuronosyltransferase 2B15 (UGT2B15) is the major enzyme responsible for sipoglitazar glucuronidation in humans: retrospective identification of the UGT isoform by in vitro analysis and the effect of UGT2B15*2 mutation.

    PubMed

    Nishihara, Mitsuhiro; Hiura, Yuto; Kawaguchi, Naohiro; Takahashi, Junzo; Asahi, Satoru

    2013-01-01

    Recently, genotyping in clinical studies has revealed that UGT2B15 genetic polymorphism has an influence on the clinical pharmacokinetics of sipoglitazar. In this study, the UGT responsible for sipoglitazar was retrospectively identified by in vitro analysis. A study using UGT-expressing supersomes revealed that sipoglitazar glucuronidation was more extensively catalyzed by UGT1A1, 1A3, 1A6, 2B4, and 2B15 than by other UGTs. Enzyme kinetic studies for sipoglitazar glucuronidation and recent findings related to mRNA expression analysis of UGTs narrowed the involved isoforms down to UGT1A1 and UGT2B15 among these five human UGTs. In a correlation study between sipoglitazar glucuronidation and UGT isoform-specific activities, the glucuronidation of S-oxazepam, a specific substrate for UGT2B15, strongly correlated with that of sipoglitazar, as compared with that of β-estradiol, a representative UGT1A1 substrate. The analysis of the species difference strengthens the possibility of UGT2B15 rather than that of UGT1A1. These in vitro findings indicate that UGT2B15 is principally responsible for sipoglitazar glucuronidation. Moreover, the UGT2B15*2 mutation significantly increased the Km value of sipoglitazar in the kinetic analysis using recombinant His-tag UGT2B15*1- or *2-membrane fractions. These results show that sipoglitazar is a good example to elucidate the relationship between phenotype and genotype for UGT2B15 from in vitro analysis.

  13. Insulin specifically regulates expression of liver and muscle phosphofructokinase isoforms.

    PubMed

    Ausina, Priscila; Da Silva, Daniel; Majerowicz, David; Zancan, Patricia; Sola-Penna, Mauro

    2018-04-11

    Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis, being considered the pacemaker of this pathway. In mammals, this enzyme exists as three different isoforms, PFKM, PFKL and PFKP, presenting different regulatory and catalytic properties. The expression of these isoforms is tissue-specific and vary according to the cell differentiation and signalization. Although it is known that the expression of the different PFK isoforms directly affects cell function, the information regarding the regulation of PFK isoforms expression is scarce. In the present work, we evaluate the role of insulin signalization on the expression of three PFK isoforms on skeletal muscle, liver, and epididymal white adipose tissue (eWAT) of mice. For this, Swiss mice were treated with streptozotocin (STZ) to disrupt pancreatic ß-cells and, thus, insulin production. Control group were treated with citrate buffer (STZ vehicle). These groups were then treated with insulin or saline twice a day for ten consecutive days when animals were euthanized and tissues used for the evaluation of PFK isoforms expression by quantitative PCR (qPCR). Our results revealed that the lack of insulin significantly impacted the expression of PFKL, presenting mild effects on PFKM and no effects on PFKP. The decrease of PFKL and PFKM mRNA levels observed on the group treated with STZ was reversed by the treatment with insulin. In conclusion, insulin, the most known regulator of glucose consumption, specifically regulates the expression of PFKL and PFKM, which impact the regulation of glycolysis in the cell. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  14. Tacrolimus strongly inhibits multiple human UDP-glucuronosyltransferase (UGT) isoforms.

    PubMed

    Liu, Xiao-You; Fang, Zhong-Ze; Dong, Pei-Pei; Shi, Xiang-Hua; Teng, Yan-Jie; Sun, Xu-Yong

    2012-09-01

    The objective of the present study is to clearly evaluate the inhibitory effects of tacrolimus (tacro) on important UGT isoforms in human liver, including determination of inhibition kinetic type and calculation of inhibition kinetic parameters. An in vitro incubation system was used to investigate the inhibitory effect of tacro on UGT isoforms. The recombinant UGT isoforms were used as enzyme source, and a nonspecific substrate 4-methylumbelliferone (4-MU) was utilized as substrate. Among the tested UGT isoforms, UGT1A1, UGT1A3, UGT2B7 and UGT2B15 were strongly inhibited by tacro in a concentration-dependent manner. Dixon and Lineweaver-Burk plots showed that the inhibition of UGT1A1, UGT1A3, and UGT2B7 was all best fit to competitive inhibition type, and the inhibition of UGT2B15 was best fit to noncompetitive type. The inhibition kinetic parameters (Ki) were determined to be 4.7, 1.3, 1.9, and 4.3 microM for UGT1A1, UGT1A3, UGT2B7, and UGT2B15, respectively. Inhibition of these important UGT isoforms in human liver might be an important reason for clinically frequent drug-drug interaction between tacro and other drugs.

  15. Characterization of Glutamine Synthetase Isoforms from Chlorella1

    PubMed Central

    Beudeker, Rob F.; Tabita, F. Robert

    1985-01-01

    Ion-exchange chromatography of extracts derived from Chlorella sorokiniana mutant strain (oxygen resistant) yielded two separate activity peaks of glutamine synthetase (GS). GSI and GSII were purified 220- and 187-fold and have molecular weights of approximately 398,000 and 360,000, respectively. Both enzymes are composed of eight identical subunits with a subunit molecular weight of 47,000 for GSI and 43,000 for GSII. The amino acid composition, catalytic, and immunological properties for both enzymes are similar. Images Fig. 1 PMID:16664139

  16. Variation in Sulfur and Selenium Accumulation Is Controlled by Naturally Occurring Isoforms of the Key Sulfur Assimilation Enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis Species Range1[W][OPEN

    PubMed Central

    Chao, Dai-Yin; Baraniecka, Patrycja; Danku, John; Koprivova, Anna; Lahner, Brett; Luo, Hongbing; Yakubova, Elena; Dilkes, Brian; Kopriva, Stanislav; Salt, David E.

    2014-01-01

    Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored. PMID:25245030

  17. Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

    PubMed Central

    Gates, Simon; Miners, John O

    1999-01-01

    Aims The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. Methods The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. Results The CYP1A2 inhibitor furafylline variably inhibited (0–65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by ≈55–60%, 35–55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. Conclusions Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans. PMID:10215755

  18. Direct Activation of Epac by Sulfonylurea is Isoform Selective

    PubMed Central

    Herbst, Katie J.; Coltharp, Carla; Amzel, L. Mario; Zhang, Jin

    2011-01-01

    Summary Commonly used as a treatment for Type II diabetes, sulfonylureas (SUs) stimulate insulin secretion from pancreatic β cells by binding to sulfonylurea receptors. Recently, SUs have been shown to also activate exchange protein directly activated by cAMP 2 (Epac2), however little is known about this molecular action. Using biosensor imaging and biochemical analysis, we show that SUs activate Epac2 and the downstream signaling via direct binding to Epac2. We further identify R447 of Epac2 to be critically involved in SU binding. This distinct binding site from cAMP points to a new mode of allosteric activation of Epac2. We also show that SUs selectively activate Epac2 isoform, but not the closely related Epac1, further establishing SUs as a new class of isoform-selective enzyme activators. PMID:21338921

  19. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    PubMed

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP 3 . In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. 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.

  1. A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms1[OPEN

    PubMed Central

    Schipper, Bert; Beekwilder, Jules

    2016-01-01

    A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments. PMID:27707890

  2. Inhibition of myo-inositol monophosphatase isoforms by aromatic phosphonates.

    PubMed

    Ganzhorn, A J; Hoflack, J; Pelton, P D; Strasser, F; Chanal, M C; Piettre, S R

    1998-10-01

    alpha-Hydroxyphosphonates are moderately potent (Ki = 6-600 microM) inhibitors of the enzyme myo-inositol monophosphatase (McLeod et al., Med. Chem. Res. 1992, 2, 96). Hydroxy-[4-(5,6,7,8-tetrahydronaphtyl-1-oxy)phenyl]methyl phosphonate (3) was resynthesized and its inhibitory potency towards the recombinant bovine brain enzyme confirmed (Ki = 20 microM). Similar aromatic difluoro-, keto-, and ketodifluorophosphonates (5, 7, 9) were inactive. Compound 3 was 15-fold less active on the human as compared to the bovine enzyme. Molecular modeling suggested that the hydrophobic part of the inhibitor interacts with amino acid side chains that are located at the interface between the enzyme subunits in an area (amino acids 175-185) with low similarity between the two isozymes. Phe-183 in the human enzyme was replaced with leucine, the corresponding residue in the bovine isoform. The three isozymes (human wild-type, bovine wild-type and human F183L) had similar kinetic properties, except that the bovine enzyme was less effectively inhibited by high concentrations of the activator Mg2+. The F183L mutant enzyme had a twofold increased affinity for compound 3 as compared to the human wild-type form. We conclude that residue 183 contributes to the binding of aromatic hydroxyphosphonates to IMPase, but it is not the only determining factor for inhibitor specificity with respect to different isozymes.

  3. 3D-localization of the a-subunit in F 0F I-ATP synthase by time resolved single-molecule FRET

    NASA Astrophysics Data System (ADS)

    Düser, Monika G.; Zarrabi, Nawid; Bi, Yumin; Zimmermann, Boris; Dunn, Stanley D.; Börsch, Michael

    2006-02-01

    F °F I-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F I part to the proton translocation through the F ° part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F ° motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ɛ-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ɛ-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ɛ resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ɛ, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F ° part.

  4. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    SciTech Connect

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms ofmore » ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.« less

  5. [Effect of industrial toxic pollutants on the activity and isoforms of acid DNase in the freshwater snail (Viviparus viviparus L.)].

    PubMed

    Popov, A P; Konichev, A S; Tsvetkov, I L

    2003-01-01

    The effect of various toxic compounds (phenol, gasoline, detergents, halogenated benzenes, and copper salts) on the activity and multiple forms of acid DNase was investigated in the liver of the widespread freshwater snail species Viviparus viviparus L. Characteristic variations in the specific activity and isoform pattern of the enzyme depending on pollutant concentration and exposure time were revealed. It was shown that the pattern of DNase isoforms in V. viviparus could be an index of water pollution.

  6. Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae

    PubMed Central

    Gorman, Maureen J.; Sullivan, Lucinda I.; Nguyen, Thi D. T.; Dai, Huaien; Arakane, Yasuyuki; Dittmer, Neal T.; Syed, Lateef U.; Li, Jun; Hua, Duy H.; Kanost, Michael R.

    2011-01-01

    Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-β-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 – 550 min−1 mM−1. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 – 30 min−1 mM−1; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min−1 mM−1. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions. PMID:22198355

  7. Friedelin in Maytenus ilicifolia Is Produced by Friedelin Synthase Isoforms.

    PubMed

    Alves, Thaís B; Souza-Moreira, Tatiana M; Valentini, Sandro R; Zanelli, Cleslei F; Furlan, Maysa

    2018-03-20

    Triterpenes are interesting compounds because they play an important role in cell homeostasis and a wide variety exhibiting defense functions is produced by plant secondary metabolism. Those same plant secondary metabolites also exhibit biological properties with promising therapeutic potential as anti-inflammatory and antitumor agents. Friedelin is a triterpene ketone with anti-inflammatory and gastroprotective activities and it is a precursor of relevant antitumor quinonemethides. Although many triterpene synthases have been described, only two friedelin synthases were characterized and there is no information about their genomic features and alleles. In the present work, we aimed to identify the gene and new isoforms of friedelin synthase in Maytenus ilicifolia leaves to be functionally characterized in Saccharomyces cerevisiae . The gene sequence analysis elucidated the exon/intron structure and confirmed the presence of single nucleotide polymorphisms with four non-synonymous mutations outside the active site of the enzyme. Therefore, two new isoforms were observed and the heterologous production of the enzymes in yeast showed similar production of friedelin. This first description of different alleles of the gene of friedelin synthase in M. ilicifolia can guide their validation as markers for friedelin-producer specimens.

  8. Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

    PubMed

    Costantin, J; Noceti, F; Qin, N; Wei, X; Birnbaumer, L; Stefani, E

    1998-02-15

    1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

  9. 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…

  10. Identification of novel RNA isoforms of LMNA.

    PubMed

    DeBoy, Emily; Puttaraju, Madaiah; Jailwala, Parthav; Kasoji, Manjula; Cam, Maggie; Misteli, Tom

    2017-09-03

    The nuclear lamina is a proteinaceous meshwork situated underneath the inner nuclear membrane and is composed of nuclear lamin proteins, which are type-V intermediate filaments. The LMNA gene gives rise to lamin A and lamin C through alternative splicing. Mutations in LMNA cause multiple diseases known as laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder caused by a point mutation that activates a cryptic 5' splice site in exon 11, resulting in a 150 bp deletion in the LMNA mRNA and the production of the dominant lamin A isoform progerin. During RNA sequencing analysis of wild type and HGPS patient skin fibroblasts, we discovered two novel LMNA isoforms. LMNAΔ447 and LMNAΔ297 use an alternative 3' splice acceptor site in the 3' untranslated region, and either the HGPS cryptic 5' splice site in exon 11 or the wild type 5' splice site. Both isoforms are present at low levels in HGPS patient and wild type cells in multiple cell types. We validate and quantify the expression levels of these novel isoforms in HGPS and wild type fibroblasts. Overexpression of either LMNAΔ447 or LMNAΔ297 is not sufficient to induce the typical HGPS cellular disease phenotypes and no significant difference in the two isoforms were found between young and old fibroblasts. These results identify and characterize two novel RNA isoforms of LMNA produced through alternative splicing.

  11. p53 Isoforms: An Intracellular Microprocessor?

    PubMed

    Khoury, Marie P; Bourdon, Jean-Christophe

    2011-04-01

    Normal function of the p53 pathway is ubiquitously lost in cancers either through mutation or inactivating interaction with viral or cellular proteins. However, it is difficult in clinical studies to link p53 mutation status to cancer treatment and clinical outcome, suggesting that the p53 pathway is not fully understood. We have recently reported that the human p53 gene expresses not only 1 but 12 different p53 proteins (isoforms) due to alternative splicing, alternative initiation of translation, and alternative promoter usage. p53 isoform proteins thus contain distinct protein domains. They are expressed in normal human tissues but are abnormally expressed in a wide range of cancer types. We have recently reported that p53 isoform expression is associated with breast cancer prognosis, suggesting that they play a role in carcinogenesis. Indeed, the cellular response to damages can be switched from cell cycle arrest to apoptosis by only manipulating p53 isoform expression. This may provide an explanation to the hitherto inconsistent relationship between p53 mutation, treatment response, and outcome in breast cancer. However, the molecular mechanism is still unknown. Recent reports suggest that it involves modulation of gene expression in a p53-dependent and -independent manner. In this review, we summarize our current knowledge about the biological activities of p53 isoforms and propose a molecular mechanism conciliating our current knowledge on p53 and integrating p63 and p73 isoforms in the p53 pathway.

  12. 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

  13. Deep understanding of the interaction between thienorphine and UDP-glucuronosyltransferase (UGT) isoforms.

    PubMed

    Dong, Rui-Hua; Fang, Zhong-Ze; Zhu, Liang-Liang; Ge, Guang-Bo; Li, Xiao-Bao; Hu, Cui-Min; Cao, Yun-Feng; Xia, Yang-Liu; Yang, Ling; Liu, Ze-Yuan

    2013-02-01

    Thienorphine has been demonstrated to be a potent, long-acting partial opioid agonist. It is being developed as a good candidate to treat opioid dependence. The thienorphine's glucuronide was detected after thienorphine was incubated with human liver microsomes (HLMs). Recombinant UGT isoforms screening experiment and enzyme kinetic study showed that UGT1A1 completely contributed to the glucuronidation of thienorphine. Among the tested UGT isoforms, UGT1A3 and UGT2B7 were inhibited by thienorphine, with other UGT isoforms negligibly influenced. The inhibition type is competitive, and inhibition kinetic parameters (K(i)) were 1.65 and 5.27 μM for UGT1A3 and UGT2B7, respectively. However, due to low plasma concentration of thienorphine, in vivo drug-drug interaction might not occur.

  14. Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue

    NASA Technical Reports Server (NTRS)

    Karuppiah, N.; Vadlamudi, B.; Kaufman, P. B.

    1989-01-01

    Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (beta-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organo-mercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, Km of 12 millimolar, and a Vmax of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, Km of millimolar, and a Vmax of 9 micromole per minute per milligram of protein.

  15. Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

    PubMed Central

    Štrbenc, Malan; Smerdu, Vika; Pogačnik, Azra; Fazarinc, Gregor

    2006-01-01

    To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (α-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4–6 weeks of age, which coincides with the increased physical activity of puppies. PMID:16879596

  16. 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.

  17. 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

  18. Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust.

    PubMed

    Martínez-Morales, Fernando; Bertrand, Brandt; Pasión Nava, Angélica A; Tinoco, Raunel; Acosta-Urdapilleta, Lourdes; Trejo-Hernández, María R

    2015-02-01

    Two laccase isoforms (lcc1 and lcc2) produced by Trametes versicolor, grown on oak sawdust under solid-state fermentation conditions, were purified and characterized. The two isoforms showed significant biochemical differences. Lcc1 and lcc2 had MWs of 60 and 100 kDa, respectively. Both isoforms had maximal activity at pH 3 with ABTS and 2,6-dimethyloxyphenol (DMP). Lcc1 was the most attractive isoform due to its greater affinity towards all the laccase substrates used. Lcc1 had Km values of 12, 10, 15 and 17 mM towards ABTS, DMP, guaiacol and syringaldazine, respectively. Lcc2 had equivalent values of 45, 47, 15 and 39 mM. The biochemical properties of lcc1 substantiate the potential of this enzyme for application in the treatment of contaminated water with low pH values and high phenolic content.

  19. Expression of Gls and Gls2 glutaminase isoforms in astrocytes.

    PubMed

    Cardona, Carolina; Sánchez-Mejías, Elisabeth; Dávila, José C; Martín-Rufián, Mercedes; Campos-Sandoval, José A; Vitorica, Javier; Alonso, Francisco J; Matés, José M; Segura, Juan A; Norenberg, Michael D; Rama Rao, Kakulavarapu V; Jayakumar, Arumugan R; Gutiérrez, Antonia; Márquez, Javier

    2015-03-01

    The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes. © 2014 Wiley Periodicals, Inc.

  20. Crystallization and Identification of the Glycosylated Moieties of Two Isoforms of the Main Allergen Hev b 2 and Preliminary X-ray Analysis of Two Polymorphs of Isoform ll

    SciTech Connect

    Fuentes-Silva,D.; Mendoza-Hernandez, G.; Stojanoff, V.

    2007-01-01

    Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a {beta}-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapor-diffusion method. Isoform Imore » crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 {angstrom} were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 {angstrom}, {beta}= 113.6{sup o}. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.« less

  1. Characterization of endogenous human promyelocytic leukemia isoforms.

    PubMed

    Condemine, Wilfried; Takahashi, Yuki; Zhu, Jun; Puvion-Dutilleul, Francine; Guegan, Sarah; Janin, Anne; de Thé, Hugues

    2006-06-15

    Promyelocytic leukemia (PML) has been implicated in a variety of functions, including control of TP53 function and modulation of cellular senescence. Sumolated PML is the organizer of mature PML bodies, recruiting a variety of proteins onto these nuclear domains. The PML gene is predicted to encode a variety of protein isoforms. Overexpression of only one of them, PML-IV, promotes senescence in human diploid fibroblasts, whereas PML-III was proposed to specifically interact with the centrosome. We show that all PML isoform proteins are expressed in cell lines or primary cells. Unexpectedly, we found that PML-III, PML-IV, and PML-V are quantitatively minor isoforms compared with PML-I/II and could not confirm the centrosomal targeting of PML-III. Stable expression of each isoform, in a pml-null background, yields distinct subcellular localization patterns, suggesting that, like in other RBCC/TRIM proteins, the COOH-terminal domains of PML are involved in interactions with specific cellular components. Only the isoform-specific sequences of PML-I and PML-V are highly conserved between man and mouse. That PML-I contains all conserved exons and is more abundantly expressed than PML-IV suggests that it is a critical contributor to PML function(s).

  2. Pancreatic Enzymes

    MedlinePlus

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  3. Possible senescence associated change in the predominant a-Na+/K+ ATP-ase isoform in the renal cortex of the rat.

    PubMed

    Potilinski, María Constanza; Moretta, Rosalía; Casal, Leonardo; García Gras, Eduardo; Amorena, Carlos E

    With aging the kidney exhibits progressive deterioration, with a decrease in renal function. Most of the filtered Na+ is actively reabsorbed in the proximal tubules through different transporters located in apical membrane. This process is possible because basolateral Na+/K+-ATP-ase generates electrochemical conditions necessary for energetically favorable Na+ transport. The a-subunit is the catalytic domain of Na+/K+-ATP-ase. There are three isoforms of the a/subunit present in rat kidney. The present study was undertaken to examine the expression pattern of rat a-Na+/K+-ATP-ase during senescence. We tested the impact of aging on mRNA expression of a-Na+/K+-ATP-ase in cortex and medulla of aged Wistar rats. We observed a significant expression decrease in mRNA levels and a possible change of isoform in the cortex of aged animals. These expression changes observed for a subunit could be contributing to affect the renal function in conditions of water and salt stress.

  4. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    PubMed

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  5. Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

    PubMed

    Castiglia, Daniela; Cardi, Manuela; Landi, Simone; Cafasso, Donata; Esposito, Sergio

    2015-08-01

    In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Differential localization of Acanthamoeba myosin I isoforms

    PubMed Central

    1992-01-01

    Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids. PMID:1447297

  7. Characterization of Rabaptin-5 γ isoform.

    PubMed

    Korobko, E V; Kiselev, S L; Korobko, I V

    2014-09-01

    Rab GTPases are key regulators of intracellular membrane traffic acting through their effector molecules. Rabaptin-5 is a Rab5 effector in early endosome fusion and connects Rab5- and Rab4-positive membrane compartments owing to its ability to interact with Rab4 GTPase. Recent studies showed that Rabaptin-5 transcript is subjected to extensive alternative splicing, thus resulting in expression of Rabaptin-5 isoforms mostly bearing short deletions in the polypeptide chain. As interactions of a Rab GTPase with different effectors lead to different responses, functional characterization of Rabaptin-5 isoforms becomes an attractive issue. Indeed, it was shown that Rab GTPase effector properties of Rabaptin-5 and its α and δ isoforms are different. This work focused on another Rabaptin-5 isoform, Rabaptin-5γ. Despite its ability to interact with Rab5, endogenously produced Rabaptin-5γ was absent from early endosomes. Rather, it was found to be tightly associated with trans-Golgi network and partially localized to a Rab4-positive membrane compartment. The revealed intracellular localization of Rabaptin-5γ indicates that it is not involved in Rab5-driven events; rather, it functions in other membrane transport steps. Our study signifies the role of alternative splicing in determination of functional activities of Rab effector molecules.

  8. Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

    PubMed

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

    2015-01-01

    Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

  9. Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries

    PubMed Central

    Chen, Shanyan; Chen, Zhenzhou; Tomlinson, Brittany N.; Wesley, Jennifer M.; Sun, Grace Y.; Whaley-Connell, Adam T.; Sowers, James R.; Cui, Jiankun; Gu, Zezong

    2015-01-01

    Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions. PMID:25859655

  10. Tandem mass spectrometry multiplex analysis of methylated and non-methylated urinary Gb3 isoforms in Fabry disease patients.

    PubMed

    Abaoui, Mona; Boutin, Michel; Lavoie, Pamela; Auray-Blais, Christiane

    2016-01-15

    Fabry disease is a lysosomal storage disorder leading to the accumulation of glycosphingolipids in biological fluids and tissues. Globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are currently used for Fabry screening and diagnosis. However, these biomarkers are not always increased in Fabry patients with residual enzyme activity. We recently identified 7 urinary methylated Gb3-related isoforms. The aims of this study were (1) to develop and validate a novel LC-MS/MS method for the relative quantification of methylated and non-methylated Gb3 isoforms normalized to creatinine, (2) to evaluate these biomarkers in Fabry patients and healthy controls, and (3) to assess correlations between biomarker urinary excretion with age, gender, treatment and genotype of patients. Urine samples from 150 Fabry patients and 95 healthy controls were analyzed. Samples were purified and injected in the tandem mass spectrometer working in positive electrospray ionization. Relative quantification was performed for 15 methylated and non-methylated Gb3 isoforms. Significant correlations (p<0.001) were established between Gb3 isoform concentrations, gender and treatment. Five patients with the late-onset cardiac mutation p.N215S showed abnormal concentrations of methylated Gb3 isoforms compared to their non-methylated homologues. Methylated Gb3 isoforms might be helpful urinary biomarkers for Fabry patients with late-onset cardiac variant mutations. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

    SciTech Connect

    Fuentes-Silva, D.; Mendoza-Hernández, G.; Stojanoff, V.

    2007-09-01

    Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoformsmore » were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.« less

  12. Integrated genomic and functional analyses of histone demethylases identify oncogenic KDM2A isoform in breast cancer.

    PubMed

    Liu, Hui; Liu, Lanxin; Holowatyj, Andreana; Jiang, Yuanyuan; Yang, Zeng-Quan

    2016-05-01

    Histone lysine demethylases (KDMs) comprise a large class of enzymes that catalyze site-specific demethylation of lysine residues on histones and other proteins. They play critical roles in controlling transcription, chromatin architecture, and cellular differentiation. However, the genomic landscape and clinical significance of KDMs in breast cancer remain poorly characterized. Here, we conducted a meta-analysis of 24 KDMs in breast cancer and identified associations among recurrent copy number alterations, gene expression, breast cancer subtypes, and clinical outcome. Two KDMs, KDM2A and KDM5B, had the highest frequency of genetic amplification and overexpression. Furthermore, among the 24 KDM genes, KDM2A had the highest correlation between copy number and mRNA expression, and high mRNA levels of KDM2A were significantly associated with shorter survival of breast cancer patients. KDM2A has two isoforms: the long isoform is comprised of a JmjC domain, CXXC-zinc finger, PHD zinc finger, F-box, and the AMN1 protein domain; whereas the short isoform of KDM2A lacks the N-terminal JmjC domain but contains all other motifs. Detailed characterization of KDM2A in breast cancer revealed that the short isoform of KDM2A is more abundant than the long isoform at DNA, mRNA, and protein levels in a subset of breast cancers. Furthermore, our data indicate that the short isoform of KDM2A has oncogenic potential and functions as an oncogenic isoform in a subset of breast cancers. Taken together, our findings suggest that amplification and overexpression of the KDM2A short isoform is critical in breast cancer progression. © 2015 Wiley Periodicals, Inc.

  13. Impaired Discrimination Learning in Mice Lacking the NMDA Receptor NR2A Subunit

    ERIC Educational Resources Information Center

    Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew

    2008-01-01

    N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…

  14. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin

    2006-06-09

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-stepmore » affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.« less

  15. Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants

    PubMed Central

    Rossano, Rocco; Larocca, Marilena; Riviello, Lea; Coniglio, Maria Gabriella; Vandooren, Jennifer; Liuzzi, Grazia Maria; Opdenakker, Ghislain; Riccio, Paolo

    2014-01-01

    The matrix metalloproteinases (MMPs) gelatinase A (MMP-2) and gelatinase B (MMP-9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2-DZ) as an extension of classic monodimensional zymography (1-DZ) to analyse the complete pattern of isoforms and post-translational modifications of both MMP-9 and MMP-2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP-2 isoforms, and at least three different isoforms and two different states of organization of MMP-9 (the multimeric MMP-9 and the N-GAL-MMP-9 complex) were observed. In addition, 2-DZ revealed for the first time that all MMP-9 and MMP-2 isoforms actually exist in the form of charge variants: four or five variants in the N-GAL complex, more charge variants in the case of MMP-9; and five to seven charge variants for MMP-2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP-9) and phosphorylation (MMP-2) contributed to molecular heterogeneity. The detection of charge variants of MMP-9 and MMP-2 in MS serum samples illustrates the power of 2-DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases. PMID:24616914

  16. Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12.

    PubMed

    Ball, Ann-Katrin; Beilstein, Kim; Wittmann, Sandra; Sürün, Duran; Saul, Meike J; Schnütgen, Frank; Flamand, Nicolas; Capelo, Ricardo; Kahnt, Astrid S; Frey, Helena; Schaefer, Liliana; Marschalek, Rolf; Häfner, Ann-Kathrin; Steinhilber, Dieter

    2017-05-01

    Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Isoform specificity of progesterone receptor antibodies.

    PubMed

    Fabris, Victoria; Abascal, María F; Giulianelli, Sebastián; May, María; Sequeira, Gonzalo R; Jacobsen, Britta; Lombès, Marc; Han, Julie; Tran, Luan; Molinolo, Alfredo; Lanari, Claudia

    2017-10-01

    Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone-dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N-terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin-fixed paraffin-embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D-YA and -YB cells expressing PRA or PRB, respectively, MDA-MB-231 cells modified to synthesize PRB, and MDA-MB-231/iPRAB cells which can bi-inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H-190, clone 636, clone 16, and Ab-6 anti-PR antibodies, the latter exclusively recognizing PRB. Except for Ab-6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H-190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA-specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer.

  18. Salt-inducible isoform of plasma membrane H+ATPase gene in rice remains constitutively expressed in natural halophyte, Suaeda maritima.

    PubMed

    Sahu, Binod Bihari; Shaw, Birendra Prasad

    2009-07-01

    To look into a possible involvement of plasma membrane H+ATPase (PM-H+ATPase, EC 3.6.3.6) in mitigation of physiological disturbances imposed by salt stress, response of the enzyme was studied in two Oryza sativa Indica cultivars, salt-tolerant Lunishri and non-tolerant Badami, and a natural halophyte Suaeda maritima after challenge of the young plants with NaCl. Significant increase in activity of the enzyme was observed in response to NaCl in all the test plants with S. maritima showing maximum increase. Protein blot analysis, however, did not show any increase in the amount of the enzyme (protein). RNA blot analysis, on the other hand, revealed significant increase in transcript level of the enzyme upon NaCl treatment. In the rice cultivars, salt treatment also induced expression of a new isoform of PM-H+ATPase gene, not reported so far. The induced transcript showed maximum homology to OSA7 (O. sativa PM-H+ATPase isoform 7). Similar transcript message, however, remained constitutively present in S. maritima, along with the transcript of another isoform of PM-H+ATPase showing resemblance to OSA3 (O. sativa PM-H+ATPase isoform 3). The latter was the only PM-H+ATPase isoform expressed in both the rice cultivars not exposed to NaCl. In the salt-treated test plants, both rice and S. maritima, the salt-inducible PM-H+ATPase isoform resembling OSA7 was expressed in much greater amount than that resembling OSA3. Appearance of a new PM-H+ATPase transcript, besides increase in the enzyme activity, indicates the important role of the enzyme in maintaining ion-homeostasis in plants under salt stress, enabling them to survive under saline conditions.

  19. A novel splicing isoform of protein arginine methyltransferase 1 (PRMT1) that lacks the dimerization arm and correlates with cellular malignancy.

    PubMed

    Patounas, Odysseas; Papacharalampous, Ioanna; Eckerich, Carmen; Markopoulos, Georgios S; Kolettas, Evangelos; Fackelmayer, Frank O

    2018-02-01

    Methylation of arginine residues is an important modulator of protein function that is involved in epigenetic gene regulation, DNA damage response and RNA maturation, as well as in cellular signaling. The enzymes that catalyze this post-translational modification are called protein arginine methyltransferases (PRMTs), of which PRMT1 is the predominant enzyme. Human PRMT1 has previously been shown to occur in seven splicing isoforms, which are differentially abundant in different tissues, and have distinct substrate specificity and intracellular localization. Here we characterize a novel splicing isoform which does not affect the amino-terminus of the protein like the seven known isoforms, but rather lacks exons 8 and 9 which encode the dimerization arm of the enzyme that is essential for enzymatic activity. Consequently, the isoform does not form catalytically active oligomers with the other endogenous PRMT1 isoforms. Photobleaching experiments reveal an immobile fraction of the enzyme in the nucleus, in accordance with earlier results from our laboratory that had shown a tight association of inhibited or inactivated PRMT1 with chromatin and the nuclear scaffold. Thus, it apparently is able to bind to the same substrates as catalytically active PRMT1. This isoform is found in a variety of cell lines, but is increased in those of cancer origin or after expression of the EMT-inducing transcriptional repressor Snail1. We discuss that the novel isoform could act as a modulator of PRMT1 activity in cancer cells by acting as a competitive inhibitor that shields substrates from access to active PRMT1 oligomers. © 2017 Wiley Periodicals, Inc.

  20. Identification of a novel transcript isoform of the TTLL12 gene in human cancers.

    PubMed

    Wen, Ruiling; Xiao, Yingying; Zhang, Yuhua; Yang, Min; Lin, Yongping; Tang, Jun

    2016-12-01

    Tubulin tyrosine ligase like 12 (TTLL12), a member of the tubulin tyrosine ligase (TTLL) family, has not been completely characterized to date. It is reported that histone methylation, tubulin modifications, mitotic duration and chromosome ploidy play crucial roles in a variety of cancers, and are related to tumorigenesis and cancer progression. A recent study showed that TTLL12 may be a pseudo-enzyme which has a SET-like domain and a TTL-like domain. In the present study, we first used 3'-rapid amplification of cDNA ends (3'-RACE) to amplify the transcripts of the TTLL12 gene from a human lung cancer cell line H1299, and unexpectedly discovered a new transcript isoform characterized with an additional 108-bp nucleotide sequence inserted at the location from 902 to 903 bases of the TTLL12 coding sequence (CDS), where it also locates between exons 5 and 6. Next, utilizing RT-PCR and Sanger sequencing, we further confirmed the existence of such a new transcript isoform of TTLL12 in more human cancer cells including lung cancer cells and other cancer cells. Moreover, several lung cancer cell lines were found to display a much higher proportion of the new isoform compared with TTLL12 wild-type transcript. These results suggest that the new TTLL12 isoform may be of importance for proper maintenance of lung cancer cells. Therefore, the new isoform of TTLL12, with the inserted sequences probably acting as a disordered region, provides a novel perspective regarding TTLL12 functions in human cancers including lung cancer.

  1. Three Isoforms of Isoamylase Contribute Different Catalytic Properties for the Debranching of Potato GlucansW⃞

    PubMed Central

    Hussain, Hasnain; Mant, Alexandra; Seale, Robert; Zeeman, Sam; Hinchliffe, Edward; Edwards, Anne; Hylton, Christopher; Bornemann, Stephen; Smith, Alison M.; Martin, Cathie; Bustos, Regla

    2003-01-01

    Isoamylases are debranching enzymes that hydrolyze α-1,6 linkages in α-1,4/α-1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism. PMID:12509527

  2. Examination of Gelatinase Isoforms in Rodent Models of Acute Neurodegenerative Diseases Using Two-Dimensional Zymography.

    PubMed

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Qu, Zhe; Cui, Jiankun; Gu, Zezong

    2017-01-01

    Pathological activation of gelatinases (matrix metalloproteinase-2 and -9; MMP-2/-9) has been shown to cause a number of detrimental outcomes in neurodegenerative diseases. In gel gelatin zymography is a highly sensitive methodology commonly used in revealing levels of gelatinase activity and in separating the proform and active form of gelatinases, based on their different molecular weights. However, this methodology is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity can be regulated at transcriptional and/or post-translational levels under in vivo conditions resulting in alternation of their isoelectric focusing (IEF) points. In this chapter, we describe an advanced methodology, termed two-dimensional zymography, combining IEF with zymographic electrophoresis under non-reducing conditions to achieve significant improvement in separation of the gelatinase isoforms in both cell-based and in vivo models for acute brain injuries and neuroinflammation.

  3. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    PubMed Central

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  4. Silencing of the major salt-dependent isoform of pectinesterase in tomato alters fruit softening.

    PubMed

    Phan, Thanh D; Bo, Wen; West, Gill; Lycett, Grantley W; Tucker, Gregory A

    2007-08-01

    Pectinesterase (PE; E.C. 3.1.1.11) is an enzyme responsible for the demethylation of galacturonyl residues in high-molecular-weight pectin and is believed to play an important role in cell wall metabolism. In this study, Pmeu1, a ubiquitously expressed PE gene, has been characterized by antisense suppression in tomato (Solanum lycopersicum). Transgenic tomato plants showed reduced PE activity levels in both green fruit and leaf tissue to around 65% and 25% of that found in wild-type plants, respectively. Pmeu1 was observed to encode a salt-dependent PE isoform that correlated with PE1 as previously described in fruit tissue. Silencing of Pmeu1 did not result in any detectable phenotype within the leaf tissue despite the gene product representing the major isoform in this tissue. In comparison, silencing in fruit resulted in an enhancement to the rate of softening during ripening. The role of PMEU1 in fruit ripening is discussed.

  5. Construction of a Baculovirus vector containing A subunit of Shiga toxin for protein delivery.

    PubMed

    Oloomi, Mana; Bouzari, Saeid; Imani, Maryam; Akhtarian, Narges

    2013-12-01

    Baculovirus can be used as a vector in gene delivery system. Viral envelope of baculovirus would display expressed protein/peptide and it could render as a potential vaccine delivery system. In this regard, the gene coding for A subunit of shiga toxin (StxA) from Escherichia coli (E. coli) strain was cloned in a baculovirus expression system. StxA subunit has the ability to inhibit protein synthesis and this ability applied in cancer therapy. In this study, expression of StxA in baculovirus as a protein delivery system was assessed in vitro. StxA gene was cloned in pTriEx™ multisystem expression vector. This vector enables the protein expression in multisystem, E. coli and baculovirus. This construct was used to express the gene in E. coli and baculovirus. The construct containing StxA gene was made in baculovirus and expression was confirmed, then baculovirus expressing STXA transfect HeLa cells. The expression of STXA peptide (32kDa) was confirmed by SDS-PAGE and western blotting in both expression systems. The A subunit challenge to human cell Lines was applied as a delivery system by baculoviruses. On the other hand, the inhibition of cell proliferation was also demonstrated by baculovirus containing STXA subunit. STXA peptide expression in baculovirus was shown in E. coli and baculovirus expression system. Furthermore, it was shown that A subunit of Shiga toxin delivered by baculovirus can inhibit cell proliferation in HeLa cells and leading to cell death. Therefore, this prototype system could be a promising model for in vivo cancer therapy and targeted protein delivery system.

  6. Metallothionein isoform expression by breast cancer cells.

    PubMed

    Barnes, N L; Ackland, M L; Cornish, E J

    2000-08-01

    Expression of metallothionein (MT) isoforms by a human breast cancer cell line, PMC42, which retains many characteristics of normal breast epithelial cells and expresses functional estrogen receptors, was examined because it has been proposed that human breast cancer cells which are estrogen receptor positive can be differentiated from those which are estrogen receptor negative, by failure to express MT-1E [J.A. Friedline, S.H. Garrett, S. Somji, J.H. Todd, D. A. Sens, Differential expression of the MT-1E gene in estrogen-receptor positive and -negative breast cancer cell lines, Am. J. Pathol. 152 (1998) 23-27]. Using RT-PCR, PMC42 cells were found to transcribe genes for the MT isoforms IE, IX and 2A but not 1A or 1H. In order to examine which of the expressed isoforms might protect against metal toxicity, the cells were challenged with high concentrations of zinc and copper. Using competitive RT-PCR, cells resistant to 500 microM zinc showed 7+/-2 fold (SD, n=3) increases in expression of MT-1X and 6+/-3 fold increases in expression of MT-2A compared to control cells in normal media. For cells resistant to 250 microM copper the corresponding increases were 37+/-13 and 60+/-20 fold, whilst for control cells treated with 250 microM copper for only 6 h, increases were 10+/-3 and 6+/-3 fold. There was only a low level of expression of MT-1E in untreated cells and but a >120 fold increase in copper- resistant cells. Thus estrogen receptor positive cells cannot, in general, be differentiated from estrogen receptor negative cells by failure to express MT-1E, as suggested by Friedline et al. (1998). Increased expression of MT-1E, as well as MT-1X and MT-2A, protects against metal toxicity in PMC42 breast cancer cells.

  7. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  8. 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.

  9. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  10. Marine enzymes.

    PubMed

    Debashish, Ghosh; Malay, Saha; Barindra, Sana; Joydeep, Mukherjee

    2005-01-01

    Marine enzyme biotechnology can offer novel biocatalysts with properties like high salt tolerance, hyperthermostability, barophilicity, cold adaptivity, and ease in large-scale cultivation. This review deals with the research and development work done on the occurrence, molecular biology, and bioprocessing of marine enzymes during the last decade. Exotic locations have been accessed for the search of novel enzymes. Scientists have isolated proteases and carbohydrases from deep sea hydrothermal vents. Cold active metabolic enzymes from psychrophilic marine microorganisms have received considerable research attention. Marine symbiont microorganisms growing in association with animals and plants were shown to produce enzymes of commercial interest. Microorganisms isolated from sediment and seawater have been the most widely studied, proteases, carbohydrases, and peroxidases being noteworthy. Enzymes from marine animals and plants were primarily studied for their metabolic roles, though proteases and peroxidases have found industrial applications. Novel techniques in molecular biology applied to assess the diversity of chitinases, nitrate, nitrite, ammonia-metabolizing, and pollutant-degrading enzymes are discussed. Genes encoding chitinases, proteases, and carbohydrases from microbial and animal sources have been cloned and characterized. Research on the bioprocessing of marine-derived enzymes, however, has been scanty, focusing mainly on the application of solid-state fermentation to the production of enzymes from microbial sources.

  11. Inhibition of COX isoforms by nutraceuticals.

    PubMed

    Seaver, Ben; Smith, Jerry Robert

    2004-01-01

    Humans have two isoforms of Prostaglandin H Synthase or cyclooxygenase: COX-1 and COX-2. COX-1 is cytoprotective. COX-2 inhibitors reduce inflammation without the risk of ulceration and kidney damage. The ideal nutraceutical would inhibit COX-2 synthesis while preserving COX-1 synthesis. The hypothesis for this research was that COX inhibitors would fall primarily into three categories: COX-2 specific inhibition, non-specific inhibition (COX-1 and COX-2), and minimal inhibition. The human Cayman COX inhibitor screening assay was used to determine the inhibitory concentration 50 (IC50) of COX-1/ COX-2 activity of each nutraceutical. The assay was run, in duplicate, with three concentrations of a suspected inhibitor, a standard curve of eight concentrations, a non-specific binding sample, and a maximum binding sample. The inhibition and concentration of each sample was then put on a multiple regression best-fit line and the IC50 determined. For comparison, ibuprofen, rofecoxib, naproxen, and indomethacin were used. Positive results were seen for ipriflavone, resveratrol, MSV-60, amentoflavone, ruscus extract and notoginseng. Glucosamine, nexrutine, and berberine did not inhibit either isoform.

  12. Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana.

    PubMed

    Chen, Qingbo; Man, Cong; Li, Danning; Tan, Huijuan; Xie, Ye; Huang, Jirong

    2016-12-05

    Anthocyanins, a group of L-phenylalanine (Phe)-derived flavonoids, have been demonstrated to play important roles in plant stress resistance and interactions between plants and insects. Although the anthocyanin biosynthetic pathway and its regulatory mechanisms have been extensively studied, it remains unclear whether the level of Phe supply affects anthocyanin biosynthesis. Here, we investigated the roles of arogenate dehydratases (ADTs), the key enzymes that catalyze the conversion of arogenate into Phe, in sucrose-induced anthocyanin biosynthesis in Arabidopsis. Genetic analysis showed that all six ADT isoforms function redundantly in anthocyanin biosynthesis but have differential contributions. ADT2 contributes the most to anthocyanin accumulation, followed by ADT1 and ADT3, and ADT4-ADT6. We found that anthocyanin content is positively correlated with the levels of Phe and sucrose-induced ADT transcripts in seedlings. Consistently, addition of Phe to the medium could dramatically increase anthocyanin content in the wild-type plants and rescue the phenotype of the adt1 adt3 double mutant regarding the anthocyanin accumulation. Moreover, transgenic plants overexpressing ADT4, which appears to be less sensitive to Phe than overexpression of ADT2, hyperaccumulate Phe and produce elevated level of anthocyanins. Taken together, our results suggest that the level of Phe is an important regulatory factor for sustaining anthocyanin biosynthesis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  13. Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

    PubMed Central

    Valero, Rebeca; Bayés, Mònica; Francisca Sánchez-Font, M; González-Angulo, Olga; Gonzàlez-Duarte, Roser; Marfany, Gemma

    2001-01-01

    Background The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes. PMID:11597335

  14. Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

    PubMed Central

    Kopka, Joachim; Pical, Christophe; Gray, Julie E.; Müller-Röber, Bernd

    1998-01-01

    Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner. PMID:9449844

  15. Cooperation between two ClpB isoforms enhances the recovery of the recombinant {beta}-galactosidase from inclusion bodies

    SciTech Connect

    Guenther, Izabela; Zolkiewski, Michal; Kedzierska-Mieszkowska, Sabina, E-mail: kedzie@biotech.ug.gda.pl

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer An important role of synergistic cooperation between the two ClpB isoforms. Black-Right-Pointing-Pointer Both ClpB isoforms are associated with IBs of {beta}-galactosidase. Black-Right-Pointing-Pointer ClpB is a key chaperone in IB protein release. -- Abstract: Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpB{Delta}N), which does not contain the N-terminalmore » domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model {beta}-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of {beta}-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of {beta}-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a

  16. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  17. Pancreatic Enzymes

    MedlinePlus

    ... Pancreatic Enzymes and Their Effects Lipase Effects: Lipase works with bile from the liver to break down fat molecules so they can be absorbed ... at the beginning of the meal and the rest at various points throughout the meal. Enzymes generally do not work well if forgotten and only taken at the ...

  18. Impact of Reduced O-Acetylserine(thiol)lyase Isoform Contents on Potato Plant Metabolism1

    PubMed Central

    Riemenschneider, Anja; Riedel, Kerstin; Hoefgen, Rainer; Papenbrock, Jutta; Hesse, Holger

    2005-01-01

    Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H2S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed. PMID:15728339

  19. The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast

    PubMed Central

    Smardon, Anne M.; Diab, Heba I.; Tarsio, Maureen; Diakov, Theodore T.; Nasab, Negin Dehdar; West, Robert W.; Kane, Patricia M.

    2014-01-01

    The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H+-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1∆ mutants exhibit a Vma− growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma− growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma− growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1–green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma− phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor. PMID:24307682

  20. Use of a subunit feline leukemia virus vaccine in exotic cats.

    PubMed

    Citino, S B

    1988-04-01

    Three adult bengal tigers, 2 immature white tigers, and 3 adult servals were vaccinated IM with three 1-ml doses of a subunit FeLV vaccine with dosage interval guidelines of the manufacturer. All cats had increased antibody titers to FeLV gp 70 capsular antigen and feline oncornavirus cell membrane-associated antigen during the vaccination trial. Three weeks after the third vaccination, 7 of the 8 cats had gp70 antibody titers greater than 0.2 (optical density), and all 8 cats had feline oncornavirus cell membrane-associated antigen antibody titers greater than 1:8.

  1. 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

  2. Expression, activation, and role of AKT isoforms in the uterus.

    PubMed

    Fabi, François; Asselin, Eric

    2014-11-01

    The three isoforms of AKT: AKT1, AKT2, and AKT3, are crucial regulators of both normal and pathological cellular processes. Each of these isoforms exhibits a high level of homology and functional redundancy with each other. However, while being highly similar and structurally homologous, a rising amount of evidence is showing that each isoform possesses specific targets as well as preferential subcellular localization. The role of AKT has been studied extensively in reproductive processes, but isoform-specific roles are yet to be fully understood. This review will focus on the role of AKT in the uterus and its function in processes related to cell death and proliferation such as embryo implantation, decidualization, endometriosis, and endometrial cancer in an isoform-centric manner. In this review, we will cover the activation of AKT in various settings, localization of isoforms in subcellular compartments, and the effect of isoform expression on cellular processes. To fully understand the dynamic molecular processes taking place in the uterus, it is crucial that we better understand the physiological role of AKT isoforms as well as their function in the emergence of diseases. © 2014 Society for Reproduction and Fertility.

  3. Seawater acclimation and inositol monophosphatase isoform expression in the European eel (Anguilla anguilla) and Nile tilapia (Orechromis niloticus).

    PubMed

    Kalujnaia, Svetlana; Gellatly, Steven A; Hazon, Neil; Villasenor, Alfredo; Yancey, Paul H; Cramb, Gordon

    2013-08-15

    Inositol monophosphatase (IMPA) is responsible for the synthesis of inositol, a polyol that can function as an intracellular osmolyte helping re-establish cell volume when exposed to hypertonic environments. Some epithelial tissues in euryhaline teleosts such as the eel and tilapia encounter considerable hyperosmotic challenge when fish move from freshwater (FW) to seawater (SW) environments; however, the roles played by organic osmolytes, such as inositol, have yet to be determined. Syntenic analysis has indicated that, as a result of whole genome- and tandem-duplication events, up to six IMPA isoforms can exist within teleost genomes. Four isoforms are homologs of the mammalian IMPA1 gene, and two isoforms are homologs of the mammalian IMPA2 gene. Although the tissue-dependent isoform expression profiles of the teleost isoforms appear to be species-specific, it was primarily mRNA for the IMPA1.1 isoform that was upregulated in epithelial tissues after fish were transferred to SW (up to 16-fold in eel and 90-fold in tilapia). Although up-regulation of IMPA1.1 expression was evident in many tissues in the eel, more substantial increases in IMPA1.1 expression were found in tilapia tissues, where SW acclimation resulted in up to 2,000-fold increases in protein expression, 16-fold increases in enzyme activity and 15-fold increases in tissue inositol contents. Immunohistochemical studies indicated that the tissue and cellular distribution of IMPA1.1 protein differed slightly between eels and tilapia; however, in both species the basal epithelial cell layers within the skin and fin, and the branchial epithelium and interstitial cells within the kidney, exhibited high levels of IMPA1.1 protein expression.

  4. Serum concentrations of apelin-17 isoform vary in accordance to blood pressure categories in individuals with obesity class 3.

    PubMed

    Cano Martínez, Luis Javier; Coral Vázquez, Ramón Mauricio; Méndez, Juan Pablo; Trejo, Silvia; Pérez Razo, Juan Carlos; Canto, Patricia

    2018-04-13

    The aim of this study was to investigate if serum concentrations of apelin-36, apelin-17, apelin-13 or apelin-12 were different in obesity class 3 individuals with hypertension, when compared to those without hypertension (normal or high-normal). Twenty six individuals with obesity class 3-related hypertension and thirty three individuals without hypertension, who were divided in individuals with normal (n = 23) or with high-normal (n = 10) blood pressure (BP) were analyzed. All individuals presented obesity class 3, without diabetes mellitus. Measurements of all apelin isoforms were performed using enzyme-linked immunosorbent assay kits. Analysis of differences between groups of Apelin isoform concentrations was performed by a One-way ANOVA, with a Tukey test post hoc. The individuals of the hypertensive group presented a slightly lower serum concentration of all apelin isoforms, but these differences were not statistically significant. These results were more evident when the group of patients without hypertension were divided based in normal and high-normal BP, observing that apelin-17 isoform were higher in individuals with high-normal BP in comparison to subjects with normal BP (P = 0.018); concentrations were also higher when compared to subjects with hypertension (P = 0.004). To our knowledge, this is the first study regarding the differences of apelin-17 isoform concentrations in individuals pertaining to different categories of BP, who presented obesity class 3. The group of patients that presented hypertension showed a lower concentration of all isoforms. This observation could be due to the fact that these patients were taking antihypertensive medication.

  5. Psychoactive substances belonging to the amphetamine class potently activate brain carbonic anhydrase isoforms VA, VB, VII, and XII.

    PubMed

    Angeli, Andrea; Vaiano, Fabio; Mari, Francesco; Bertol, Elisabetta; Supuran, Claudiu T

    2017-12-01

    Identifying possible new biological activities of psychoactive substances belonging to various chemical classes may lead to a better understanding of their mode of action and side effects. We report here that amines structurally related to amphetamine, a widely used psychoactive substance, such as amphetamine, methamphetamine, phentermine, mephentermine, and chlorphenteramine, potently activate several carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in important physiological functions. Of the 11 investigated human (h) isoforms, the widespread hCA I and II, the secreted hCA VI, as well as the cytosolic hCA XIII, and membrane-bound hCA IX and XIV were poorly activated by these amines, whereas the extracellular hCA IV, the mitochondrial enzymes hCA VA/VB, the cytosolic hCA VII, and the transmembrane isoform hCA XII were potently activated. Some of these enzymes are abundant in the brain, raising the possibility that some of the cognitive effects of such psychoactive substances might be related to their activation of these enzymes.

  6. The role of tissue factor isoforms in cancer biology.

    PubMed

    Leppert, Ulrike; Eisenreich, Andreas

    2015-08-01

    Tissue Factor (TF) is an evolutionary conserved glycoprotein, which is of immense importance for a variety of biologic processes. TF is expressed in two naturally occurring protein isoforms, membrane-bound "full-length" (fl)TF and soluble alternatively spliced (as)TF. The TF isoform expression is differentially modulated on post-transcriptional level via regulatory factors, such as serine/arginine-rich (SR) proteins, SR protein kinases and micro (mi)RNAs. Both isoforms mediate a variety of physiologic- and pathophysiologic-relevant functions, such as thrombogenicity, angiogenesis, cell signaling, tumor cell proliferation and metastasis. In this review, we will depict the main mechanisms regulating the TF isoform expression in cancer and under other pathophysiologic-relevant conditions. Moreover, we will summarize and discuss the latest findings regarding the role of TF and its isoforms in cancer biology. © 2014 UICC.

  7. Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.

    PubMed

    Niedźwiecka, Natalia; Grzyb, Katarzyna; Nona-Mołdawa, Agnieszka; Gronczewska, Jadwiga; Skorkowski, Edward F

    2015-07-01

    Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Resolving Transferrin Isoforms via Agarose Gel Electrophoresis.

    PubMed

    Anani, Waseem Q; Ojerholm, Eric; Shurin, Michael R

    2015-01-01

    Cerebrospinal fluid (CSF) leaks may present as rhinorrhea or otorrhea and can lead to life-threatening complications if not detected. The usefulness of the morpholinopropanesulfonic acid (MOPS)-histidine buffer in detecting β₂-transferrin, which is only found in the cerebrospinal fluid, was compared with the standard barbital buffer. We evaluated 20 aural or nasal patient specimens submitted for CSF testing via agarose electrophoresis with barbital and MOPS-histidine buffers. The MOPS-histidine and barbital buffers revealed 5 transferrin bands and 2 transferrin bands with CSF, respectively. Seventeen of 20 patient specimens had concordant results. The 3 discrepant specimens initially tested negative with the barbital buffer and positive with the MOPS-histidine buffer. Two of the 3 patient specimens later tested positive with the barbital buffer when a new specimen was submitted. Agarose electrophoresis with the MOPS-histidine buffer increases the resolution of transferrin isoforms. Copyright© by the American Society for Clinical Pathology (ASCP).

  9. Crystal Structures of Vegetative Soybean Lipoxygenase VLX-B and VLX-D, and Comparisons with Seed Isoforms LOX-1 and LOX-3

    PubMed Central

    Youn, Buhyun; Sellhorn, George E.; Mirchel, Ryan J.; Gaffney, Betty J.; Grimes, Howard D.; Kang, ChulHee

    2009-01-01

    The lipoxygenase family of lipidperoxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX-A, VLX-B, VLX-C, VLX-D, VLX-E, and four seed isoforms LOX-1, LOX-2, LOX-3a, LOX-3b have been identified. In this study, we determined the crystal structures of the substrate-free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX-B and VLX-D. Their structures are similar to the two seed isoforms, LOX-1 and LOX-3, having two domains with similar secondary structural elements: a β-barrel N-terminal domain containing highly flexible loops and an α-helix-rich C-terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX-1 and LOX-3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate-binding and O2 access, differs among the isoforms in a manner that reflects the differences in positional specificities. PMID:17022084

  10. Developmental Analysis of Spliceosomal snRNA Isoform Expression

    PubMed Central

    Lu, Zhipeng; Matera, A. Gregory

    2014-01-01

    Pre-mRNA splicing is a critical step in eukaryotic gene expression that contributes to proteomic, cellular, and developmental complexity. Small nuclear (sn)RNAs are core spliceosomal components; however, the extent to which differential expression of snRNA isoforms regulates splicing is completely unknown. This is partly due to difficulties in the accurate analysis of the spatial and temporal expression patterns of snRNAs. Here, we use high-throughput RNA-sequencing (RNA-seq) data to profile expression of four major snRNAs throughout Drosophila development. This analysis shows that individual isoforms of each snRNA have distinct expression patterns in the embryo, larva, and pharate adult stages. Expression of these isoforms is more heterogeneous during embryogenesis; as development progresses, a single isoform from each snRNA subtype gradually dominates expression. Despite the lack of stable snRNA orthologous groups during evolution, this developmental switching of snRNA isoforms also occurs in distantly related vertebrate species, such as Xenopus, mouse, and human. Our results indicate that expression of snRNA isoforms is regulated and lays the foundation for functional studies of individual snRNA isoforms. PMID:25416704

  11. Developmental analysis of spliceosomal snRNA isoform expression.

    PubMed

    Lu, Zhipeng; Matera, A Gregory

    2014-11-21

    Pre-mRNA splicing is a critical step in eukaryotic gene expression that contributes to proteomic, cellular, and developmental complexity. Small nuclear (sn)RNAs are core spliceosomal components; however, the extent to which differential expression of snRNA isoforms regulates splicing is completely unknown. This is partly due to difficulties in the accurate analysis of the spatial and temporal expression patterns of snRNAs. Here, we use high-throughput RNA-sequencing (RNA-seq) data to profile expression of four major snRNAs throughout Drosophila development. This analysis shows that individual isoforms of each snRNA have distinct expression patterns in the embryo, larva, and pharate adult stages. Expression of these isoforms is more heterogeneous during embryogenesis; as development progresses, a single isoform from each snRNA subtype gradually dominates expression. Despite the lack of stable snRNA orthologous groups during evolution, this developmental switching of snRNA isoforms also occurs in distantly related vertebrate species, such as Xenopus, mouse, and human. Our results indicate that expression of snRNA isoforms is regulated and lays the foundation for functional studies of individual snRNA isoforms. Copyright © 2015 Lu and Matera.

  12. Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism

    PubMed Central

    Silvera, Katia; Winter, Klaus; Rodriguez, B. Leticia; Albion, Rebecca L.; Cushman, John C.

    2014-01-01

    Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins. PMID:24913627

  13. Antiangiogenic isoforms of vascular endothelial growth factor predominate in subretinal fluid of patients with rhegmatogenous retinal detachment and proliferative vitreoretinopathy.

    PubMed

    Ricker, Lukas J A G; Dieudonné, Suzanne C; Kessels, Alfons G H; Rennel, Emma S; Berendschot, Tos T J M; Hendrikse, Fred; Kijlstra, Aize; La Heij, Ellen C

    2012-01-01

    In proliferative vitreoretinopathy (PVR), a nonangiogenic eye disease that is characterized by the formation of mainly avascular membranes, vascular endothelial growth factor (VEGF) levels are found to be upregulated. Recently, it was discovered that VEGF is alternatively spliced to form the angiogenic (VEGF xxx) and antiangiogenic (VEGF xxx b) family of isoforms. Previous studies on expression of VEGF in PVR samples have not distinguished between the two families of isoforms. We measured total VEGF and VEGF xxx b levels in subretinal fluid of patients with PVR (n = 10) and in patients with uncomplicated rhegmatogenous retinal detachment (n = 27) using enzyme-linked immunosorbent assay. : We found total VEGF levels to be 2- to 3-fold elevated in the PVR group as compared with the rhegmatogenous retinal detachment group (P = 0.047). Antiangiogenic VEGF xxx b isoforms predominated (>60% of total VEGF) in the majority of rhegmatogenous retinal detachment and PVR samples investigated, although a wide variability of isoform ratios was observed within both groups. The absence of an increased ratio of VEGF xxx to VEGF xxx b in patients with PVR as compared with patients with uncomplicated rhegmatogenous retinal detachment may explain a lack of blood vessels in PVR membranes. Elevated VEGF levels indicate that this cytokine may play a role in the pathogenesis of PVR that is not related to angiogenesis.

  14. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

    PubMed

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-11-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

  15. Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus.

    PubMed

    Kulisz, Andre; Dowal, Louisa; Scarlata, Suzanne; Shen, Sheldon S

    2005-06-01

    Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.

  16. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats

    PubMed Central

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-01-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases. PMID:27882225

  17. Detection of VEGF-Axxxb Isoforms in Human Tissues

    PubMed Central

    Bates, David O.; Mavrou, Athina; Qiu, Yan; Carter, James G.; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V.; Millar, Ann B.; Salmon, Andrew H. J.; Oltean, Sebastian; Harper, Steven J.

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls. PMID:23935865

  18. A novel isoform of pantothenate synthetase in the Archaea.

    PubMed

    Ronconi, Silvia; Jonczyk, Rafal; Genschel, Ulrich

    2008-06-01

    The linear biosynthetic pathway leading from alpha-ketoisovalerate to pantothenate (vitamin B5) and on to CoA comprises eight steps in the Bacteria and Eukaryota. Genes for up to six steps of this pathway can be identified by sequence homology in individual archaeal genomes. However, there are no archaeal homologs to known isoforms of pantothenate synthetase (PS) or pantothenate kinase. Using comparative genomics, we previously identified two conserved archaeal protein families as the best candidates for the missing steps. Here we report the characterization of the predicted PS gene from Methanosarcina mazei, which encodes a hypothetical protein (MM2281) with no obvious homologs outside its own family. When expressed in Escherichia coli, MM2281 partially complemented an auxotrophic mutant without PS activity. Purified recombinant MM2281 showed no PS activity on its own, but the enzyme enabled substantial synthesis of [14C]4'-phosphopantothenate from [14C]beta-alanine, pantoate and ATP when coupled with E. coli pantothenate kinase. ADP, but not AMP, was detected as a coproduct of the coupled reaction. MM2281 also transferred the 14C-label from [14C]beta-alanine to pantothenate in the presence of pantoate and ADP, presumably through isotope exchange. No exchange took place when pantoate was removed or ADP replaced with AMP. Our results indicate that MM2281 represents a novel type of PS that forms ADP and is strongly inhibited by its product pantothenate. These properties differ substantially from those of bacterial PS, and may explain why PS genes, in contrast to other pantothenate biosynthetic genes, were not exchanged horizontally between the Bacteria and Archaea.

  19. Sleep-inducing effect of substance P-cholera toxin A subunit in mice.

    PubMed

    Zielinski, Mark R; Gerashchenko, Dmitry

    2017-10-17

    Evidence indicates that the neuropeptide substance P (SP) can act through neurokinin receptors to alter sleep and/or non-rapid eye movement (NREM) sleep slow-wave activity. Consequently, drugs acting on SP receptors could potentially be used as a novel treatment for sleep-related disorders. In the present study, we used SP conjugated with cholera toxin A subunit (SP-CTA), which enhances its duration of activity on SP receptor-expressing cells, to determine the effects of selectively activating SP receptor-expressing brain cells on sleep regulation in mice. Herein, we found that intracerebroventricular administration of SP-CTA enhanced amounts of NREM sleep which was highly fragmented. This result suggests that the activation of SP receptor-expressing cells in the brain can produce not only arousal effects as shown in previous studies but also sleep-inducing effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    PubMed

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  1. Actin isoforms in amphioxus Branchiostoma lanceolatum.

    PubMed

    Fagotti, A; Di Rosa, I; Simoncelli, F; Chaponnier, C; Gabbiani, G; Pascolini, R

    1998-04-01

    Actin is a highly conserved cytoskeletal protein that is ubiquitous in all eukaryotes. Little is known about actin expression in amphioxus, the closest living relative of the vertebrates. In the present study, involving Western blotting and indirect immunofluorescence, we report the characterization and localization of various actin isoforms in amphioxus (Branchiostoma lanceolatum) tissues. Three antibodies against vertebrate actins were used: a polyclonal antibody recognizing beta-cytoplasmic actin (anti-beta actin), a monoclonal antibody against sarcomeric actins (anti-alphaSR-1), and a monoclonal antibody specific for alpha-smooth actin (anti-alphaSM-1). Western blot analysis of amphioxus extracts immunodecorated with these antibodies showed a 43-kDa-positive band co-migrating with respective controls. The amphioxus isoactin expression patterns recognized by these antibodies were similar to those of vertebrates, i.e., anti-beta actin showed positive staining mainly in non-muscle cells, anti-alphaSR-1 labelled dorsolateral myotomal muscles, and anti-alphaSM-1 stained ventral muscles. These results demonstrate that at least two muscle actins are present in amphioxus, suggesting that muscle actin gene duplication events began before vertebrate divergence from the amphioxus lineage.

  2. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  3. Engineering enzymes

    PubMed Central

    Moser, Christopher C.

    2014-01-01

    Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of structural and energetic engineering tolerances of the mechanism. Significant barriers to achieving an engineering understanding of enzyme mechanisms arise from natural protein complexity. In certain cases we can surmount these barriers to understanding, such as natural electron tunneling, coupling of electron tunneling to light capture and proton exchange as well as simpler bond breaking redox catalysis. Hope for similar solutions of more complex bioinorganic enzymes is indicated in several papers presented in this Discussion. Armed with an engineering understanding of mechanism, the current serious frustrations to successful creation of functional artificial proteins that are rooted in protein complexity can fall away. Here we discuss the genetic and biological roots of protein complexity and show how to dodge and minimize the effects of complexity. In the best-understood cases, artificial enzymes can be designed from scratch using the simplest of protein scaffolds. PMID:21322497

  4. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  5. Expression and modulation of CD44 variant isoforms in humans

    PubMed Central

    1994-01-01

    CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines. PMID:7507492

  6. Expression and modulation of CD44 variant isoforms in humans.

    PubMed

    Mackay, C R; Terpe, H J; Stauder, R; Marston, W L; Stark, H; Günthert, U

    1994-01-01

    CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.

  7. Characterisation of Cdkl5 transcript isoforms in rat.

    PubMed

    Hector, Ralph D; Dando, Owen; Ritakari, Tuula E; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2017-03-01

    CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically and experimentally, the rat Cdkl5 gene structure and its associated transcript isoforms. New exonic regions, splice sites and UTRs are described, confirming the presence of four distinct transcript isoforms. The predominant isoform in the brain, which we name rCdkl5_1, is orthologous to the human hCDKL5_1 and mouse mCdkl5_1 isoforms and is the most highly expressed isoform across all brain regions tested. This updated gene model of Cdkl5 in rat provides a framework for studies into its protein products and provides a reference for the development of molecular therapies for testing in rat models of CDKL5 disorder. Copyright © 2016 Elsevier B.V. All rights reserved.

  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. Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification.

    PubMed

    Simůnek, J; Tishchenko, G; Rozhetsky, K; Bartonová, H; Kopecný, J; Hodrová, B

    2004-01-01

    A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.

  10. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects.

    PubMed

    Watanabe, Takayuki; Takeuchi, Hideaki; Kubo, Takeo

    2010-02-12

    The ecdysone receptor (EcR) regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF)-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional regulation mechanisms.

  11. Transcreener: screening enzymes involved in covalent regulation.

    PubMed

    Lowery, Robert G; Kleman-Leyer, Karen

    2006-02-01

    Enzymes that catalyse group transfer reactions comprise a significant fraction of the human proteome and are a rich source of drug targets because of their role in covalent regulatory cycles. Phosphorylation, glycosylation, sulfonation, methylation and acetylation represent some of the key types of group transfer reactions that modulate the function of diverse biomolecules through covalent modification. Development of high-throughput screening methods for these enzymes has been problematic because of the diversity of acceptor substrates. Recently, the authors developed a novel assay platform called Transcreener that relies upon fluorescence detection of the invariant reaction product of a group transfer reaction, usually a nucleotide. This platform enables screening of any isoform in a family of group transfer enzymes, with any acceptor substrate, using the same assay reagents.

  12. A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

    PubMed

    Kurdyumov, Alexey S; Manuvera, Valentin A; Baskova, Isolda P; Lazarev, Vassili N

    2015-11-21

    Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

  13. Cooperation between two ClpB isoforms enhances the recovery of the recombinant β-galactosidase from inclusion bodies.

    PubMed

    Guenther, Izabela; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2012-10-05

    Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpBΔN), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model β-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of β-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of β-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic cooperation between the two isoforms of ClpB chaperone. In addition, no significant recovery of the β-galactosidase from IBs in ΔclpB mutant cells suggests that ClpB is a key chaperone in IB protein release. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Molecular characterization of two isoforms of a farnesyl pyrophosphate synthase gene in wheat and their roles in sesquiterpene synthesis and inducible defence against aphid infestation.

    PubMed

    Zhang, Yan; Li, Zhi-Xia; Yu, Xiu-Dao; Fan, Jia; Pickett, John A; Jones, Huw D; Zhou, Jing-Jiang; Birkett, Michael A; Caulfield, John; Napier, Johnathan A; Zhao, Guang-Yao; Cheng, Xian-Guo; Shi, Yi; Bruce, Toby J A; Xia, Lan-Qin

    2015-05-01

    Aphids are important pests of wheat (Triticum aestivum) that affect crop production globally. Herbivore-induced emission of sesquiterpenes can repel pests, and farnesyl pyrophosphate synthase (FPS) is a key enzyme involved in sesquiterpene biosynthesis. However, fps orthologues in wheat and their functional roles in sesquiterpene synthesis and defence against aphid infestation are unknown. Here, two fps isoforms, Tafps1 and Tafps2, were identified in wheat. Quantitative real-time polymerase chain reaction (qRT-PCR) and in vitro catalytic activity analyses were conducted to investigate expression patterns and activity. Heterologous expression of these isoforms in Arabidopsis thaliana, virus-induced gene silencing (VIGS) in wheat and aphid behavioural assays were performed to understand the functional roles of these two isoforms. We demonstrated that Tafps1 and Tafps2 played different roles in induced responses to aphid infestation and in sesquiterpene synthesis. Heterologous expression in A. thaliana resulted in repulsion of the peach aphid (Myzus persicae). Wheat plants with these two isoforms transiently silenced were significantly attractive to grain aphid (Sitobion avenae). Our results provide new insights into induced defence against aphid herbivory in wheat, in particular, the different roles of the two Tafps isoforms in both sesquiterpene biosynthesis and defence against aphid infestation. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  15. Characterization of the Sucrose Phosphate Phosphatase (SPP) Isoforms from Arabidopsis thaliana and Role of the S6PPc Domain in Dimerization.

    PubMed

    Albi, Tomás; Ruiz, M Teresa; de Los Reyes, Pedro; Valverde, Federico; Romero, José M

    2016-01-01

    Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.

  16. The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch.

    PubMed

    Doan; Rudi; Olsen

    1999-11-01

    We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed.

  17. The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch1

    PubMed Central

    Doan, Danny N.P.; Rudi, Heidi; Olsen, Odd-Arne

    1999-01-01

    We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed. PMID:10557246

  18. In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan.

    PubMed

    Matsumoto, Takashi; Kushida, Hirotaka; Maruyama, Takeshi; Nishimura, Hiroaki; Watanabe, Junko; Maemura, Kazuya; Kase, Yoshio

    2016-01-01

    1. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. 2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. 3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes. 4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.

  19. Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

    PubMed

    Barchiesi, Julieta; Hedin, Nicolás; Gomez-Casati, Diego F; Ballicora, Miguel A; Busi, María V

    2015-10-28

    Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding. The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.

  20. Arabidopsis RIBA Proteins: Two out of Three Isoforms Have Lost Their Bifunctional Activity in Riboflavin Biosynthesis

    PubMed Central

    Hiltunen, Hanna-Maija; Illarionov, Boris; Hedtke, Boris; Fischer, Markus; Grimm, Bernhard

    2012-01-01

    Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution. PMID:23203051

  1. Expression of various sarcomeric tropomyosin isoforms in equine striated muscles

    PubMed Central

    Dube, Syamalima; Chionuma, Henry; Matoq, Amr; Alshiekh-Nasany, Ruham; Abbott, Lynn; Poiesz, Bernard J.; Dube, Dipak K.

    2017-01-01

    In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle. PMID:28717602

  2. Expression of various sarcomeric tropomyosin isoforms in equine striated muscles.

    PubMed

    Dube, Syamalima; Chionuma, Henry; Matoq, Amr; Alshiekh-Nasany, Ruham; Abbott, Lynn; Poiesz, Bernard J; Dube, Dipak K

    2017-01-01

    In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

  3. Expression and distribution of cellulase, amylase and peptidase isoforms along the midgut of Morimus funereus L. (Coleoptera: Cerambycidae) larvae is dependent on nutrient substrate composition.

    PubMed

    Dojnov, Biljana; Pavlović, Ratko; Božić, Nataša; Margetić, Aleksandra; Nenadović, Vera; Ivanović, Jelisaveta; Vujčić, Zoran

    2013-04-01

    The influence of diet composition--two substrates, wheat bran and sawdust--on isoform expression of digestive enzymes (cellulase, amylase and peptidase) in the midgut of Morimus funereus larvae was examined. Their impact on larval development was demonstrated by measuring the increase of larval weight during development and by analysis of digestive enzymes zymographic profiles, where the expression of cellulase isoforms from M. funereus larvae midgut has been examined for the first time in this study. Larvae reared on wheat bran had higher body weight between day 60 and day 100 than larvae reared on sawdust; however, both groups achieved similar body weight after day 110. Wheat bran as substrate induced different cellulase and amylase isoforms. Oak sawdust in substrate acted as inducer of peptidases. The highest cellulase activity and the greatest isoform variability were detected in the midgut extracts of larvae reared on wheat bran. From our results it can be assumed that M. funereus endocellulase, amylase and peptidase are secreted in the anterior midgut, and their concentration gradually decreases towards the hindgut. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    SciTech Connect

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} formore » fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.« less

  6. Silencing of the Major Salt-Dependent Isoform of Pectinesterase in Tomato Alters Fruit Softening1

    PubMed Central

    Phan, Thanh D.; Bo, Wen; West, Gill; Lycett, Grantley W.; Tucker, Gregory A.

    2007-01-01

    Pectinesterase (PE; E.C. 3.1.1.11) is an enzyme responsible for the demethylation of galacturonyl residues in high-molecular-weight pectin and is believed to play an important role in cell wall metabolism. In this study, Pmeu1, a ubiquitously expressed PE gene, has been characterized by antisense suppression in tomato (Solanum lycopersicum). Transgenic tomato plants showed reduced PE activity levels in both green fruit and leaf tissue to around 65% and 25% of that found in wild-type plants, respectively. Pmeu1 was observed to encode a salt-dependent PE isoform that correlated with PE1 as previously described in fruit tissue. Silencing of Pmeu1 did not result in any detectable phenotype within the leaf tissue despite the gene product representing the major isoform in this tissue. In comparison, silencing in fruit resulted in an enhancement to the rate of softening during ripening. The role of PMEU1 in fruit ripening is discussed. PMID:17556513

  7. Tropomyosin isoforms bias actin track selection by vertebrate myosin Va

    PubMed Central

    Sckolnick, Maria; Krementsova, Elena B.; Warshaw, David M.; Trybus, Kathleen M.

    2016-01-01

    Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin–Tpm3.1 and actin–Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin–Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an “actin–Tpm code” that allows for spatial and temporal sorting of actomyosin function in the cell. PMID:27535431

  8. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  9. Neuroprotection by the NR3A Subunit of the NMDA Receptor

    PubMed Central

    Nakanishi, Nobuki; Tu, Shichun; Shin, Yeonsook; Cui, Jiankun; Kurokawa, Toru; Zhang, Dongxian; Chen, H.-S. Vincent; Tong, Gary; Lipton, Stuart A.

    2009-01-01

    Hyperactivation of N-methyl-D-aspartate-type glutamate receptors (NMDARs) results in excitotoxicity, contributing to damage in stroke and neurodegenerative disorders. NMDARs are generally comprised of NR1/NR2 subunits but may contain modulatory NR3 subunits. Inclusion of NR3 subunits reduces the amplitude and dramatically decreases the Ca2+ permeability of NMDAR-associated channels in heterologous expression systems and in transgenic mice. Since excessive Ca2+ influx into neurons is a crucial step for excitotoxicity, we asked whether NR3A subunits are neuroprotective. To address this question, we subjected neurons genetically lacking NR3A to various forms of excitotoxic insult. We found that cultured neurons prepared from NR3A knockout (KO) mice displayed greater sensitivity to damage by NMDA application than wild-type (WT) neurons. In vivo, neonatal, but not adult, WT mice contain NR3A in the cortex, and neonatal NR3A KO mice manifested more damage than WT following hypoxia-ischemia. In adult retina, one location where high levels of NR3A normally persist into adulthood, injection of NMDA into the eye killed more retinal ganglion cells in adult NR3A KO than WT mice. These data suggest that endogenous NR3A is neuroprotective. We next asked whether we could decrease excitotoxicity by overexpressing NR3A. We found that cultured neurons expressing transgenic (TG) NR3A displayed greater resistance to NMDA-mediated neurotoxicity than WT neurons. Similarly in vivo, adult NR3A TG mice subjected to focal cerebral ischemia manifested less damage than WT mice. These data suggest that endogenous NR3A protects neurons, and exogenously added NR3A increases neuroprotection and could be potentially exploited as a therapeutic. PMID:19386922

  10. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  11. Dynamin isoforms decode action potential firing for synaptic vesicle recycling.

    PubMed

    Tanifuji, Shota; Funakoshi-Tago, Megumi; Ueda, Fumihito; Kasahara, Tadashi; Mochida, Sumiko

    2013-06-28

    Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.

  12. One protein, two enzymes revisited: A structural entropy switch interconverts the two isoforms of acireductone dioxygenase

    PubMed Central

    Ju, Tingting; Goldsmith, Rachel Beaulieu; Chai, Sergio C.; Maroney, Michael J.; Pochapsky, Susan Sondej; Pochapsky, Thomas C.

    2006-01-01

    Summary Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni+2–ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe+2 is bound (ARD′), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe+2 renders the active site of ARD′ inaccessible to standard NMR methods. The structure of ARD′ has been determined using Fe+2 binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD′. ARD′ retains the β-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the β-sandwich and decreases order at the other upon interconversion of ARD and ARD′, causing loss of the C-terminal helix in ARD′ and rearrangements of residues involved in substrate orientation in the active site. PMID:16989860

  13. Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

    PubMed

    Mazzone, Pellegrino; Scudiero, Ivan; Coccia, Elena; Ferravante, Angela; Paolucci, Marina; D'Andrea, Egildo Luca; Varricchio, Ettore; Pizzulo, Maddalena; Reale, Carla; Zotti, Tiziana; Vito, Pasquale; Stilo, Romania

    2015-01-01

    The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

  14. Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex.

    PubMed Central

    Schrader, M; Fendler, K; Bamberg, E; Gassel, M; Epstein, W; Altendorf, K; Dröse, S

    2000-01-01

    Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate. Similarities to mutations in the GYG motif of K(+) channels are discussed. PMID:10920013

  15. Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus , Sulfolobus tokodaii and Methanocaldococcus jannaschii

    SciTech Connect

    Watanabe, Yuzo; Yanai, Hisaaki; Kanagawa, Mayumi

    2016-07-27

    The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, fromThermus thermophilus,Sulfolobus tokodaiiandMethanocaldococcus jannaschiiwere determined and their structural characteristics were analyzed. For PurS fromT. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations ofmore » the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.« less

  16. Oxygenation properties and isoform diversity of snake hemoglobins.

    PubMed

    Storz, Jay F; Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E

    2015-11-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. Copyright © 2015 the American Physiological Society.

  17. Dexamethasone and sex regulate placental glucocorticoid receptor isoforms in mice.

    PubMed

    Cuffe, James S M; Saif, Zarqa; Perkins, Anthony V; Moritz, Karen M; Clifton, Vicki L

    2017-08-01

    Maternal dexamethasone exposure in the mouse impairs placental development and programs adult disease in a sexually dimorphic manner. Glucocorticoids bind to different glucocorticoid receptor (GR) isoforms to regulate gene transcription and cellular signaling. We hypothesized that sexually dimorphic placental responses to glucocorticoids are due to differences in GR isoforms present in the placenta. Pregnant C57Bl6 mice were exposed to saline or dexamethasone from E12.5 until E14.5 (1 µg/kg/h) before the collection of placentae. Cytoplasmic and nuclear protein fractions were extracted from placentae of male and female fetuses for Western blot analysis of GR isoforms. Eight known isoforms of the GR were detected in the mouse placenta including the translational isoforms GRα-A, B, C and D1-3 and the splice variants GRA and GRP. The expression of GRA, GRP and each of the GRα isoforms were altered by dexamethasone in relation to fetal sex and cellular location. Placentae of female fetuses had higher GRα-A and GRP expression in the cytoplasm than males, and GRα-C was more highly expressed in the nucleus of females than that in males. Dexamethasone significantly increased the cytoplasmic expression of GRα-A, but reduced the expression of GRα-C in placentae of males. Dexamethasone increased the expression of the GRα-C-regulated genes Sgk1 and Bcl2l11 , particularly in females. The cleaved caspase-3 staining in placental sections indicated GRα-C may mediate sex differences in dexamethasone-induced apoptosis. These findings may underlie the sex-specific placental adaptations that regulate different growth profiles in males and females and different risks for programmed disease outcomes in offspring. © 2017 Society for Endocrinology.

  18. Oxygenation properties and isoform diversity of snake hemoglobins

    PubMed Central

    Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G.; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E.

    2015-01-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. PMID:26354849

  19. WT1 Alternative Splicing: Role of Its Isoforms in Neuroblastoma.

    PubMed

    Rasà, Daniela Maria; D'Amico, Agata Grazia; Maugeri, Grazia; Cavallaro, Sebastiano; D'Agata, Velia

    2017-06-01

    Wilms tumor 1 (WT1), a tumor suppressor gene, was originally identified in the homonymous renal neoplasm but is also involved in other cancers. Its function is still unclear, since it acts both as a pro- and an anti-oncogene. At least 14 WT1 transcriptional variants have been described; yet most investigations have focused on a small number of isoforms. We describe their structural features and review the evidence of their involvement in cancer with emphasis on neuroblastoma. In future, full characterization of all WT1 isoforms is expected to identify new molecular tumor markers and/or therapeutic targets.

  20. Modulation of neuronal differentiation by CD40 isoforms

    SciTech Connect

    Hou Huayu; Obregon, Demian; Lou, Deyan

    2008-05-02

    Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40more » isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40{sup -/-} deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40{sup -/-} mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling

  1. Chemoproteomic analysis of intertissue and interspecies isoform diversity of AMP-activated protein kinase (AMPK).

    PubMed

    Wu, Jiang; Puppala, Dinesh; Feng, Xidong; Monetti, Mara; Lapworth, Amanda Lee; Geoghegan, Kieran F

    2013-12-13

    AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that senses and governs changes in the cellular energy balance represented by concentrations of AMP, ADP, and ATP. Each of its three chains (α, β, and γ) exists as either two or three subtypes, theoretically allowing up to 12 different forms of the complete enzyme. Tissue specificity in the distribution of AMPK subtypes is believed to underpin a range of biological functions for AMPK, a central regulator of metabolic function and response. It is of particular interest for drug discovery purposes to compare AMPK isoforms that are most prevalent in human liver and muscle with isoforms present in key preclinical species. To complement immunocapture/immunodetection methods, which for AMPK are challenged by sequence similarities and difficulties of obtaining accurate relative quantitation, AMPK was captured from lysates of a range of cells and tissues using the ActivX ATP probe. This chemical probe covalently attaches desthiobiotin to one or more conserved lysyl residues in the ATP-binding sites of protein kinases, including AMPK, while also labeling a wide range of ATP-utilizing proteins. Affinity-based recovery of labeled proteins followed by gel-based fractionation of the captured sample was followed by proteomic characterization of AMPK polypeptides. In agreement with transcript-based analysis and previous indications from immunodetection, the results indicated that the predominant AMPK heterotrimer in human liver is α1β2γ1 but that dog and rat livers mainly contain the α1β1γ1 and α2β1γ1 forms, respectively. Differences were not detected between the AMPK profiles of normal and diabetic human liver tissues.

  2. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    NASA Astrophysics Data System (ADS)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  3. Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4 System of Hydrilla verticillata1

    PubMed Central

    Rao, Srinath K.; Magnin, Noël C.; Reiskind, Julia B.; Bowes, George

    2002-01-01

    The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C4 plant. It typically exhibits C3 photosynthetic characteristics, but exposure to low [CO2] induces a C4 system in which the C4 and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C4 induction. This and enzyme kinetic data were consistent with it being the C4 photosynthesis isoform. However, the C4 signature serine of terrestrial plant C4 isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C3 sequences, was present. Western analyses of C3 and C4 leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C3, non-graminaceous C4, and Crassulacean acid metabolism PEPCs but not with the graminaceous C4, and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C4 angiosperms. PMID:12376652

  4. PROTEOLYTIC ENZYMES

    PubMed Central

    Grob, David

    1946-01-01

    1. The literature on conditions affecting the activity of proteolytic enzymes has been reviewed. 2. Experimental data on the control of the activity of trypsin, leucoprotease, papain, serum antiprotease, leucopeptidase, and pancreatic peptidase have been presented. These data indicate that: (a) The polymorphonuclearleucocytes of the cat contain abundant proteinase and peptidase active at neutral pH; those of the rabbit lack proteinase active at neutral pH. (b) Reducing agents, including several biologically important thiol-sulfhydryl compounds and ascorbic acid, inhibit the activity of leucoprotease and trypsin. For each reductant the degree of inhibition is proportional to the reducing capacity of the medium. (c) p-Aminobenzoic acid, sulfonamides (especially sulfathiazole), and many diphenyl sulfones inhibit the activity of leucoprotease. (d) Serum, plasma, several heavy metals, ammonium salts, asparagine, thiourea, heparin, glutamic acid, tyrothricin, calcium chloride, and bile salts and bile acids also inhibit the activity of leucoprotease, and in most cases of trypsin too. (e) Preparations of tryptic digests of proteins, and egg white trypsin inhibitor, inhibit trypsin to a much greater degree than leucoprotease. (f) Mild oxidizing agents increase the activity of leucoprotease and trypsin. (g) Oxidizing agents and some inhibitors of sulfhydryl groups inhibit the antiproteolytic activity of the serum. It is suggested that serum antiprotease may consist (chiefly) of reducing agents, including thiol-sulfhydryl peptides which exert their antiproteolytic activity by virtue of the presence of sulfhydryl groups. (h) The antiproteolytic activity of the serum is progressively weakened by exposure to a hydrogen ion concentration below pH 6.5 or above pH 9.7. Because of this the pH optima of leucoprotease and trypsin are shifted in the presence of serum from pH of 7 and 8 to pH of 6 to 6.5, and the range of activity of these enzymes is slightly widened, in both acid and

  5. Amino- and Carboxy-Terminal PEST Domains Mediate Gastrin Stabilization of Rat l-Histidine Decarboxylase Isoforms

    PubMed Central

    Fleming, John V.; Wang, Timothy C.

    2000-01-01

    Control of enzymatic function by peptide hormones can occur at a number of different levels and can involve diverse pathways that regulate cleavage, intracellular trafficking, and protein degradation. Gastrin is a peptide hormone that binds to the cholecystokinin B-gastrin receptor and regulates the activity of l-histidine decarboxylase (HDC), the enzyme that produces histamine. Here we show that gastrin can increase the steady-state levels of at least six HDC isoforms without affecting HDC mRNA levels. Pulse-chase experiments indicated that HDC isoforms are rapidly degraded and that gastrin-dependent increases are due to enhanced isoform stability. Deletion analysis identified two PEST domains (PEST1 and PEST2) and an intracellular targeting domain (ER2) which regulate HDC protein expression levels. Experiments with PEST domain fusion proteins demonstrated that PEST1 and PEST2 are strong and portable degradation-promoting elements which are positively regulated by both gastrin stimulation and proteasome inhibition. A chimeric protein containing the PEST domain of ornithine decarboxylase was similarly affected, indicating that gastrin can regulate the stability of other PEST domain-containing proteins and does so independently of antizyme/antizyme inhibitor regulation. At the same time, endoplasmic reticulum localization of a fluorescent chimera containing the ER2 domain of HDC was unaltered by gastrin stimulation. We conclude that gastrin stabilization of HDC isoforms is dependent upon two transferable and sequentially unrelated PEST domains that regulate degradation. These experiments revealed a novel regulatory mechanism by which a peptide hormone such as gastrin can disrupt the degradation function of multiple PEST-domain-containing proteins. PMID:10848618

  6. Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crab Callinectes sapidus.

    PubMed

    Serrano, Laetitia; Halanych, Kenneth M; Henry, Raymond P

    2007-07-01

    Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill, CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill, and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the alpha-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative ;housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity, but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.

  7. Conformational Flexibility Differentiates Naturally Occurring Bet v 1 Isoforms.

    PubMed

    Grutsch, Sarina; Fuchs, Julian E; Ahammer, Linda; Kamenik, Anna S; Liedl, Klaus R; Tollinger, Martin

    2017-06-03

    The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform's higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.

  8. p63 isoforms regulate metabolism of cancer stem cells.

    PubMed

    D'Aguanno, Simona; Barcaroli, Daniela; Rossi, Claudia; Zucchelli, Mirco; Ciavardelli, Domenico; Cortese, Claudio; De Cola, Antonella; Volpe, Silvia; D'Agostino, Daniela; Todaro, Matilde; Stassi, Giorgio; Di Ilio, Carmine; Urbani, Andrea; De Laurenzi, Vincenzo

    2014-04-04

    p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.

  9. Identification and characterization of novel NuMA isoforms.

    PubMed

    Wu, Jin; Xu, Zhe; He, Dacheng; Lu, Guanting

    2014-11-21

    The large nuclear mitotic apparatus (NuMA) has been investigated for over 30years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two "hotspot" exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA's various functions. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Method for the Simultaneous Quantitation of Apolipoprotein E Isoforms using Tandem Mass Spectrometry

    PubMed Central

    Wildsmith, Kristin R.; Han, Bomie; Bateman, Randall J.

    2009-01-01

    Using Apolipoprotein E (ApoE) as a model protein, we developed a protein isoform analysis method utilizing Stable Isotope Labeling Tandem Mass Spectrometry (SILT MS). ApoE isoforms are quantitated using the intensities of the b and y ions of the 13C-labeled tryptic isoform-specific peptides versus unlabeled tryptic isoform-specific peptides. The ApoE protein isoform analysis using SILT allows for the simultaneous detection and relative quantitation of different ApoE isoforms from the same sample. This method provides a less biased assessment of ApoE isoforms compared to antibody-dependent methods, and may lead to a better understanding of the biological differences between isoforms. PMID:19653990

  11. Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth.

    PubMed

    Navarro Gallón, Sandra M; Elejalde-Palmett, Carolina; Daudu, Dimitri; Liesecke, Franziska; Jullien, Frédéric; Papon, Nicolas; Dugé de Bernonville, Thomas; Courdavault, Vincent; Lanoue, Arnaud; Oudin, Audrey; Glévarec, Gaëlle; Pichon, Olivier; Clastre, Marc; St-Pierre, Benoit; Atehortùa, Lucia; Yoshikawa, Nobuyuki; Giglioli-Guivarc'h, Nathalie; Besseau, Sébastien

    2017-07-01

    The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

  12. N Termini of apPDE4 Isoforms Are Responsible for Targeting the Isoforms to Different Cellular Membranes

    ERIC Educational Resources Information Center

    Jang, Deok-Jin; Park, Soo-Won; Lee, Jin-A; Lee, Changhoon; Chae, Yeon-Su; Park, Hyungju; Kim, Min-Jeong; Choi, Sun-Lim; Lee, Nuribalhae; Kim, Hyoung; Kaang, Bong-Kiun

    2010-01-01

    Phosphodiesterases (PDEs) are known to play a key role in the compartmentalization of cAMP signaling; however, the molecular mechanisms underlying intracellular localization of different PDE isoforms are not understood. In this study, we have found that each of the supershort, short, and long forms of apPDE4 showed distinct localization in the…

  13. Advantages of human hepatocyte-derived transformants expressing a series of human cytochrome p450 isoforms for genotoxicity examination.

    PubMed

    Hashizume, Tsuneo; Yoshitomi, Sumie; Asahi, Satoru; Uematsu, Rieko; Matsumura, Shigeo; Chatani, Fumio; Oda, Hiroaki

    2010-08-01

    Metabolites of chemicals can often be ultimate genotoxic species; thus, in vitro routine testing requires the use of rat liver S9. However, there is a question as to whether this represents an appropriate surrogate for human metabolism. We have previously demonstrated the usefulness of HepG2 transformants expressing major human cytochrome P450 (CYP) isoforms to assess the genotoxicity of metabolites. We further assessed the advantages of these transformants from the following three aspects. First, the sensitivity of these transformants was confirmed with micronucleus (MN) induction by 7,12-dimethylbenz[a]anthracene or ifosfamide in transformants expressing the corresponding CYP1A1 or CYP2B6 and CYP2C9, respectively. Second, by using these transformants, beta-endosulfan, a chemical for which the CYP isoforms contributing to its genotoxicity are unknown, was found to induce MN through the CYP3A4-mediated pathway. This result was confirmed by the facts that the decreased CYP3A4 activity using a inhibitor or short interfering RNA (siRNA) repressed MN induction by beta-endosulfan and that endosulfan sulfate, one of the metabolites produced by CYP3A4, induced MN in the transformants harboring an empty vector. Third, the interaction between phase I and II drug-metabolizing enzymes was demonstrated by MN induction with inhibitors of uridine diphosphate (UDP)-glucuronosyltransferases in tamoxifen-treated transformants harboring the corresponding CYP3A4 or with inhibitors of glutathione S-transferase in safrole-treated transformants harboring the corresponding CYP2D6, whereas neither tamoxifen nor safrole alone induced MN in any transformant. These advantages provide the benefits of newly established transformants for in vitro genotoxicity testing that reflects comprehensive metabolic pathways including not only human CYP isoforms but also the phase II enzymes.

  14. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize

    USDA-ARS?s Scientific Manuscript database

    Cytokinin dehydrogenase (CKX, EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a t...

  15. Differential processing of mammalian L-histidine decarboxylase enzymes.

    PubMed

    Fennell, Lilian M; Fleming, John V

    2014-03-07

    In the mammalian species studied so far, the L-histidine decarboxylase (HDC) enzyme responsible for histamine biosynthesis has been shown to undergo post-translational processing. The processing is best characterized for the mouse enzyme, where di-asparate DD motifs mediate the production of active ~55 and ~60 kDa isoforms from the ~74 kDa precursor in a caspase-9 dependent manner. The identification of conserved di-aspartate motifs at similar locations in the rat and human HDC protein sequences has led to proposals that these may represent important processing sites in these species also. Here we used transfected Cos7 cells to demonstrate that the rat and human HDC proteins undergo differential processing compared to each other, and found no evidence to suggest that conserved di-aspartate motifs are required absolutely for processing in this cell type. Instead we identified SKD and EEAPD motifs that are important for caspase-6 dependent production of ~54 and ~59 kDa isoforms in the rat and human proteins, respectively. The addition of staurosporine, which is known to pharmacologically activate caspase enzymes, increased processing of the human HDC protein. We propose that caspase-dependent processing is a conserved feature of mammalian HDC enzymes, but that proteolysis may involve different enzymes and occur at diverse sites and sequences. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Carbonic anhydrase activators: gold nanoparticles coated with derivatized histamine, histidine, and carnosine show enhanced activatory effects on several mammalian isoforms.

    PubMed

    Saada, Mohamed-Chiheb; Montero, Jean-Louis; Vullo, Daniela; Scozzafava, Andrea; Winum, Jean-Yves; Supuran, Claudiu T

    2011-03-10

    Lipoic acid moieties were attached to amine or amino acids showing activating properties against the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The obtained lipoic acid conjugates of histamine, L-histidine methyl ester, and L-carnosine methyl ester were attached to gold nanoparticles (NPs) by reaction with Au(III) salts in reducing conditions. The CA activators (CAAs)-coated NPs showed low nanomolar activation (K(A)s of 1-9 nM) of relevant cytosolic, membrane-bound, mitochondrial, and transmembrane CA isoforms, such as CA I, II, IV, VA, VII, and XIV. These NPs also effectively activated CAs ex vivo, in whole blood experiments, with an increase of 200-280% of the CA activity. This is the first example of enzyme activation with nanoparticles and may lead to biomedical applications for conditions in which the CA activity is diminished, such as aging, Alzheimer's disease, or CA deficiency syndrome.

  17. Molecular Dynamics Simulations and Structural Analysis to Decipher Functional Impact of a Twenty Residue Insert in the Ternary Complex of Mus musculus TdT Isoform.

    PubMed

    Mutt, Eshita; Sowdhamini, Ramanathan

    2016-01-01

    Insertions/deletions are common evolutionary tools employed to alter the structural and functional repertoire of protein domains. An insert situated proximal to the active site or ligand binding site frequently impacts protein function; however, the effect of distal indels on protein activity and/or stability are often not studied. In this paper, we have investigated a distal insert, which influences the function and stability of a unique DNA polymerase, called terminal deoxynucleotidyl transferase (TdT). TdT (EC:2.7.7.31) is a monomeric 58 kDa protein belonging to family X of eukaryotic DNA polymerases and known for its role in V(D)J recombination as well as in non-homologous end-joining (NHEJ) pathways. Two murine isoforms of TdT, with a length difference of twenty residues and having different biochemical properties, have been studied. All-atom molecular dynamics simulations at different temperatures and interaction network analyses were performed on the short and long-length isoforms. We observed conformational changes in the regions distal to the insert position (thumb subdomain) in the longer isoform, which indirectly affects the activity and stability of the enzyme through a mediating loop (Loop1). A structural rationale could be provided to explain the reduced polymerization rate as well as increased thermosensitivity of the longer isoform caused by peripherally located length variations within a DNA polymerase. These observations increase our understanding of the roles of length variants in introducing functional diversity in protein families in general.

  18. 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

    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.more » 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.« less

  19. Non-homologous isofunctional enzymes: a systematic analysis of alternative solutions in enzyme evolution.

    PubMed

    Omelchenko, Marina V; Galperin, Michael Y; Wolf, Yuri I; Koonin, Eugene V

    2010-04-30

    Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity.

  20. Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) 1

    PubMed Central

    McCue, Kent F.; Conn, Eric E.

    1990-01-01

    Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID:16667741

  1. The Inhibition of UDP-Glucuronosyltransferase (UGT) Isoforms by Praeruptorin A and B.

    PubMed

    Liu, Xin; Chen, Da-Wei; Wu, Xue; Zhao, Zhenying; Fu, Zhi-Wei; Huang, Chun-Ting; Ye, Li-Xin; Du, Zuo; Yu, Yang; Fang, Zhong-Ze; Sun, Hong-Zhi

    2016-11-01

    Praeruptorin A (PA) and B (PB) are two important compounds isolated from Bai-hua Qian-hu and have been reported to exert multiple biochemical and pharmacological activities. The present study aims to determine the inhibition of PA and PB on the activity of important phase II drug-metabolizing enzymes uridine 5'-diphospho-glucuronosyltransferase (UGTs) isoforms. In vitro UGT incubation system was used to determine the inhibition potential of PA and PB on the activity of various UGT isoforms. In silico docking was performed to explain the inhibition difference between PA and PB towards the activity of UGT1A6. Inhibition behaviour was determined, and in vitro-in vivo extrapolation was performed by using the combination of in vitro inhibition kinetic parameter (K i ) and in vivo exposure level of PA. Praeruptorin A (100 μM) exhibited the strongest inhibition on the activity of UGT1A6 and UGT2B7, with 97.8% and 90.1% activity inhibited by 100 μM of PA, respectively. In silico docking study indicates the significant contribution of hydrogen bond interaction towards the stronger inhibition of PA than PB towards UGT1A6. Praeruptorin A noncompetitively inhibited the activity of UGT1A6 and competitively inhibited the activity of UGT2B7. The inhibition kinetic parameter (K i ) of PA towards UGT1A6 and UGT2B7 was calculated to be 1.2 and 3.3 μM, respectively. The [I]/K i value was calculated to be 15.8 and 5.8 for the inhibition of PA on UGT1A6 and UGT2B7, indicating high inhibition potential of PA towards these two UGT isoforms in vivo. Therefore, closely monitoring the interaction between PA and drugs mainly undergoing UGT1A6 or UGT2B7-catalyzed metabolism is very necessary. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Heavy-metal stress induced accumulation of chitinase isoforms in plants.

    PubMed

    Békésiová, Beata; Hraska, Stefan; Libantová, Jana; Moravcíková, Jana; Matusíková, Ildikó

    2008-12-01

    Plant chitinases belong to so-called pathogenesis related proteins and have mostly been detected in plants exposed to phytopathogenic viruses, bacteria or fungi. A few studies revealed that they might also be involved in plant defence against heavy metals. This work was undertaken to monitor the accumulation of chitinases in a set of heavy-metal stressed plants and bring evidence on their involvement during this kind of stress. Roots of different plant species including Vicia faba cvs. Astar and Piestanský, Pisum sativum, Hordeum vulgare, Zea mays and Glycine max were exposed to different concentrations of lead (300 and 500 mg l(-1) Pb(2+)), cadmium (100 and 300 mg l(-1) Cd(2+)) and arsenic (50 and 100 mg l(-1) As(3+)). In each case, the toxicity effects were reflected in root growth retardation to 80-10% of control values. The most tolerant were beans, most sensitive was barley. Extracts from the most stressed roots were further assayed for chitinase activity upon separation on polyacrylamide gels. Our data showed that in each combination of genotype and metal ion there were 2-5 different chitinase isoforms significantly responsive to toxic environment when compared with water-treated controls. This confirms that chitinases are components of plant defence against higher concentrations of heavy metals. In addition, accumulation of some isoforms in response to one but not to other metal ions suggests that these enzymes might also be involved in a more (metal) specific mechanism in affected plants and their biological role is more complex than expected.

  3. Hyperpolarized [(13)C]ketobutyrate, a molecular analog of pyruvate with modified specificity for LDH isoforms.

    PubMed

    von Morze, Cornelius; Bok, Robert A; Ohliger, Michael A; Zhu, Zihan; Vigneron, Daniel B; Kurhanewicz, John

    2016-05-01

    The purpose of this study was to investigate (13) C hyperpolarization of α-ketobutyrate (αKB), an endogenous molecular analog of pyruvate, and its in vivo enzymatic conversion via lactate dehydrogenase (LDH) using localized MR spectroscopy. Hyperpolarized (HP) (13) C MR experiments were conducted using [(13) C]αKB with rats in vivo and with isolated LDH enzyme in vitro, along with comparative experiments using [(13) C]pyruvate. Based on differences in the kinetics of its reaction with individual LDH isoforms, HP [(13) C]αKB was investigated as a novel MR probe, with added specificity for activity of LDHB-expressed H ("heart"-type) subunits of LDH (e.g., constituents of LDH-1 isoform). Comparable T1 and polarization values to pyruvate were attained (T1  = 52 s at 3 tesla [T], polarization = 10%, at C1 ). MR experiments showed rapid enzymatic conversion with substantially increased specificity. Formation of product HP [(13) C]α-hydroxybutyrate (αHB) from αKB in vivo was increased 2.7-fold in cardiac slabs relative to liver and kidney slabs. In vitro studies resulted in 5.0-fold higher product production from αKB with bovine heart LDH-1, as compared with pyruvate. HP [(13) C]αKB may be a useful MR probe of cardiac metabolism and other applications where the role of H subunits of LDH is significant (e.g., renal cortex and brain). © 2015 Wiley Periodicals, Inc.

  4. Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations‡

    PubMed Central

    Wagner, Jason; Avvaru, Balendu Sankara; Robbins, Arthur H.; Scozzafava, Andrea; Supuran, Claudiu T.; McKenna, Robert

    2010-01-01

    We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide- tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (KIs of 73 – 131 nM), effective hCA II inhibition (KIs of 9.1 – 36 nM) and less effective hCA IX and XII inhibition (KIs of 55-128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with KIs of 5.9 – 14.2 nM, although it was less effective as hCA I and II inhibitor (KIs of 36-120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (KI of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum. PMID:20598552

  5. The glial phosphorylase of glycogen isoform is reduced in the dorsolateral prefrontal cortex in chronic schizophrenia.

    PubMed

    Pinacho, Raquel; Vila, Elia; Prades, Roger; Tarragó, Teresa; Castro, Elena; Ferrer, Isidre; Ramos, Belén

    2016-11-01

    Reduced glutamatergic activity and energy metabolism in the dorsolateral prefrontal cortex (DLPFC) have been described in schizophrenia. Glycogenolysis in astrocytes is responsible for providing neurons with lactate as a transient energy supply helping to couple glutamatergic neurotransmission and glucose utilization in the brain. This mechanism could be disrupted in schizophrenia. The aim of this study was to explore whether the protein levels of the astrocyte isoform of glycogen phosphorylase (PYGM), key enzyme of glycogenolysis, and the isoform A of Ras-related C3 botulinum toxin substrate 1 (RAC1), a kinase that regulates PYGM activity, are altered in the postmortem DLPFC of chronic schizophrenia patients (n=23) and matched controls (n=23). We also aimed to test NMDAR blockade effect on these proteins in the mouse cortex and cortical astrocytes and antipsychotic treatments in rats. Here we report a reduction in PYGM and RAC1 protein levels in the DLPFC in schizophrenia. We found that treatment with the NMDAR antagonist dizocilpine in mice as a model of psychosis increased PYGM and reduced RAC1 protein levels. The same result was observed in rat cortical astroglial-enriched cultures. 21-day haloperidol treatment increased PYGM levels in rats. These results show that PYGM and RAC1 are altered in the DLPFC in chronic schizophrenia and are controlled by NMDA signalling in the rodent cortex and cortical astrocytes suggesting an altered NMDA-dependent glycogenolysis in astrocytes in schizophrenia. Together, this study provides evidence of a NMDA-dependent transient local energy deficit in neuron-glia crosstalk in schizophrenia, contributing to energy deficits of the disorder. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Skeletal muscle overexpression of short isoform Sirt3 altered mitochondrial cardiolipin content and fatty acid composition.

    PubMed

    Chabi, Béatrice; Fouret, Gilles; Lecomte, Jérome; Cortade, Fabienne; Pessemesse, Laurence; Baati, Narjès; Coudray, Charles; Lin, Ligen; Tong, Qiang; Wrutniak-Cabello, Chantal; Casas, François; Feillet-Coudray, Christine

    2018-03-27

    Cardiolipin (CL) is a phospholipid at the heart of mitochondrial metabolism, which plays a key role in mitochondrial function and bioenergetics. Among mitochondrial activity regulators, SIRT3 plays a crucial role in controlling the acetylation status of many enzymes participating in the energy metabolism in particular concerning lipid metabolism and fatty acid oxidation. Data suggest that possible connection may exist between SIRT3 and CL status that has not been evaluated in skeletal muscle. In the present study, we have characterized skeletal muscle lipids as well as mitochondrial lipids composition in mice overexpressing long (SIRT3-M1) and short (SIRT3-M3) isoforms of SIRT3. Particular attention has been paid for CL. We reported no alteration in muscle lipids content and fatty acids composition between the two mice SIRT3 strains and the control mice. However, mitochondrial CL content was significantly decreased in SIRT3-M3 mice and associated to an upregulation of tafazzin gene expression. In addition, mitochondrial phospholipids and fatty acids composition was altered with an increase in the PC/PE ratio and arachidonic acid content and a reduction in the MUFA/SFA ratio. These modifications in mitochondrial membrane composition are associated with a reduction in the enzymatic activities of mitochondrial respiratory chain complexes I and IV. In spite of these mitochondrial enzymatic alterations, skeletal muscle mitochondrial respiration remained similar in SIRT3-M3 and control mice. Surprisingly, none of those metabolic alterations were detected in mitochondria from SIRT3-M1 mice. In conclusion, our data indicate a specific action of the shorter SIRT3 isoform on lipid mitochondrial membrane biosynthesis and functioning.

  7. Differential Modulation of SERCA2 Isoforms by Calreticulin

    PubMed Central

    John, Linu M.; Lechleiter, James D.; Camacho, Patricia

    1998-01-01

    In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function. PMID:9722609

  8. Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification

    PubMed Central

    Simpson, Jeffrey P.; Di Leo, Rosa; Dhanoa, Preetinder K.; Allan, Wendy L.; Makhmoudova, Amina; Clark, Shawn M.; Hoover, Gordon J.; Mullen, Robert T.; Shelp, Barry J.

    2008-01-01

    Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (Km glyoxylate=34 μM), and SSA to γ-hydroxybutyrate (Km SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (kcat/Km). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response. PMID:18495639

  9. Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification.

    PubMed

    Simpson, Jeffrey P; Di Leo, Rosa; Dhanoa, Preetinder K; Allan, Wendy L; Makhmoudova, Amina; Clark, Shawn M; Hoover, Gordon J; Mullen, Robert T; Shelp, Barry J

    2008-01-01

    Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (K(m) glyoxylate=34 microM), and SSA to gamma-hydroxybutyrate (K(m) SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (k(cat)/K(m)). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.

  10. Elevated Liver Enzymes

    MedlinePlus

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  11. The plethora of PMCA isoforms: Alternative splicing and differential expression.

    PubMed

    Krebs, Joachim

    2015-09-01

    In this review the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to their tissue distribution, their differences during development and their importance for regulating Ca²⁺ homeostasis under different conditions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling.

    PubMed

    Singhal, Hari; Greene, Marianne E; Zarnke, Allison L; Laine, Muriel; Al Abosy, Rose; Chang, Ya-Fang; Dembo, Anna G; Schoenfelt, Kelly; Vadhi, Raga; Qiu, Xintao; Rao, Prakash; Santhamma, Bindu; Nair, Hareesh B; Nickisch, Klaus J; Long, Henry W; Becker, Lev; Brown, Myles; Greene, Geoffrey L

    2018-01-12

    Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR

  13. Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling

    PubMed Central

    Singhal, Hari; Greene, Marianne E.; Zarnke, Allison L.; Laine, Muriel; Al Abosy, Rose; Chang, Ya-Fang; Dembo, Anna G.; Schoenfelt, Kelly; Vadhi, Raga; Qiu, Xintao; Rao, Prakash; Santhamma, Bindu; Nair, Hareesh B.; Nickisch, Klaus J.; Long, Henry W.; Becker, Lev; Brown, Myles; Greene, Geoffrey L.

    2018-01-01

    Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR

  14. Characterization of the human LPIN1-encoded phosphatidate phosphatase isoforms.

    PubMed

    Han, Gil-Soo; Carman, George M

    2010-05-07

    The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work, we characterized human lipin 1 alpha, beta, and gamma isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the alpha, beta, and gamma isoforms were dependent on Mg(2+) or Mn(2+) ions at pH 7.5 at 37 degrees C. The activities were inhibited by concentrations of Mg(2+) and Mn(2+) above their optimums and by Ca(2+), Zn(2+), N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 degrees C. The alpha, beta, and gamma activities followed saturation kinetics with respect to the molar concentration of PA (K(m) values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number approximately 2) kinetics with respect to the surface concentration of PA (K(m) values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (k(cat)) for the alpha, beta, and gamma isoforms were 68.8 + or - 3.5, 42.8 + or - 2.5, and 5.7 + or - 0.2 s(-1), respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity.

  15. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    PubMed

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  16. Integrative gene set enrichment analysis utilizing isoform-specific expression.

    PubMed

    Li, Lie; Wang, Xinlei; Xiao, Guanghua; Gazdar, Adi

    2017-09-01

    Gene set enrichment analysis (GSEA) aims at identifying essential pathways, or more generally, sets of biologically related genes that are involved in complex human diseases. In the past, many studies have shown that GSEA is a very useful bioinformatics tool that plays critical roles in the innovation of disease prevention and intervention strategies. Despite its tremendous success, it is striking that conclusions of GSEA drawn from isolated studies are often sparse, and different studies may lead to inconsistent and sometimes contradictory results. Further, in the wake of next generation sequencing technologies, it has been made possible to measure genome-wide isoform-specific expression levels, calling for innovations that can utilize the unprecedented resolution. Currently, enormous amounts of data have been created from various RNA-seq experiments. All these give rise to a pressing need for developing integrative methods that allow for explicit utilization of isoform-specific expression, to combine multiple enrichment studies, in order to enhance the power, reproducibility, and interpretability of the analysis. We develop and evaluate integrative GSEA methods, based on two-stage procedures, which, for the first time, allow statistically efficient use of isoform-specific expression from multiple RNA-seq experiments. Through simulation and real data analysis, we show that our methods can greatly improve the performance in identifying essential gene sets compared to existing methods that can only use gene-level expression. © 2017 WILEY PERIODICALS, INC.

  17. Differential expression of ryanodine receptor isoforms after spinal cord injury.

    PubMed

    Pelisch, Nicolas; Gomes, Cynthia; Nally, Jacqueline M; Petruska, Jeffrey C; Stirling, David P

    2017-11-01

    Ryanodine receptors (RyRs) are highly conductive intracellular Ca 2+ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca 2+ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. The specificity of the protein kinase C alpha, betaII and gamma isoforms as assessed by an unnatural alcohol-appended peptide library.

    PubMed Central

    Yan, X; Curley, K; Lawrence, D S

    2000-01-01

    Previous studies using conventional peptide-based libraries have demonstrated that homologous protein-processing enzymes [e.g. the alpha, betaII and gamma isoforms of protein kinase (PKC)] typically display identical amino acid consensus sequences. These observations have hampered the acquisition of selective synthetic substrates for the individual members of these enzyme families. We describe here a parallel synthesis strategy, readily adaptable to the preparation of large libraries, that has led to the emergence of the first examples of selective substrates for the conventional PKC isoforms. In addition, we have found that a wide variety of structurally diverse N-appended alcohol-containing residues, including tyrosine, serve as substrates for the PKC alpha, betaII and gamma isoforms. This broad active-site substrate specificity with respect to both natural and unnatural residues may prove to be especially applicable to the construction of transition-state analogues and suicide substrates, species that often require the presence of structurally elaborate functionality. PMID:10903131

  19. Relationship between the hippocampal expression of selected cytochrome P450 isoforms and the animal performance in the hippocampus-dependent learning task.

    PubMed

    Gjota-Ergin, Sena; Gökçek-Saraç, Çiğdem; Adalı, Orhan; Jakubowska-Doğru, Ewa

    2018-04-23

    Despite very extensive studies on the molecular mechanisms of memory formation, relatively little is known about the molecular correlates of individual variation in the learning skills within a random population of young normal subjects. The role of cytochrome P450 (CYP) enzymes in the brain also remains poorly understood. On the other hand, these enzymes are known to be related to the metabolism of substances important for neural functions including steroids, fatty acids, and retinoic acid. In the present study, we examined the potential correlation between the animals' performance in a place learning task and the levels of selected CYP isoforms (CYP2E1, CYP2D1 and CYP7A1) in the rat hippocampus. According to their performance, rats were classified as "good" learners (percent error/number of trials to criterion ≤ group mean - 3SEM) or "poor" learners (percent error/number of trials to criterion ≥ group mean + 3SEM). The CYP enzyme levels were determined by Western Blot at the early, intermediary and advanced stages of the task acquisition (day 4, day 8 and after reaching a performance criterion of 83% correct responses). In this study, as expected, CYP2E1 and CYP2D1 isoforms have been found in the rat hippocampus. However, a putative CYP7A1 isoform was also visualized. Hippocampal expression of these enzymes was shown to be dependent on the stage of learning and animals' cognitive status. In "good" learners compared to "poor" learners, significantly higher levels of CYP2E1 were found at the early stage of training, significantly higher levels of CYP2D1 were found at the intermediate stage of training, and significantly higher levels of CYP7A1-like protein were found after reaching the acquisition criterion. These findings suggest that the differential expression of some CYP isoforms in the hippocampus may have impact on individual learning skills and that different CYP isoforms may play different roles during the learning process. Copyright © 2018

  20. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

    PubMed

    Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M; Kumar, Manish; Nixon, B Tracy; Bulone, Vincent; Zimmer, Jochen

    2016-10-04

    Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

  1. Factor XIII: congenital deficiency factor XIII, acquired deficiency, factor XIII A-subunit, and factor XIII B-subunit.

    PubMed

    Tahlan, Anita; Ahluwalia, Jasmina

    2014-02-01

    Factor XIII (FXIII) is a transglutaminase consisting of 2 catalytic A subunits and 2 noncatalytic B subunits in plasma. The noncatalytic B subunits protect the catalytic A subunits from clearance. Congenital FXIII deficiency may manifest as a lifelong bleeding tendency, abnormal wound healing, and recurrent miscarriage. Acquired FXIII deficiency, with significant reductions in FXIII levels, has been reported in several medical conditions. The routine screening tests for coagulopathies-prothrombin time, activated partial thromboplastin time, and thrombin time-do not show abnormalities in cases of FXIII deficiency. A quantitative, functional, FXIII activity assay that detects all forms of FXIII deficiency should be used as a first-line screening test. Treatment consists of recombinant FXIII or FXIII concentrate. If these are unavailable, then fresh-frozen plasma and cryoprecipitates may be used. Factor XIII has a long half-life; therefore, the patients can lead near-normal lives with regular replacements. Patients with acquired FXIII deficiency with inhibitors need immunosuppressive therapy in addition to factor replacements.

  2. A Novel Isoform of Sucrose Synthase Is Targeted to the Cell Wall during Secondary Cell Wall Synthesis in Cotton Fiber[C][W][OA

    PubMed Central

    Brill, Elizabeth; van Thournout, Michel; White, Rosemary G.; Llewellyn, Danny; Campbell, Peter M.; Engelen, Steven; Ruan, Yong-Ling; Arioli, Tony; Furbank, Robert T.

    2011-01-01

    Sucrose (Suc) synthase (Sus) is the major enzyme of Suc breakdown for cellulose biosynthesis in cotton (Gossypium hirsutum) fiber, an important source of fiber for the textile industry. This study examines the tissue-specific expression, relative abundance, and temporal expression of various Sus transcripts and proteins present in cotton. A novel isoform of Sus (SusC) is identified that is expressed at high levels during secondary cell wall synthesis in fiber and is present in the cell wall fraction. The phylogenetic relationships of the deduced amino acid sequences indicate two ancestral groups of Sus proteins predating the divergence of monocots and dicots and that SusC sequences form a distinct branch in the phylogeny within the dicot-specific clade. The subcellular location of the Sus isoforms is determined, and it is proposed that cell wall-localized SusC may provide UDP-glucose for cellulose and callose synthesis from extracellular sugars. PMID:21757635

  3. Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition

    PubMed Central

    Limongelli, Vittorio; Bonomi, Massimiliano; Marinelli, Luciana; Gervasio, Francesco Luigi; Cavalli, Andrea; Novellino, Ettore; Parrinello, Michele

    2010-01-01

    The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity. PMID:20215464

  4. The distribution of myosin heavy chain isoforms among rat extraocular muscle fiber types.

    PubMed

    Rubinstein, N A; Hoh, J F

    2000-10-01

    To determine the distribution of myosin heavy chain isoforms in each extraocular muscle (EOM) fiber type. Serial sections of adult rat EOMs were stained with isoform-specific monoclonal antibodies against an array of myosin heavy chains. Immunofluorescent antibody staining of whole adult rat EOMs, examined by confocal microscopy, demonstrated the longitudinal variations of isoforms along individual fibers. Each global fiber type reacted predominantly with a single isoform-specific antibody and showed no longitudinal variation. Two major orbital fibers were defined, and both contained multiple myosin heavy chains. Both orbital singly and multiply innervated fibers stained proximal and distal to the neuromuscular junction with antibody to embryonic myosin heavy chain, but this isoform was sharply and completely excluded from the domain of the neuromuscular junction. Orbital singly innervated fibers also contained the EOM-specific isoform at the neuromuscular junction. Orbital multiply innervated fibers did not contain the EOM-specific isoform, but additionally contained a slow isoform along their entire length. Adult rat EOMs show unique fiber types with arrangements of myosin heavy chain isoforms not seen in other skeletal muscles. Moreover, unique cellular mechanisms must exist to target each isoform to its proper domain along individual orbital fibers.

  5. Tryptophan-Catabolizing Enzymes – Party of Three

    PubMed Central

    Ball, Helen J.; Jusof, Felicita F.; Bakmiwewa, Supun M.; Hunt, Nicholas H.; Yuasa, Hajime J.

    2014-01-01

    Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that have independently evolved to catalyze the first step in tryptophan catabolism via the kynurenine pathway (KP). The depletion of tryptophan and formation of KP metabolites modulates the activity of the mammalian immune, reproductive, and central nervous systems. IDO and TDO enzymes can have overlapping or distinct functions depending on their expression patterns. The expression of TDO and IDO enzymes in mammals differs not only by tissue/cellular localization but also by their induction by distinct stimuli. To add to the complexity, these genes also have undergone duplications in some organisms leading to multiple isoforms of IDO or TDO. For example, many vertebrates, including all mammals, have acquired two IDO genes via gene duplication, although the IDO1-like gene has been lost in some lower vertebrate lineages. Gene duplications can allow the homologs to diverge and acquire different properties to the original gene. There is evidence for IDO enzymes having differing enzymatic characteristics, signaling properties, and biological functions. This review analyzes the evolutionary convergence of IDO and TDO enzymes as tryptophan-catabolizing enzymes and the divergent evolution of IDO homologs to generate an enzyme family with diverse characteristics not possessed by TDO enzymes, with an emphasis on the immune system. PMID:25346733

  6. New titin (connectin) isoforms and their functional role in striated muscles of mammals: facts and suppositions.

    PubMed

    Vikhlyantsev, I M; Podlubnaya, Z A

    2012-12-01

    This review summarizes results of our studies on titin isoform composition in vertebrate striated muscles under normal conditions, during hibernation, real and simulated microgravity, and under pathological conditions (stiff-person syndrome, post-apoplectic spasticity, dilated cardiomyopathy, cardiac hypertrophy). Experimental evidence for the existence in mammalian striated muscles of higher molecular weight isoforms of titin (NT-isoforms) in addition to the known N2A-, N2BA-, and N2B-titin isoforms was obtained. Comparative studies of changes in titin isoform composition and structure-functional properties of human and animal striated muscles during adaptive and pathological processes led to a conclusion about the key role of NT-isoforms of titin in maintenance of sarcomere structure and contractile function of these muscles.

  7. PSA Isoforms' Velocities for Early Diagnosis of Prostate Cancer.

    PubMed

    Heidegger, Isabel; Klocker, Helmut; Pichler, Renate; Horninger, Wolfgang; Bektic, Jasmin

    2015-06-01

    Free prostate-specific antigen (fPSA) and its molecular isoforms are suggested for enhancement of PSA testing in prostate cancer (PCa). In the present study we evaluated whether PSA isoforms' velocities might serve as a tool to improve early PCa diagnosis. Our study population included 381 men who had undergone at least one ultrasound-guided prostate biopsy whose pathologic examination yielded PCa or showed no evidence of prostatic malignancy. Serial PSA, fPSA, and proPSA measurements were performed on serum samples covering 7 years prior to biopsy using Beckmann Coulter Access immunoassays. Afterwards, velocities of PSA (PSAV), fPSA% (fPSA%V), proPSA% (proPSA%V) and the ratio proPSA/PSA/V were calculated and their ability to discriminate cancer from benign disease was evaluated. Among 381 men included in the study, 202 (53%) were diagnosed with PCa and underwent radical prostatectomy at our Department. PSAV, fPSA%V, proPSA%V as well as proPSA/PSA/V were able to differentiate significantly between PCa and non-cancerous prostate. The highest discriminatory power between cancer and benign disease has been observed two and one year prior to diagnosis with all measured parameters. Among all measured parameters, fPSA%V showed the best cancer specificity of 45.3% with 90% of sensitivity. In summary, our results highlight the value of PSA isoforms' velocity for early detection of PCa. Especially fPSA%V should be used in the clinical setting to increase cancer detection specificity. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    PubMed Central

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

  9. The Folylpolyglutamate Synthetase Plastidial Isoform Is Required for Postembryonic Root Development in Arabidopsis1[W][OA

    PubMed Central

    Srivastava, Avinash C.; Ramos-Parra, Perla A.; Bedair, Mohamed; Robledo-Hernández, Ana L.; Tang, Yuhong; Sumner, Lloyd W.; Díaz de la Garza, Rocío I.; Blancaflor, Elison B.

    2011-01-01

    A recessive Arabidopsis (Arabidopsis thaliana) mutant with short primary roots and root hairs was identified from a forward genetic screen. The disrupted gene in the mutant encoded the plastidial isoform of folylpolyglutamate synthetase (FPGS), previously designated as AtDFB, an enzyme that catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. The short primary root of atdfb was associated with a disorganized quiescent center, dissipated auxin gradient in the root cap, bundled actin cytoskeleton, and reduced cell division and expansion. The accumulation of monoglutamylated forms of some folate classes in atdfb was consistent with impaired FPGS function. The observed cellular defects in roots of atdfb underscore the essential role of folylpolyglutamates in the highly compartmentalized one-carbon transfer reactions (C1 metabolism) that lead to the biosynthesis of compounds required for metabolically active cells found in the growing root apex. Indeed, metabolic profiling uncovered a depletion of several amino acids and nucleotides in atdfb indicative of broad alterations in metabolism. Methionine and purines, which are synthesized de novo in plastids via C1 enzymatic reactions, were particularly depleted. The root growth and quiescent center defects of atdfb were rescued by exogenous application of 5-formyl-tetrahydrofolate, a stable folate that was readily converted to metabolically active folates. Collectively, our results indicate that AtDFB is the predominant FPGS isoform that generates polyglutamylated folate cofactors to support C1 metabolism required for meristem maintenance and cell expansion during postembryonic root development in Arabidopsis. PMID:21233333

  10. NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

    PubMed

    Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

    2009-08-15

    Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

  11. Male-Specific Fruitless Isoforms Target Neurodevelopmental Genes to Specify a Sexually Dimorphic Nervous System

    PubMed Central

    Neville, Megan C.; Nojima, Tetsuya; Ashley, Elizabeth; Parker, Darren J.; Walker, John; Southall, Tony; Van de Sande, Bram; Marques, Ana C.; Fischer, Bettina; Brand, Andrea H.; Russell, Steven; Ritchie, Michael G.; Aerts, Stein; Goodwin, Stephen F.

    2014-01-01

    Summary Background In Drosophila, male courtship behavior is regulated in large part by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation or the role that isoform diversity plays in the formation of a male-specific nervous system. Results To characterize the roles of sex-specific Fru isoforms in specifying male behavior, we generated novel isoform-specific mutants and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specified by either a single isoform or a combination of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genomic enhancers and novel downstream regulators of male sexual behavior. Conclusions These findings suggest that Fru isoform diversity facilitates both redundancy and specificity in gene expression, and that the regulation of neuronal developmental genes may be the most ancient and conserved role of fru in the specification of a male-specific nervous system. PMID:24440396

  12. Novel protein isoforms of carcinoembryonic antigen are secreted from pancreatic, gastric and colorectal cancer cells

    PubMed Central

    2013-01-01

    Background Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines. Results We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns. Conclusions This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker. PMID:24070190

  13. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  14. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    SciTech Connect

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren

    2008-04-09

    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold duringmore » lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.« less

  15. An NAD(+) biosynthetic pathway enzyme functions cell non-autonomously in C. elegans development.

    PubMed

    Crook, Matt; Mcreynolds, Melanie R; Wang, Wenqing; Hanna-Rose, Wendy

    2014-08-01

    Disruption of cellular metabolite levels can adversely impact development. Specifically, loss-of-function of the C. elegans NAD(+) salvage biosynthesis gene PNC-1 results in an array of developmental phenotypes. Intriguingly, PNC-1 and its functional equivalent in vertebrates are secreted, but the contributions of the extracellular enzymes are poorly understood. We sought to study the tissue-specific requirements for PNC-1 expression and to examine the role of the secreted isoform. A thorough analysis of PNC-1 expression did not detect expression in tissues that require PNC-1 function. Limited expression of both the secreted and intracellular PNC-1 isoforms provided function at a distance from the tissues with phenotypes. We also find that the secreted isoform contributes to in vivo PNC-1 activity. Furthermore, uv1 cell survival has the most stringent requirements in terms of PNC-1 expression pattern or level. Using careful promoter analysis and a restricted expression approach, we have shown that both the secreted and the intracellular PNC-1 isoforms function cell non-autonomously, and that the PNC-1a isoform is functionally relevant in vivo. Our work suggests a model where PNC-1 function is provided cell non-autonomously by a mix of intra and extracellular activity, most likely requiring NAD(+) salvage metabolite transport between tissues. © 2014 Wiley Periodicals, Inc.

  16. An NAD+ biosynthetic pathway enzyme functions cell non-autonomously in C. elegans development

    PubMed Central

    Crook, Matt; McReynolds, Melanie R.; Wang, Wenqing; Hanna-Rose, Wendy

    2017-01-01

    Background Disruption of cellular metabolite levels can adversely impact development. Specifically, loss-of-function of the C. elegans NAD+ salvage biosynthesis gene PNC-1 results in an array of developmental phenotypes. Intriguingly, PNC-1 and its functional equivalent in vertebrates are secreted, but the contributions of the extracellular enzymes are poorly understood. We sought to study the tissue-specific requirements for PNC-1 expression and to examine the role of the secreted isoform. Results A thorough analysis of PNC-1 expression did not detect expression in tissues that require PNC-1 function. Limited expression of both the secreted and intracellular PNC-1 isoforms provided function at a distance from the tissues with phenotypes. We also find that the secreted isoform contributes to in vivo PNC-1 activity. Furthermore, uv1 cell survival has the most stringent requirements in terms of PNC-1 expression pattern or level. Conclusion Using careful promoter analysis and a restricted expression approach we have shown that both the secreted and the intracellular PNC-1 isoforms function cell non-autonomously, and that the PNC-1a isoform is functionally relevant in vivo. Our work suggests a model where PNC-1 function is provided cell non-autonomously by a mix of intra and extracellular activity, most likely requiring NAD+ salvage metabolite transport between tissues. PMID:24753121

  17. 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.

  18. Evaluating PI3 kinase isoforms using Transcreener ADP assays.

    PubMed

    Klink, Tony A; Kleman-Leyer, Karen M; Kopp, Andrew; Westermeyer, Thane A; Lowery, Robert G

    2008-07-01

    Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Transcreener ADP Assay relies on homogeneous immunodetection of adenosine diphosphate (ADP), using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP--the invariant product of all kinase reactions--provides complete flexibility for varying lipid substrate parameters. The authors used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The nonphysiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP- and TR-FRET-based assays, and there was excellent concordance (r(2)=0.93) in the IC(50) values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. Adenosine triphosphate (ATP) hydrolysis in the absence of substrate was detected with the PI3Kalpha isoform, and inhibitors affected PI3Kalpha intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.

  19. Evaluating PI3 Kinase Isoforms Using Transcreener™ ADP Assays

    PubMed Central

    KLINK, TONY A.; KLEMAN-LEYER, KAREN M.; KOPP, ANDREW; WESTERMEYER, THANE A.; LOWERY, ROBERT G.

    2009-01-01

    Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Trancreener™ ADP Assay relies on homogenous immunodetection of ADP, using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP - the invariant product of all kinase reactions - provides complete flexibility for varying lipid substrate parameters. We used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The non-physiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP and TR-FRET based assays and there was excellent concordance (r2 = 0.93) in the IC50 values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. ATP hydrolysis in the absence of substrate was detected with the PI3Kα isoform, and inhibitors affected PI3Kα intrinsic ATP hydrolysis activity similarly to lipid phosphorylation. PMID:18566477

  20. Toward β-Secretase-1 Inhibitors with Improved Isoform Selectivity.

    PubMed

    Johansson, Patrik; Kaspersson, Karin; Gurrell, Ian K; Bäck, Elisabeth; Eketjäll, Susanna; Scott, Clay W; Cebers, Gvido; Thorne, Philip; McKenzie, Michael J; Beaton, Haydn; Davey, Paul; Kolmodin, Karin; Holenz, Jörg; Duggan, Mark E; Budd Haeberlein, Samantha; Bürli, Roland W

    2018-04-10

    BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aβ production, and has been indicated to play a key role in the pathogenesis of Alzheimer's disease. The related isoform BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Cocrystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methylcyclohexyl element resulted in lead molecule 28, which exhibited ∼50-fold selectivity. Compared to a nonselective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.

  1. Analysis of Histone Modifications from Tryptic Peptides of Deuteroacetylated Isoforms

    PubMed Central

    Hersman, Elisabeth; Nelson, Dwella M.; Griffith, Wendell P.; Jelinek, Christine; Cotter, Robert J.

    2011-01-01

    The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species. PMID:22389584

  2. Nuclear localization of a novel human syntaxin 1B isoform.

    PubMed

    Pereira, Sandrine; Massacrier, Annick; Roll, Patrice; Vérine, Alain; Etienne-Grimaldi, Marie-Christine; Poitelon, Yannick; Robaglia-Schlupp, Andrée; Jamali, Sarah; Roeckel-Trevisiol, Nathalie; Royer, Barbara; Pontarotti, Pierre; Lévêque, Christian; Seagar, Michael; Lévy, Nicolas; Cau, Pierre; Szepetowski, Pierre

    2008-11-01

    The syntaxins are proteins associated with various intracellular membrane compartments. They are major participants in a large variety of physiological processes where membrane fusion occurs, including exocytosis. We have identified a novel syntaxin isoform generated by alternative splicing of the human STX1B gene. In contrast with the canonical syntaxins, this isoform (STX1B-DeltaTMD) lacked the classical C-terminal transmembrane domain and localized to the nucleus of various tumoral and non-tumoral cell types including human brain cortical neurons in vivo. The reversible blockade of STX1B-DeltaTMD nuclear import demonstrated that nuclear import occurred via a Ran-dependent pathway. A specific and glycine-rich C-terminus of 15 amino acids served as an unconventional nuclear localization signal. STX1B-DeltaTMD colocalized with Lamin A/C and NuMA (NUclear Mitotic Apparatus protein) in interphasic nuclei, and with NuMA and gamma-tubulin in the pericentrosomal region of the mitotic spindle in dividing cells. In a series of 37 human primary brain tumors, the ratio of STX1B-DeltaTMD to Lamin A/C transcripts was a significant prognostic marker of survival, independent of tumor staging. The characterization of STX1B-DeltaTMD as the first nucleoplasmic syntaxin with no transmembrane domain, illustrates the importance of alternative splicing in the emergence of unsuspected properties of the syntaxins in human cells, in both physiological and pathological conditions.

  3. Characterization of heparan sulfate N-deacetylase/N-sulfotransferase isoform 4 using synthetic oligosaccharide substrates.

    PubMed

    Li, Yi-Jun; Yin, Feng-Xin; Zhang, Xin-Ke; Yu, Jie; Zheng, Shuang; Song, Xin-Lei; Wang, Feng-Shan; Sheng, Ju-Zheng

    2018-03-01

    The final structure of heparan sulfate chains is strictly regulated in vivo, though the biosynthesis is not guided by a template process. N-deacetylase/N-sulfotransferase (NDST) is the first modification enzyme in the HS biosynthetic pathway. The N-sulfo groups introduced by NDST are reportedly involved in determination of the susceptibility to subsequent processes catalyzed by C 5 -epimerse and 3-O-sulfotransferases. Understanding the substrate specificities of the four human NDST isoforms has become central to uncovering the regulatory mechanism of HS biosynthesis. Highly-purified recombinant NDST-4 (rNDST-4) and a selective library of structurally-defined oligosaccharides were employed to determine the substrate specificity of rNDST-4. Full-length rNDST-4 lacks obvious N-deacetylase activity, and displays only N-sulfotransferase activity. Unlike NDST-1, NDST-4 did not show directional N-sulfotransferase activity while the N-deacetylase domain was inactive. Individual NDST-4 could not effectively assume the key role in the distribution of N-S domains and N-Ac domains in HS biosynthesis in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Characterization of Non-Nitrocatechol Pan and Isoform Specific Catechol-O-methyltransferase Inhibitors and Substrates

    PubMed Central

    2011-01-01

    Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity. PMID:22860182

  5. In vitro inhibition of human UGT isoforms by ritonavir and cobicistat.

    PubMed

    Algeelani, Sara; Alam, Novera; Hossain, Md Amin; Mikus, Gerd; Greenblatt, David J

    2017-09-11

    1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer's data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat. 2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors. 3. The 50% inhibitory concentrations (IC 50 ) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC 50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC 50 17 µM). 4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

  6. Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

    PubMed Central

    Madan, Namrata; Xu, Yunhui; Duan, Qiming; Banerjee, Moumita; Larre, Isabel; Pierre, Sandrine V.

    2017-01-01

    The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na+ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na+ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1. PMID:27903584

  7. Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase.

    PubMed

    Madan, Namrata; Xu, Yunhui; Duan, Qiming; Banerjee, Moumita; Larre, Isabel; Pierre, Sandrine V; Xie, Zijian

    2017-03-01

    The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na + affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na + was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1. Copyright © 2017 the American Physiological Society.

  8. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

    PubMed Central

    Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

    2016-01-01

    Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

  9. Isoform-selective HDAC1/6/8 inhibitors with an imidazo-ketopiperazine cap containing stereochemical diversity.

    PubMed

    Lecointre, Bertrand; Narozny, Remy; Borrello, Maria Teresa; Senger, Johanna; Chakrabarti, Alokta; Jung, Manfred; Marek, Martin; Romier, Christophe; Melesina, Jelena; Sippl, Wolfgang; Bischoff, Laurent; Ganesan, A

    2018-06-05

    A series of hydroxamic acids linked by different lengths to a chiral imidazo-ketopiperazine scaffold were synthesized. The compounds with linker lengths of 6 and 7 carbon atoms were the most potent in histone deacetylase (HDAC) inhibition, and were specific submicromolar inhibitors of the HDAC1, HDAC6 and HDAC8 isoforms. A docking model for the binding mode predicts binding of the hydroxamic acid to the active site zinc cation and additional interactions between the imidazo-ketopiperazine and the enzyme rim. The compounds were micromolar inhibitors of the MV4-11, THP-1 and U937 cancer cell lines. Increased levels of histone H3 and tubulin acetylation support a cellular mechanism of action through HDAC inhibition.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

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

    SciTech Connect

    Silver, Kristopher S.; Soderlund, David M.

    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}1more » 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

  11. The study of NAD-malic enzyme in Amaranthus cruentus L. under drought.

    PubMed

    Babayev, Hasan; Mehvaliyeva, Ulduza; Aliyeva, Minakhanym; Feyziyev, Yashar; Guliyev, Novruz

    2014-08-01

    Decarboxylating NAD-malate dehydrogenase (NAD-malic enzyme, NAD-ME, EC 1.1.1.39) has been investigated under a long-term drought during pre-anthesis, anthesis and seed-formation phases of ontogenesis of a NAD-ME type C4 plant Amaranthus cruentus L. using cytosol, chloroplast and mitochondrial fractions of mesophyll (M) and bundle sheath (BS) cells. We detected several molecular forms of NAD-ME with different subcellular localization patterns in the studied phases of amaranth ontogenesis. However, no enzyme activity was observed experimentally in chloroplasts of M and BS cells. In the pre-anthesis phase NAD-ME isoform with molecular weight of ∼115 kDa was found in cytosol of M and BS cells of control and drought-exposed plants. One of NAD-ME isoforms with molecular weight of 110 kDa was located in mitochondria of BS cells of control and drought-exposed plants, and a new isoform of ∼121 kDa was formed in mitochondria of BS cells under the influence of drought. After resuming watering this isoform (∼121 kDa) disappeared again. Approximately 90.6% and 9.4% of the total NAD-ME activity were localized in mitochondrial stroma and cytosol of BS cells, respectively, while in mesophyll cells 100% activity was found in cytosol fractions. The reaction catalyzed by NAD-ME follows Michaelis-Menten equation. NAD(+), l-malate and Mn(2+) activate this enzyme in mitochondria. Appearance of the ∼121 kDa isoform of NAD-ME in the mitochondrial fraction of BS cells under drought and its disappearance after resuming watering could be attributed to one of the protection functions of plants. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  12. A novel high throughput screening assay for binding affinities of perfluoroalkyl iodide for estrogen receptor alpha and beta isoforms.

    PubMed

    Song, Wenting; Zhao, Lixia; Sun, Zhendong; Yang, Xiaoxi; Zhou, Qunfang; Jiang, Guibin

    2017-12-01

    Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) α or β isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered β-galactosidase enzyme (β-gal) for the binding to ERα or β, thus quantitatively altering the formation of enzymatically active β-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ERα or β concentrations, it was found that luminescent signals were sustainably emitted for 9h, and 40nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E 2 ) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on β-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ERα or β isoforms. The binding affinities of PFIs for ERα were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure

  13. Evaluation of the non-catalytic binding function of Ts26GST a glutathione transferase isoform of Taenia solium.

    PubMed

    Plancarte, A; Romero, J R; Nava, G; Reyes, H; Hernández, M

    2014-03-01

    Taenia solium glutathione transferase isoform of 26.5 kDa (Ts26GST) was observed to bind non-catalytically to porphyrins, trans-trans-dienals, bile acids and fatty acids, as assessed by inhibition kinetics, fluorescence spectroscopy and competitive fluorescence assays with 8-anilino-1-naphthalene sulfonate (ANS). The quenching of Ts26GST intrinsic fluorescence allowed for the determination of the dissociation constants (KD) for all ligands. Obtained data indicate that Ts26GST binds to all ligands but with different affinity. Porphyrins and lipid peroxide products inhibited Ts26GST catalytic activity up to 100% in contrast with only 20-30% inhibition observed for bile acids and two saturated fatty acids. Non-competitive type inhibition was observed for all enzyme inhibitor ligands except for trans-trans-2,4-decadienal, which exhibited uncompetitive type inhibition. The dissociation constant value KD = 0.7 μM for the hematin ligand, determined by competitive fluorescence assays with ANS, was in good agreement with its inhibition kinetic value Ki = 0.3 μM and its intrinsic fluorescence quenching KD = 0.7 μM. The remaining ligands did not displace ANS from the enzyme suggesting the existence of different binding sites. In addition to the catalytic activity of Ts26GST the results obtained suggest that the enzyme exhibits a ligandin function with broad specificity towards nonsubstrate ligands. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Altered levels of LIL3 isoforms in Arabidopsis lead to disturbed pigment-protein assembly and chlorophyll synthesis, chlorotic phenotype and impaired photosynthetic performance.

    PubMed

    Lohscheider, Jens N; Rojas-Stütz, Marc C; Rothbart, Maxi; Andersson, Ulrica; Funck, Dietmar; Mendgen, Kurt; Grimm, Bernhard; Adamska, Iwona

    2015-10-01

    Light-harvesting complex (LHC)-like (LIL) proteins contain two transmembrane helices of which the first bears a chlorophyll (Chl)-binding motif. They are widespread in photosynthetic organisms, but almost nothing is known about their expression and physiological functions. We show that two LIL3 paralogues (LIL3:1 and LIL3:2) in Arabidopsis thaliana are expressed in photosynthetically active tissues and their expression is differentially influenced by light stress. Localization studies demonstrate that both isoforms are associated with subcomplexes of LHC antenna of photosystem II. Transgenic plants with reduced amounts of LIL3:1 exhibited a slightly impaired growth and have reduced Chl and carotenoid contents as compared to wild-type plants. Ectopic overexpression of either paralogue led to a developmentally regulated switch to co-suppression of both LIL3 isoforms, resulting in a circular chlorosis of the leaf rosettes. Chlorotic sectors show severely diminished levels of LIL3 isoforms and other proteins, and thylakoid morphology was changed. Additionally, the levels of enzymes involved in Chl biosynthesis are altered in lil3 mutant plants. Our data support a role of LIL3 paralogues in the regulation of Chl biosynthesis under light stress and under standard growth conditions as well as in a coordinated ligation of newly synthesized and/or rescued Chl molecules to their target apoproteins. © 2015 John Wiley & Sons Ltd.

  15. The effects of salt stress cause a diversion of basal metabolism in barley roots: possible different roles for glucose-6-phosphate dehydrogenase isoforms.

    PubMed

    Cardi, Manuela; Castiglia, Daniela; Ferrara, Myriam; Guerriero, Gea; Chiurazzi, Maurizio; Esposito, Sergio

    2015-01-01

    In this study the effects of salt stress and nitrogen assimilation have been investigated in roots of hydroponically-grown barley plants exposed to 150 mM NaCl, in presence or absence of ammonium as the sole nitrogen source. Salt stress determines a diversion of root metabolism towards the synthesis of osmolytes, such as glycine betaine and proline, and increased levels of reduced glutathione. The metabolic changes triggered by salt stress result in a decrease in both activities and protein abundance of key enzymes, namely GOGAT and PEP carboxylase, and in a slight increase in HSP70. These variations would enhance the requirement for reductants supplied by the OPPP, consistently with the observed increase in total G6PDH activity. The involvement and occurrence of the different G6PDH isoforms have been investigated, and the kinetic properties of partially purified cytosolic and plastidial G6PDHs determined. Bioinformatic analyses examining co-expression profiles of G6PDHs in Arabidopsis and barley corroborate the data presented. Moreover, the gene coding for the root P2-G6PDH isoform was fully sequenced; the biochemical properties of the corresponding protein were examined experimentally. The results are discussed in the light of the possible distinct roles and regulation of the different G6PDH isoforms during salt stress in barley roots. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  16. Ginger extract mitigates ethanol-induced changes of alpha and beta - myosin heavy chain isoforms gene expression and oxidative stress in the heart of male wistar rats.

    PubMed

    Shirpoor, Alireza; Zerehpoosh, Mitra; Ansari, Mohammad Hasan Khadem; Kheradmand, Fatemeh; Rasmi, Yousef

    2017-09-01

    The association between ethanol consumption and heart abnormalities, such as chamber dilation, myocyte damage, ventricular hypertrophy, and hypertension is well known. However, underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. The aim of this study was to investigate the effect of chronic ethanol exposure on alpha and beta - myosin heavy chain (MHC) isoforms gene expression transition and oxidative stress in rats' heart. It was also planned to find out whether ginger extract mitigated the abnormalities induced by ethanol in rats' heart. Male wistar rats were divided into three groups of eight animals as follows: Control, ethanol, and ginger extract treated ethanolic (GETE) groups. After six weeks of treatment, the results revealed a significant increase in the β-MHC gene expression, 8- OHdG amount, and NADPH oxidase level. Furthermore, a significant decrease in the ratio of α-MHC/β-MHC gene expression to the amount of paraoxonase enzyme in the ethanol group compared to the control group was found. The consumption of Ginger extract along with ethanol ameliorated the changes in MHC isoforms gene expression and reduced the elevated amount of 8-OHdG and NADPH oxidase. Moreover, compared to the consumption of ethanol alone, it increased the paraoxonase level significantly. These findings indicate that ethanol-induced heart abnormalities may in part be associated with MHC isoforms changes mediated by oxidative stress, and that these effects can be alleviated by using ginger extract as an antioxidant molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. NOX isoforms in the development of abdominal aortic aneurysm.

    PubMed

    Siu, Kin Lung; Li, Qiang; Zhang, Yixuan; Guo, Jun; Youn, Ji Youn; Du, Jie; Cai, Hua

    2017-04-01

    Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H 4 B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX

  18. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  19. Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications.

    PubMed

    Yang, Zhang-Min; Yu, Hui; Liu, Zhen-Zhen; Pei, Jian-Zhu; Yang, Yu-E; Yan, Su-Xian; Zhang, Cui; Zhao, Wen-Long; Wang, Zhe-Zhi; Wang, Ying-Ming; Tsai, Inn-Ho

    2017-07-05

    Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. Full sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and

  20. Cardiac Arrest and Therapeutic Hypothermia Decrease Isoform-Specific Cytochrome P450 Drug Metabolism

    PubMed Central

    Zhou, Jiangquan; Empey, Philip E.; Bies, Robert R.; Kochanek, Patrick M.

    2011-01-01

    Mild therapeutic hypothermia is emerging clinically as a neuroprotection therapy for individuals experiencing cardiac arrest (CA); however, its effects combined with disease pathogenesis on drug disposition and response have not been fully elucidated. We determined the activities of four major hepatic-metabolizing enzymes (CYP3A, CYP2C, CYP2D, and CYP2E) during hypothermia after experimental CA in rats by evaluating the pharmacokinetics of their probe drugs as a function of altered body temperature. Animals were randomized into sham normothermia (37.5–38°C), CA normothermia, sham hypothermia (32.5–33°C), and CA hypothermia groups. Probe drugs (midazolam, diclofenac, dextromethorphan, and chlorzoxazone) were given simultaneously by intravenous bolus after temperature stabilization. Multiple blood samples were collected between 0 and 8 h after drug administration. Pharmacokinetic (PK) analysis was conducted using a noncompartmental approach and population PK modeling. Noncompartmental analysis showed that the clearance of midazolam (CYP3A) in CA hypothermia was reduced from sham normothermia rats (681.6 ± 190.0 versus 1268.8 ± 348.9 ml · h−1 · kg−1, p < 0.05). The clearance of chlorzoxazone (CYP2E) in CA hypothermia was also reduced from sham normothermia rats (229.6 ± 75.6 versus 561.89 ± 215.9 ml · h−1 · kg−1, p < 0.05). Population PK analysis further demonstrated the decreased clearance of midazolam (CYP3A) was associated with CA injury (p < 0.05). The decreased clearance of chlorzoxazone (CYP2E1) was also associated with CA injury (p < 0.01). Hypothermia was found to be associated with the decreased volume of distribution of midazolam (V1), dextromethorphan (V1), and peripheral compartment for chlorzoxazone (V2) (p < 0.05, p < 0.05, and p < 0.01, respectively). Our data indicate that hypothermia, CA, and their interaction alter cytochrome P450-isoform specific activities in an isoform-specific manner. PMID:21868471

  1. Myosin heavy chain isoform content and energy metabolism can be uncoupled in pig skeletal muscle.

    PubMed

    Park, S K; Gunawan, A M; Scheffler, T L; Grant, A L; Gerrard, D E

    2009-02-01

    Genetic selection for improved growth and overall meatiness has resulted in the occurrence of 2 major mutations in pigs, the Rendement Napole (RN) and Halothane (Hal) gene mutations. At the tissue level, these mutations influence energy metabolism in skeletal muscle and muscle fiber type composition, yet also influence total body composition. The RN mutation affects the adenosine monophosphate-activated protein kinase gamma subunit and results in increased glycogen deposition in the muscle, whereas the Hal mutation alters sarcoplasmic calcium release mechanisms and results in altered energy metabolism. From a meat quality standpoint, these mutations independently influence the extent and rate of muscle energy metabolism postmortem, respectively. Even though these mutations alter overall muscle energy metabolism and histochemically derived muscle fiber type independently, their effects have not been yet fully elucidated in respect to myosin heavy chain (MyHC) isoform content and those enzymes responsible for defining energetics of the tissue. Therefore, the objective of this study was to determine the collective effects of the RN and Hal genes on genes and gene products associated with different muscle fiber types in pig skeletal muscle. To overcome potential pitfalls associated with traditional muscle fiber typing, real-time PCR, gel electrophoresis, and Western blotting were used to evaluate MyHC composition and several energy-related gene expressions in muscles from wild-type, RN, Hal, and Hal-RN mutant pigs. The MyHC mRNA levels displayed sequential transitions from IIb to IIx and IIa in pigs bearing the RN mutation. In addition, our results showed MyHC protein isoform abundance is correlated with mRNA level supporting the hypothesis that MyHC genes are transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism, including lactate dehydrogenase, citrate synthase, glycogen synthase, and peroxisome proliferator

  2. Targeting of the Nuclear Receptor Coativator Isoform Delta 3aib1 in Breast Cancer. Addendum

    DTIC Science & Technology

    2007-07-01

    using a regulatable AIB1 directed ribozyme , resulted in reduced tumor growth in vivo. Overall, these data indicate a major role for AIB1 and its isoform...regulatable AIB1 directed ribozyme , resulted in reduced tumor growth in vivo. Overall, these data indicate a major role for AIB1 and its isoform ∆3AIB1 in

  3. Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2.

    PubMed

    Peach, Chloe J; Mignone, Viviane W; Arruda, Maria Augusta; Alcobia, Diana C; Hill, Stephen J; Kilpatrick, Laura E; Woolard, Jeanette

    2018-04-23

    Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGF xxx a or VEGF xxx b isoforms. Alternative splicing events at exons 5⁻7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF 165 a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology.

  4. Comprehensive Analysis of Tropomyosin Isoforms in Skeletal Muscles by Top-down Proteomics

    PubMed Central

    Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A.; Larsson, Lars; Ge, Ying

    2016-01-01

    Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases. PMID:27090236

  5. Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics.

    PubMed

    Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A; Larsson, Lars; Ge, Ying

    2016-04-01

    Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases.

  6. Differential expression and localization of Ankrd2 isoforms in human skeletal and cardiac muscles.

    PubMed

    Jasnic-Savovic, Jovana; Krause, Sabine; Savic, Slobodan; Kojic, Ana; Kovcic, Vlado; Boskovic, Srdjan; Nestorovic, Aleksandra; Rakicevic, Ljiljana; Schreiber-Katz, Olivia; Vogel, Johannes G; Schoser, Benedikt G; Walter, Maggie C; Valle, Giorgio; Radojkovic, Dragica; Faulkner, Georgine; Kojic, Snezana

    2016-11-01

    Four human Ankrd2 transcripts, reported in the Ensembl database, code for distinct protein isoforms (360, 333, 327 and 300 aa), and so far, their existence, specific expression and localization patterns have not been studied in detail. Ankrd2 is preferentially expressed in the slow fibers of skeletal muscle. It is found in both the nuclei and the cytoplasm of skeletal muscle cells, and its localization is prone to change during differentiation and upon stress. Ankrd2 has also been detected in the heart, in ventricular cardiomyocytes and in the intercalated disks (ICDs). The main objective of this study was to distinguish between the Ankrd2 isoforms and to determine the contribution of each one to the general profile of Ankrd2 expression in striated muscles. We demonstrated that the known expression and localization pattern of Ankrd2 in striated muscle can be attributed to the isoform of 333 aa which is dominant in both tissues, while the designated cardiac and canonical isoform of 360 aa was less expressed in both tissues. The 360 aa isoform has a distinct nuclear localization in human skeletal muscle, as well as in primary myoblasts and myotubes. In contrast to the isoform of 333 aa, it was not preferentially expressed in slow fibers and not localized to the ICDs of human cardiomyocytes. Regulation of the expression of both isoforms is achieved at the transcriptional level. Our results set the stage for investigation of the specific functions and interactions of the Ankrd2 isoforms in healthy and diseased human striated muscles.

  7. Top-down Targeted Proteomics for Deep Sequencing of Tropomyosin Isoforms

    PubMed Central

    Peng, Ying; Chen, Xin; Zhang, Han; Xu, Qingge; Hacker, Timothy A.; Ge, Ying

    2013-01-01

    SUMMARY Tropomyosins (Tm) constitute a family of ubiquitous and highly conserved actin-binding proteins, playing essential roles in a variety of biological processes. Tm isoforms produced by multiple Tm encoding genes and alternatively expressed exons along with post-translational modifications (PTMs) regulate Tm function. Therefore, to gain a better understanding of the functional role of Tm, it is essential to fully characterize Tm isoforms. Herein, we developed a top-down high-resolution mass spectrometry (MS) based targeted proteomics method for comprehensive characterization of Tm isoforms. α–Tm was identified to be the predominant isoform in swine cardiac muscle. We further characterized its sequence and localized the PTMs such as acetylation and phosphorylation as well as amino acid polymorphisms. Interestingly, we discovered a “novel” Tm isoform that does not match with any of the currently available swine Tm sequences. A deep sequencing of this isoform by top-down MS revealed an exact match with mouse β–Tm sequence, suggesting that this “novel” isoform is swine β–Tm which is 100% conserved between swine and mouse. Taken together, we demonstrated that top-down targeted proteomics provides a powerful tool for deep sequencing of Tm isoforms from genetic variations together with complete mapping of the PTM sites. PMID:23256820

  8. Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium

    PubMed Central

    Wang, Lu; Harris, Sean M.; Espinoza, Herbert M.; McClain, Valerie; Gallagher, Evan P.

    2013-01-01

    Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival, and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active-sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7 ppb) and high (347 ppb) environmental Cd concentrations for 24–48 hrs significantly decreased gpx4a

  9. Human adenylosuccinate lyase (ADSL), cloning and characterization of full-length cDNA and its isoform, gene structure and molecular basis for ADSL deficiency in six patients.

    PubMed

    Kmoch, S; Hartmannová, H; Stibůrková, B; Krijt, J; Zikánová, M; Sebesta, I

    2000-06-12

    Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.

  10. Profiling the orphan enzymes.

    PubMed

    Sorokina, Maria; Stam, Mark; Médigue, Claudine; Lespinet, Olivier; Vallenet, David

    2014-06-06

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

  11. Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in Grewia asiatica L

    PubMed Central

    Wani, Tareq A.; Pandith, Shahzad A.; Gupta, Ajai P.; Chandra, Suresh; Sharma, Namrata

    2017-01-01

    Chalcone synthase constitutes a functionally diverse gene family producing wide range of flavonoids by catalyzing the initial step of the phenylpropanoid pathway. There is a pivotal role of flavonoids in pollen function as they are imperative for pollen maturation and pollen tube growth during sexual reproduction in flowering plants. Here we focused on medicinally important fruit-bearing shrub Grewia asiatica. It is a rich repository of flavonoids. The fruits are highly acclaimed for various putative health benefits. Despite its importance, full commercial exploitation is hampered due to two drawbacks which include short shelf life of its fruits and larger seed volume. To circumvent these constraints, seed abortion is one of the viable options. Molecular interventions tested in a number of economic crops have been to impair male reproductive function by disrupting the chalcone synthase (CHS) gene activity. Against this backdrop the aim of the present study included cloning and characterization of two full-length cDNA clones of GaCHS isoforms from the CHS multigene family. These included GaCHS1 (NCBI acc. KX129910) and GaCHS2 (NCBI acc. KX129911) with an ORF of 1176 and 1170 bp, respectively. GaCHSs were heterologously expressed and purified in E. coli to validate their functionality. Functionality of CHS isoforms was also characterized via enzyme kinetic studies using five different substrates. We observed differential substrate specificities in terms of their Km and Vmax values. Accumulation of flavonoid constituents naringenin and quercetin were also quantified and their relative concentrations corroborated well with the expression levels of GaCHSs. Further, our results demonstrate that GaCHS isoforms show differential expression patterns at different reproductive phenological stages. Transcript levels of GaCHS2 were more than its isoform GaCHS1 at the anthesis stage of flower development pointing towards its probable role in male reproductive maturity. PMID

  12. Molecular Dynamics Simulations and Structural Analysis to Decipher Functional Impact of a Twenty Residue Insert in the Ternary Complex of Mus musculus TdT Isoform

    PubMed Central

    Mutt, Eshita; Sowdhamini, Ramanathan

    2016-01-01

    Insertions/deletions are common evolutionary tools employed to alter the structural and functional repertoire of protein domains. An insert situated proximal to the active site or ligand binding site frequently impacts protein function; however, the effect of distal indels on protein activity and/or stability are often not studied. In this paper, we have investigated a distal insert, which influences the function and stability of a unique DNA polymerase, called terminal deoxynucleotidyl transferase (TdT). TdT (EC:2.7.7.31) is a monomeric 58 kDa protein belonging to family X of eukaryotic DNA polymerases and known for its role in V(D)J recombination as well as in non-homologous end-joining (NHEJ) pathways. Two murine isoforms of TdT, with a length difference of twenty residues and having different biochemical properties, have been studied. All-atom molecular dynamics simulations at different temperatures and interaction network analyses were performed on the short and long-length isoforms. We observed conformational changes in the regions distal to the insert position (thumb subdomain) in the longer isoform, which indirectly affects the activity and stability of the enzyme through a mediating loop (Loop1). A structural rationale could be provided to explain the reduced polymerization rate as well as increased thermosensitivity of the longer isoform caused by peripherally located length variations within a DNA polymerase. These observations increase our understanding of the roles of length variants in introducing functional diversity in protein families in general. PMID:27311013

  13. Cloning and characterization of the neural isoforms of human dystonin

    SciTech Connect

    Brown, A.; Dalpe, G.; Mathieu, M.

    1995-10-10

    Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5{prime} exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequencesmore » of the neural-specific exons in the mouse and human genes are over 96% identical. 17 refs., 2 figs.« less

  14. Protein kinase C isoforms in neutrophil adhesion and activation.

    PubMed

    Bertram, Anna; Ley, Klaus

    2011-04-01

    Neutrophils are the first line of defense against bacterial and mycotic pathogens. In order to reach the pathogens, neutrophils need to transmigrate through the vascular endothelium and migrate to the site of infection. Defense strategies against pathogens include phagocytosis, production and release of oxygen radicals through the oxidative burst, and degranulation of antimicrobial and inflammatory molecules. Protein kinase C (PKC)-δ is required for full assembly of NADPH oxidase and activation of the respiratory burst. Neutrophils also express PKC-α and -β, which may be involved in adhesion, degranulation and phagocytosis, but the evidence is not conclusive yet. This review focuses on the potential impact of protein kinase C isoforms on neutrophil adhesion and activation.

  15. Progesterone Receptors, their Isoforms and Progesterone Regulated Transcription

    PubMed Central

    Jacobsen, Britta M.; Horwitz, Kathryn B.

    2011-01-01

    This review discusses mechanisms by which progesterone receptors (PR) regulate transcription. We examine available data in different species and tissues regarding: 1) regulation of PR levels; and 2) expression profiling of progestin-regulated genes by total PRs, or their PRA and PRB isoforms. 3) We address current views about the composition of progesterone response elements, and postulate that PR monomers acting through “half-site” elements are common, entailing cooperativity with neighboring DNA-bound transcription factors. 4) We summarize transcription data for multiple progestin-regulated promoters as directed by total PR, or PRA vs. PRB. We conclude that current models and methods used to study PR function are problematical, and recommend that future work employ cells and receptors appropriate to the species, focusing on analyses of the effects of endogenous receptors targeting endogenous genes in native chromatin. PMID:21952082

  16. Interactions of different carrageenan isoforms and flour components in breadmaking.

    PubMed

    León, A E; Ribotta, P D; Ausar, S F; Fernández, C; Lanada, C A; Beltramo, D M

    2000-07-01

    The aim of this study was to compare the effects of carrageenans with different sulfate contents on bread volume and dough rheological properties. Results showed that only lambda carrageenan, the most sulfated isoform, produced a significant increase in bread volume. In contrast, the different carrageenans induced a negative effect on the cookie factor. Alveographic and farinographic analyses indicated that dough rheological properties were differentially modified depending on whether lambda carrageenan was added to flour and then hydrated or vice versa. Analysis of the interaction between lambda carrageenan and flour components by infrared spectroscopy and SDS-PAGE indicated that a pool of low molecular weight hydrophobic gluten proteins interact with carrageenan. This interaction drastically changes their physicochemical properties since carrageenan-gluten protein complexes show a hydrophilic behavior. In addition, the results indicate that carrageenan sulfate groups and probably the amino groups of glutamines present in the primary structure of gluten proteins are involved in the interaction.

  17. Identification of a novel splicing isoform of murine CGI-58

    PubMed Central

    Yang, Xingyuan; Lu, Xin; Liu, Jun

    2010-01-01

    The CGI-58 gene, mutations of which are linked to Chanarin-Dorfman syndrome, encodes a protein of the α/β hydrolase domain subfamily. We report here a new alternative splicing isoform of the murine CGI-58 gene, termed mCGI-58S. Sequence comparison indicates the lack of second and third exons in this cDNA variant. While the full length protein displayed perilipin-dependent localization to lipid droplets, mCGI-58S showed a predominant cytoplasmic staining when expressed in cells. mCGI-58S was incapable of activating ATGL but retained the capacity to acylate lysophosphatidic acid. Overexpression of mCGI-58S failed to promote lipid droplet turnover and loss of intracellular triacylglycerols. These results suggest that this splicing event may be involved in the regulation of lipid homeostasis. PMID:20083112

  18. 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

  19. Extracellular regulation of VEGF: isoforms, proteolysis, and vascular patterning

    PubMed Central

    Vempati, Prakash; Popel, Aleksander S.; Mac Gabhann, Feilim

    2014-01-01

    The regulation of vascular endothelial growth factor A (VEGF) is critical to neovascularization in numerous tissues under physiological and pathological conditions. VEGF has multiple isoforms, created by alternative splicing or proteolytic cleavage, and characterized by different receptor-binding and matrix-binding properties. These isoforms are known to give rise to a spectrum of angiogenesis patterns marked by differences in branching, which has functional implications for tissues. In this review, we detail the extensive extracellular regulation of VEGF and the ability of VEGF to dictate the vascular phenotype. We explore the role of VEGF-releasing proteases and soluble carrier molecules on VEGF activity. While proteases such as MMP9 can ‘release’ matrix-bound VEGF and promote angiogenesis, for example as a key step in carcinogenesis, proteases can also suppress VEGF’s angiogenic effects. We explore what dictates pro- or anti-angiogenic behavior. We also seek to understand the phenomenon of VEGF gradient formation. Strong VEGF gradients are thought to be due to decreased rates of diffusion from reversible matrix binding, however theoretical studies show that this scenario cannot give rise to lasting VEGF gradients in vivo. We propose that gradients are formed through degradation of sequestered VEGF. Finally, we review how different aspects of the VEGF signal, such as its concentration, gradient, matrix-binding, and NRP1-binding can differentially affect angiogenesis. We explore how this allows VEGF to regulate the formation of vascular networks across a spectrum of high to low branching densities, and from normal to pathological angiogenesis. A better understanding of the control of angiogenesis is necessary to improve upon limitations of current angiogenic therapies. PMID:24332926

  20. 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.

  1. Marker-assisted selection for elevated concentrations of the a' subunit of B-conglycinin and its influence on agronomic and seed traits of soybean

    USDA-ARS?s Scientific Manuscript database

    Soybean [Glycine max (L.) Merr.] cultivars with elevated concentrations of the a' subunit of ß-conglycinin (BC) may provide health benefits to soy protein consumers. Two Monsanto single nucleotide polymorphism markers were used to classify F2 plants in four segregating populations as having elevate...

  2. Enzymes for improved biomass conversion

    DOEpatents

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  3. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  4. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  5. Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

    PubMed Central

    Zoch, Ansgar; Mayerl, Steffen; Schulz, Alexander; Greither, Thomas; Frappart, Lucien; Rübsam, Juliane; Heuer, Heike; Giovannini, Marco; Morrison, Helen

    2015-01-01

    The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2. PMID:26258444

  6. Vacuolar-type H+-ATPase V1A subunit is a molecular partner of Wolfram syndrome 1 (WFS1) protein, which regulates its expression and stability.

    PubMed

    Gharanei, Seley; Zatyka, Malgorzata; Astuti, Dewi; Fenton, Janine; Sik, Attila; Nagy, Zsuzsanna; Barrett, Timothy G

    2013-01-15

    Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that also localizes to secretory granules in pancreatic beta cells. Although its precise functions are unknown, WFS1 protein deficiency affects the unfolded protein response, intracellular ion homeostasis, cell cycle progression and granular acidification. In this study, immunofluorescent and electron-microscopy analyses confirmed that WFS1 also localizes to secretory granules in human neuroblastoma cells. We demonstrated a novel interaction between WFS1 and the V1A subunit of the H(+) V-ATPase (proton pump) by co-immunoprecipitation in human embryonic kidney (HEK) 293 cells and with endogenous proteins in human neuroblastoma cells. We mapped the interaction to the WFS1-N terminal, but not the C-terminal domain. V1A subunit expression was reduced in WFS1 stably and transiently depleted human neuroblastoma cells and depleted NT2 (human neuron-committed teratocarcinoma) cells. This reduced expression was not restored by adenoviral overexpression of BiP (immunoglobulin-binding protein) to correct the ER stress. Protein stability assays demonstrated that the V1A subunit was degraded more rapidly in WFS1 depleted neuroblastoma cells compared with wild-type; however, proteosomal inhibition did not restore the expression of the V1A subunit. Cell cycle assays measuring p21(cip) showed reduced levels in WFS1 depleted cells, and an inverse association between p21(cip) expression and apoptosis. We conclude that WFS1 has a specific interaction with the V1A subunit of H(+) ATPase; this interaction may be important both for pump assembly in the ER and for granular acidification.

  7. Profiling the orphan enzymes

    PubMed Central

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  8. Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo.

    PubMed

    Okegawa, Yuki; Motohashi, Ken

    2015-12-01

    Thioredoxins (Trxs) regulate the activity of various chloroplastic proteins in a light-dependent manner. Five types of Trxs function in different physiological processes in the chloroplast of Arabidopsis thaliana. Previous in vitro experiments have suggested that the f-type Trx (Trx f) is the main redox regulator of chloroplast enzymes, including Calvin cycle enzymes. To investigate the in vivo contribution of each Trx isoform to the redox regulatory system, we first quantified the protein concentration of each Trx isoform in the chloroplast stroma. The m-type Trx (Trx m), which consists of four isoforms, was the most abundant type. Next, we analyzed several Arabidopsis Trx-m-deficient mutants to elucidate the physiological role of Trx m in vivo. Deficiency of Trx m impaired plant growth and decreased the CO2 assimilation rate. We also determined the redox state of Trx target enzymes to examine their photo-reduction, which is essential for enzyme activation. In the Trx-m-deficient mutants, the reduction level of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase was lower than that in the wild type. Inconsistently with the historical view, our in vivo study suggested that Trx m plays a more important role than Trx f in the activation of Calvin cycle enzymes. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  9. Enzymes from extremophiles.

    PubMed

    Demirjian, D C; Morís-Varas, F; Cassidy, C S

    2001-04-01

    The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.

  10. Identification and evolutionary analysis of tissue-specific isoforms of mitochondrial complex I subunit NDUFV3.

    PubMed

    Guerrero-Castillo, Sergio; Cabrera-Orefice, Alfredo; Huynen, Martijn A; Arnold, Susanne

    2017-03-01

    Mitochondrial complex I is the largest respiratory chain complex. Despite the enormous progress made studying its structure and function in recent years, potential regulatory roles of its accessory subunits remained largely unresolved. Complex I gene NDUFV3, which occurs in metazoa, contains an extra exon that is only present in vertebrates and thereby evolutionary even younger than the rest of the gene. Alternative splicing of this extra exon gives rise to a short NDUFV3-S and a long NDUFV3-L protein isoform. Complexome profiling revealed that the two NDUFV3 isoforms are constituents of the multi-subunit complex I. Further mass spectrometric analyses of complex I from different murine and bovine tissues showed a tissue-specific expression pattern of NDUFV3-S and NDUFV3-L. Hence, NDUFV3-S was identified as the only isoform in heart and skeletal muscle, whereas in liver, brain, and lung NDUFV3-L was expressed as the dominant isoform, together with NDUFV3-S present in all tissues analyzed. Thus, we identified NDUFV3 as the first out of 30 accessory subunits of complex I present in vertebrate- and tissue-specific isoforms. Interestingly, the tissue-specific expression pattern of NDUFV3-S and NDUFV3-L isoforms was paralleled by changes in kinetic parameters, especially the substrate affinity of complex I. This may indicate a regulatory role of the NDUFV3 isoforms in different vertebrate tissues. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Regulation of Cardiac Remodeling by Cardiac Na+/K+-ATPase Isoforms

    PubMed Central

    Liu, Lijun; Wu, Jian; Kennedy, David J.

    2016-01-01

    Cardiac remodeling occurs after cardiac pressure/volume overload or myocardial injury during the development of heart failure and is a determinant of heart failure. Preventing or reversing remodeling is a goal of heart failure therapy. Human cardiomyocyte Na+/K+-ATPase has multiple α isoforms (1–3). The expression of the α subunit of the Na+/K+-ATPase is often altered in hypertrophic and failing hearts. The mechanisms are unclear. There are limited data from human cardiomyocytes. Abundant evidences from rodents show that Na+/K+-ATPase regulates cardiac contractility, cell signaling, hypertrophy and fibrosis. The α1 isoform of the Na+/K+-ATPase is the ubiquitous isoform and possesses both pumping and signaling functions. The α2 isoform of the Na+/K+-ATPase regulates intracellular Ca2+ signaling, contractility and pathological hypertrophy. The α3 isoform of the Na+/K+-ATPase may also be a target for cardiac hypertrophy. Restoration of cardiac Na+/K+-ATPase expression may be an effective approach for prevention of cardiac remodeling. In this article, we will overview: (1) the distribution and function of isoform specific Na+/K+-ATPase in the cardiomyocytes. (2) the role of cardiac Na+/K+-ATPase in the regulation of cell signaling, contractility, cardiac hypertrophy and fibrosis in vitro and in vivo. Selective targeting of cardiac Na+/K+-ATPase isoform may offer a new target for the prevention of cardiac remodeling. PMID:27667975

  12. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    PubMed Central

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  13. High Molecular Weight Isoforms of Growth Hormone In Cells of the Immune System

    PubMed Central

    Weigent, Douglas A.

    2013-01-01

    A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100 kDa, 65 kDa, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 kDa and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm whereas the lower molecular weight 65 kDa and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost three-fold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress. PMID:21741628

  14. Inulin isoforms differ by repeated additions of one crystal unit cell

    PubMed Central

    Cooper, Peter D.; Barclay, Thomas G.; Ginic-Markovic, Milena; Gerson, Andrea R.; Petrovsky, Nikolai

    2014-01-01

    Inulin isoforms, especially delta inulin, are important biologically as immune activators and clinically as vaccine adjuvants. In exploring action mechanisms, we previously found regular increments in thermal properties of the seven-member inulin isoform series that suggested regular additions of some energetic structural unit. Because the previous isolates carried additional longer chains that masked defining ranges, these were contrasted with new isoform isolates comprising only inulin chain lengths defining that isoform. The new series began with 19 fructose units per chain (alpha-1 inulin), increasing regularly by 6 fructose units per isoform. Thus the ‘energetic unit’ equates to 6 fructose residues per chain. All isoforms showed indistinguishable X-ray diffraction patterns that were also identical with known inulin crystals. We conclude that an ‘energetic unit’ equates to one helix turn of 6 fructose units per chain as found in one unit cell of the inulin crystal. Each isoform chain comprised progressively more helix turns plus one additional fructose and glucose residues per chain. PMID:24528745

  15. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

    PubMed

    Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2016-05-15

    Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. © 2016. Published by The Company of Biologists Ltd.

  16. Biophysical, histopathological and pharmacological characterization of crotamine isoforms F22 and F32.

    PubMed

    Toyama, Marcos H; Marangoni, Sérgio; Novello, José C; Leite, Gildo B; Prado-Franceschi, Julia; da Cruz-Höfling, Maria Alice; Rodrigues-Simioni, Léa

    2003-03-01

    Two major crotamine isoforms (F22 and F32) were obtained after three chromatographic steps and were assayed in mouse phrenic nerve-diaphragm preparations. F32 and F22 (0.5 microg/ml, n=4) produced a facilitatory effect, which increased isometric twitch-tension by 300 and 230%, respectively, after a 120 min incubation. At a concentration of 0.1 microg/ml, both isoforms increased the twitch-tension by about 160%. However, when the isoforms were co-incubated (final concentration, 0.5 microg/ml) for 30 min prior to testing, they did not cause the facilitation seen with > or =0.1 microg/ml of each isoform alone. Histologically, F32 and F22 at 0.5 and 1 microg/ml were quantitatively alike in inducing tissue myonecrosis. However, a mixture of the two isoforms (final concentration, 0.5 microg/ml) significantly attenuated the damage seen with either toxin alone. Mass spectrometry analysis showed that the isoforms had the same molecular mass (4.8 kDa) and that they existed as monomers with a highly stable structure. These results indicate that F22 and F32 acted on muscle cells of the mouse phrenic-nerve diaphragm preparation through similar mechanisms. Since the isoforms did not produce the expected summation in the increase in muscle twitch-tension, it is possible that they may have different affinities for the sodium channel subunits.

  17. The role of isoforms in the evolution of cryptic coloration in Peromyscus mice.

    PubMed

    Mallarino, Ricardo; Linden, Tess A; Linnen, Catherine R; Hoekstra, Hopi E

    2017-01-01

    A central goal of evolutionary biology is to understand the molecular mechanisms underlying phenotypic adaptation. While the contribution of protein-coding and cis-regulatory mutations to adaptive traits has been well documented, additional sources of variation - such as the production of alternative RNA transcripts from a single gene, or isoforms - have been understudied. Here, we focus on the pigmentation gene Agouti, known to express multiple alternative transcripts, to investigate the role of isoform usage in the evolution of cryptic colour phenotypes in deer mice (genus Peromyscus). We first characterize the Agouti isoforms expressed in the Peromyscus skin and find two novel isoforms not previously identified in Mus. Next, we show that a locally adapted light-coloured population of P. maniculatus living on the Nebraska Sand Hills shows an upregulation of a single Agouti isoform, termed 1C, compared with their ancestral dark-coloured conspecifics. Using in vitro assays, we show that this preference for isoform 1C may be driven by isoform-specific differences in translation. In addition, using an admixed population of wild-caught mice, we find that variation in overall Agouti expression maps to a region near exon 1C, which also has patterns of nucleotide variation consistent with strong positive selection. Finally, we show that the independent evolution of cryptic light pigmentation in a different species, P. polionotus, has been driven by a preference for the same Agouti isoform. Together, these findings present an example of the role of alternative transcript processing in adaptation and demonstrate molecular convergence at the level of isoform regulation. © 2016 John Wiley & Sons Ltd.

  18. PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation

    PubMed Central

    Hands, Katherine J.; Cuchet-Lourenco, Delphine; Everett, Roger D.; Hay, Ronald T.

    2014-01-01

    SUMMARY Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment. PMID:24190887

  19. The polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions

    PubMed Central

    Cooper, Peter D; Barclay, Thomas G; Ginic-Markovic, Milena; Petrovsky, Nikolai

    2013-01-01

    In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous → alpha-1 (AI-1) → alpha-2 (AI-2) → gamma (GI) → delta (DI) → zeta (ZI) → epsilon (EI) → omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) “melting” or “freezing” points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators. PMID:23853206

  20. Myosin isoform fiber type and fiber size in the tail of the Virginia opossum (Didelphis virginiana).

    PubMed

    Hazimihalis, P J; Gorvet, M A; Butcher, M T

    2013-01-01

    Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. Copyright © 2012 Wiley Periodicals, Inc.

  1. Detection of VEGF-A(xxx)b isoforms in human tissues.

    PubMed

    Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

  2. Urinary human chorionic gonadotropin isoform concentrations in doping control samples.

    PubMed

    Butch, Anthony W; Woldemariam, Getachew A

    2016-11-01

    Anti-doping laboratories routinely use immunoassays to measure urinary concentrations of human chorionic gonadotropin (hCG). To minimize immunoassay differences and false positive screen results from inactive isoforms (free β-subunit (hCGβ), β-subunit core fragment (hCGβcf)) laboratories now use intact hCG instead of total hCG immunoassays to measure hCG. To determine the distribution of hCG isoforms in urine, we determined the concentrations of intact hCG, hCGβ, and hCGβcf in male urine samples based on immunoassay total hCG concentrations using a sequential immunoextraction and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. hCG was isolated using antibody-conjugated magnetic beads and unique tryptic peptides were quantified by LC-MS/MS. Negative samples with detectable but low total hCG concentrations (1.2-3.5 pmol/L) had intact and hCGβ concentrations <1.2 pmol/L, and hCGβcf concentrations <2.3 pmol/L by LC-MS/MS. Urine samples from an athlete receiving hCG had intact hCG concentrations ranging from 18.8 to 57.6 pmol/L, hCGβ concentrations <0.7 pmol/L, and hCGβcf concentrations ranging from 94 to 243% of the intact hCG concentration. In 27 atypical samples with total hCG concentrations ranging from 16.7 to 412.7 pmol/L with intact hCG <2.7 pmol/L by immunoassay, all samples had intact hCG concentrations <3.8 pmol/L and hCGβ concentrations <6.2 pmol/L by LC-MS/MS. hCGβcf concentrations by LC-MS/MS varied widely and ranged from 1.03 to 21.9 pmol/L. In summary, total hCG immunoassays significantly overestimate hCG concentrations and can produce false positive results. Although the intact hCG immunoassay slightly overestimates hCG concentrations compared to LC-MS/MS, it can distinguish between cases of hCG use and atypical cases with elevated total hCG concentrations. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. [Selective regulation of laccase isoform production by the Lentinus strigosus 1566 fungus].

    PubMed

    Myasoedova, N M; Gasanov, N B; Chernykh, A M; Kolomytseva, M P; Golovleva, L A

    2015-01-01

    The effects of a number of culture medium components, such as peptone, yeast extract, mono- and disaccharides, copper ions, 2,6-dimethylphenol, and polycaproamide fiber, on the laccase activity dynamics in the culture liquid and laccase isoform production by the Lentinus strigosus 1566 fungus were studied. It was demonstrated that some saccharides selectively induced or inhibited the synthesis of different laccase isoforms. Similar action was exerted by copper ions, 2,6-dimethylphenol, and polycaproamide fiber, as well as by their combination. Selective in vivo regulation of the production of certain laccase isoforms by basidial fungi by means of altering the culturing medium composition can be utilised for various biotechnological purposes.

  4. Protocol for high-resolution electrophoresis separation of myosin heavy chain isoforms in bovine skeletal muscle.

    PubMed

    Picard, Brigitte; Barboiron, Christiane; Chadeyron, David; Jurie, Catherine

    2011-07-01

    In this short communication we describe a specific protocol for SDS-PAGE separation of adult bovine myosin heavy-chain (MyHC) isoforms. The conditions defined in this protocol allow a good separation with a good reproducibility of the four MyHC isoforms (MyHC I, IIa, IIx, IIb) identified in adult skeletal muscle of this species. This procedure uses mini-gel electrophoresis system and does not involve preparation of gradient separating gels. In addition, this protocol can also be applied to the electrophoretic separation of ovine and camel MyHC isoforms. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Seven novel mutations in the factor XIII A-subunit gene causing hereditary factor XIII deficiency in 10 unrelated families.

    PubMed

    Vysokovsky, A; Saxena, R; Landau, M; Zivelin, A; Eskaraev, R; Rosenberg, N; Seligsohn, U; Inbal, A

    2004-10-01

    Hereditary factor (F)XIII deficiency is a rare bleeding disorder mostly due to mutations in FXIII A subunit. We studied the molecular basis of FXIII deficiency in patients from 10 unrelated families originating from Israel, India and Tunisia. Exons 2-15 of genomic DNA consisting of coding regions and intron/exon boundaries were amplified and sequenced. Structural analysis of the mutations was undertaken by computer modeling. Seven novel mutations were identified in the FXIIIA gene. The propositus from the Ethiopian-Jewish family was found to be a compound heterozygote for two novel mutations: a 10-bp deletion in exon 12 at nucleotides 1652-1661 (followed by 22 altered amino acids and termination codon) and Ala318Val mutation. The propositus of the Tunisian family was homozygous for C insertion after nucleotide 863 within a stretch of six cytosines of exon 7. This insertion results in generation of eight altered amino acids followed by a termination codon downstream. The propositus from Indian-Jewish origin was found to be homozygous for G to T substitution at IVS 11 [+1] resulting in skipping of exons 10 and 11. In addition to the Ala318Val mutation, three of the novel mutations identified are missense mutations: Arg260Leu, Thr398Asn and Gly210Arg each occurring in a homozygous state in an Israeli-Arab and two Indian families, respectively. Structure-function correlation analysis by computer modeling of the new missense mutations predicted that Gly210Arg will cause protein misfolding, Ala318Val and Thr398Asn will interfere with the catalytic process or protein stability, and Arg260Leu will impair dimerization.

  6. Development of a subunit vaccine containing recombinant chicken anemia virus VP1 and pigeon IFN-γ.

    PubMed

    Shen, Sin Ying; Chang, Wei Chun; Yi, Hsiang Heng; Tsai, Shinn-Shong; Liu, Hung Jen; Liao, Pei-Chun; Chuang, Kuo Pin

    2015-10-15

    Chicken anemia virus (CAV) is a severe threat to the chicken industry and causes heavy economic losses worldwide. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant VP1 (rVP1) and pigeon interferon-γ (rPiIFN-γ). Results indicated that rPiIFN-γ enhanced humoral immunity elicited by rVP1 as early as 10 day after primary immunization and reach the high titer after secondary immunization. When compared to chickens immunized with rVP1, inactivated vaccine, chickens immunized with rVP1+rPiIFN-γ showed faster and higher levels (p<0.05) of antibody titer. The CAV challenge result showed that the rVP1+rPiIFN-γ vaccine prevent the reducing of hematocrit values in comparison with the rVP1 or inactivated groups. The relative fold inductions of mRNA expression of Th1-type (IFN-γ), but not Th2-type (IL-4) cytokines in splenocytes isolated from chickens immunized with rVP1+rPiIFN-γ were significantly higher than those of the rVP1 or inactivated vaccine groups. In conclusion, our study found that rPiIFN-γ can enhance both humoral and cellular immunity elicited by an rVP1 vaccine. The rVP1+rPiIFN-γ vaccine may provide a new strategy vaccine against CAV in chicken. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Biallelic Mutations in ATP5F1D , which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder

    DOE PAGES

    Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle; ...

    2018-02-22

    ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1F O ATP synthase andmore » subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.« less

  8. Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

    PubMed

    Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle; Jangam, Sharayu; Fernandez, Liliana; Davidson, Jean M; Kyle, Jennifer E; Grove, Megan E; Fisk, Dianna G; Kohler, Jennefer N; Holmes, Matthew; Dries, Annika M; Huang, Yong; Zhao, Chunli; Contrepois, Kévin; Zappala, Zachary; Frésard, Laure; Waggott, Daryl; Zink, Erika M; Kim, Young-Mo; Heyman, Heino M; Stratton, Kelly G; Webb-Robertson, Bobbie-Jo M; Snyder, Michael; Merker, Jason D; Montgomery, Stephen B; Fisher, Paul G; Feichtinger, René G; Mayr, Johannes A; Hall, Julie; Barbosa, Ines A; Simpson, Michael A; Deshpande, Charu; Waters, Katrina M; Koeller, David M; Metz, Thomas O; Morris, Andrew A; Schelley, Susan; Cowan, Tina; Friederich, Marisa W; McFarland, Robert; Van Hove, Johan L K; Enns, Gregory M; Yamamoto, Shinya; Ashley, Euan A; Wangler, Michael F; Taylor, Robert W; Bellen, Hugo J; Bernstein, Jonathan A; Wheeler, Matthew T

    2018-03-01

    ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1 F O ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae.

    PubMed

    Cura, Carolina I; Corradi, Gerardo R; Rinaldi, Débora E; Adamo, Hugo P

    2008-12-01

    The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.

  10. Co-ordinate activation of lipogenic enzymes in hepatocellular carcinoma.

    PubMed

    Yahagi, Naoya; Shimano, Hitoshi; Hasegawa, Kiyoshi; Ohashi, Kenichi; Matsuzaka, Takashi; Najima, Yuho; Sekiya, Motohiro; Tomita, Sachiko; Okazaki, Hiroaki; Tamura, Yoshiaki; Iizuka, Yoko; Ohashi, Ken; Nagai, Ryozo; Ishibashi, Shun; Kadowaki, Takashi; Makuuchi, Masatoshi; Ohnishi, Shin; Osuga, Jun-ichi; Yamada, Nobuhiro

    2005-06-01

    Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or C are prevalent. Small hepatocellular carcinoma lesions detected by ultrasonography at an early stage are often hyperechoic because they are composed of well-differentiated cancer cells that are rich in triglyceride droplets. The triglyceride content of hepatocytes depends in part on the rate of lipogenesis. Key lipogenic enzymes, such as fatty acid synthase, are co-ordinately regulated at the transcriptional level. We therefore examined the mRNA expression of lipogenic enzymes in human hepatocellular carcinoma samples from 10 patients who had undergone surgical resection. All of the samples exhibited marked elevation of expression of mRNA for lipogenic enzymes, such as fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase, compared with surrounding non-cancerous liver tissue. In contrast, the changes in mRNA expression of SREBP-1, a transcription factor that regulates a battery of lipogenic enzymes, did not show a consistent trend. In some cases where SREBP-1 was elevated, the main contributing isoform was SREBP-1c rather than SREBP-1a. Thus, lipogenic enzymes are markedly induced in hepatocellular carcinomas, and in some cases SREBP-1c is involved in this activation.

  11. Annotation of Alternatively Spliced Proteins and Transcripts with Protein-Folding Algorithms and Isoform-Level Functional Networks.

    PubMed

    Li, Hongdong; Zhang, Yang; Guan, Yuanfang; Menon, Rajasree; Omenn, Gilbert S

    2017-01-01

    Tens of thousands of splice isoforms of proteins have been catalogued as predicted sequences from transcripts in humans and other species. Relatively few have been characterized biochemically or structurally. With the extensive development of protein bioinformatics, the characterization and modeling of isoform features, isoform functions, and isoform-level networks have advanced notably. Here we present applications of the I-TASSER family of algorithms for folding and functional predictions and the IsoFunc, MIsoMine, and Hisonet data resources for isoform-level analyses of network and pathway-based functional predictions and protein-protein interactions. Hopefully, predictions and insights from protein bioinformatics will stimulate many experimental validation studies.

  12. Enzymes in Fermented Fish.

    PubMed

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  13. Enzyme electrochemcial sensor electrode

    SciTech Connect

    Rishpon, J.; Zawodzinski, T.A.; Gottesfeld, S.

    1989-12-11

    An electrochemical sensor electrode is formed from an electronic conductor coated with a casting solution containing a perfluorosulfonic acid ionomer and a selected enzyme. The selected enzyme catalyzes a reaction between a predetermined substance in a solution and oxygen to form an electrochemically active compound that is detected at the electronic conductor. The resulting perfluorosulfonic acid polymer provides a stable matrix for the enzyme for long lived enzyme activity, wherein only thin coatings are required on the metal conductor. The polymer also advantageously repels interfering substances from contacting the enzyme and contains quantities of oxygen to maintain a sensing capabilitymore » during conditions of oxygen depletion in the sample. In one particular embodiment, glucose oxidase is mixed with the perfluorosulfonic acid ionomer to form an electrode for glucose detection. 3 figs.« less

  14. Industrial Enzymes and Biocatalysis

    NASA Astrophysics Data System (ADS)

    McAuliffe, Joseph C.; Aehle, Wolfgang; Whited, Gregory M.; Ward, Donald E.

    All life processes are the result of enzyme activity. In fact, life itself, whether plant or animal, involves a complex network of enzymatic reactions. An enzyme is a protein that is synthesized in a living cell. It catalyzes a thermodynamically possible reaction so that the rate of the reaction is compatible with the numerous biochemical processes essential for the growth and maintenance of a cell. The synthesis of an enzyme thus is under tight metabolic regulations and controls that can be genetically or environmentally manipulated sometimes to cause the overproduction of an enzyme by the cell. An enzyme, like chemical catalysts, in no way modifies the equilibrium constant or the free energy change of a reaction.

  15. Application of a coupled enzyme assay to characterize nicotinamide riboside kinases.

    PubMed

    Dölle, Christian; Ziegler, Mathias

    2009-02-15

    The recently identified nicotinamide riboside kinases (Nrks) constitute a distinct pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Here we present the combination of an established optical adenosine triphosphatase (ATPase) test, the pyruvate kinase/lactate dehydrogenase system, with the Nrk-catalyzed reaction to determine kinetic properties of these enzymes, in particular affinities for ATP. The assay allows variation of both nucleoside and phosphate donor substrates, thereby providing major advantages for the characterization of these enzymes. We confirm previously established kinetic parameters and identify differences in substrate selectivity between the two human Nrk isoforms. The proposed assay is inexpensive and may be applied for high-throughput screening.

  16. DEEPre: sequence-based enzyme EC number prediction by deep learning.

    PubMed

    Li, Yu; Wang, Sheng; Umarov, Ramzan; Xie, Bingqing; Fan, Ming; Li, Lihua; Gao, Xin

    2018-03-01

    Annotation of enzyme function has a broad range of applications, such as metagenomics, industrial biotechnology, and diagnosis of enzyme deficiency-caused diseases. However, the time and resource required make it prohibitively expensive to experimentally determine the function of every enzyme. Therefore, computational enzyme function prediction has become increasingly important. In this paper, we develop such an approach, determining the enzyme function by predicting the Enzyme Commission number. We propose an end-to-end feature selection and classification model training approach, as well as an automatic and robust feature dimensionality uniformization method, DEEPre, in the field of enzyme function prediction. Instead of extracting manually crafted features from enzyme sequences, our model takes the raw sequence encoding as inputs, extracting convolutional and sequential features from the raw encoding based on the classification result to directly improve the prediction performance. The thorough cross-fold validation experiments conducted on two large-scale datasets show that DEEPre improves the prediction performance over the previous state-of-the-art methods. In addition, our server outperforms five other servers in determining the main class of enzymes on a separate low-homology dataset. Two case studies demonstrate DEEPre's ability to capture the functional difference of enzyme isoforms. The server could be accessed freely at http://www.cbrc.kaust.edu.sa/DEEPre. xin.gao@kaust.edu.sa. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  17. Deletion of the NR2A subunit prevents developmental changes of NMDA-mEPSCs in cultured mouse cerebellar granule neurones

    PubMed Central

    Fu, Zhanyan; Logan, Stephen M; Vicini, Stefano

    2005-01-01

    We investigated the role N-methyl-d-aspartate (NMDA) receptor subunits play in shaping excitatory synaptic currents in cultures of cerebellar granule cells (CGCs) from NR2A knockout (NR2A−/−) and wild-type (+/+) mice. Cultures were maintained in a condition that facilitates the occurrence of functional synapses, allowing us to record NMDA-miniature excitatory postsynaptic currents (mEPSCs) in addition to NMDA receptor-mediated whole-cell currents at three ages in vitro. Whole-cell NMDA current density decreased with development in both strains though currents from NR2A−/− neurones demonstrated greater sensitivity to CP101 606, an NR2B subunit specific blocker. Sensitivity to Mg2+ blockade decreased with age in vitro in +/+ but not in NR2A−/− CGCs. Immunocytochemistry revealed that dendrites and somas displayed distinct NR1 and NR2A subunit clusters which became increasingly colocalized in +/+ neurones. Qualitatively the overall NR2B subunit staining pattern was similar in +/+ and NR2A−/− neurones throughout development, suggesting that the NR2B subunit distribution is not mediated by the NR2A subunit. In addition, staining with markers for excitatory synapses showed that expression of NR2A subunit (but not NR2B) increases at both synaptic and extrasynaptic sites in +/+ neurones during development. In parallel, NMDA-mEPSCs were faster in +/+ compared with NR2A−/− neurones at all time points studied, suggesting that the NR2A subunit begins to replace NR2B-rich NMDA receptors even at early stages of development. Many NR2A−/− neurones were devoid of NMDA-mEPSCs at the later time point, and transfection of the NR2A subunit in these neurones restored fast decay and the occurrence of NMDA-mEPSCs. Taken together, our results indicate that the NR2A subunit is mainly responsible for the developmental changes observed in the maturation of excitatory synapses. PMID:15649973

  18. A single Drosophila melanogaster myosin light chain kinase gene produces multiple isoforms whose activities are differently regulated.

    PubMed

    Kojima, S; Mishima, M; Mabuchi, I; Hotta, Y

    1996-09-01

    Myosin light chain kinase (MLCK) specifically phosphorylates the myosin regulatory light chain in a calcium/calmodulin (Ca2+/CaM)-dependent manner in animal cells. The roles of MLCK are not fully understood, particularly in nonmuscle cells. Therefore, we cloned and characterized a Drosophila MLCK gene as the first step in a genetic analysis of this process. Four transcripts are produced from this gene. These transcripts encode at least three isoforms (isoform-I, -II and -III), which share a kinase domain, a fibronectin type III motif and an immunoglobulin C2 motif. However, regulatory regions differ between isoform-I/II and -III due to the alternative splicing of the exon encoding a CaM-binding domain. As a result, isoform-I and -II are Ca2+/CaM-dependent forms, whereas isoform-III is a Ca2+/CaM-independent form. Northern blotting and in situ hybridization showed that the expressions of these isoforms are distinctly regulated in stage- and tissue-dependent manners. Isoform-I seems to be expressed ubiquitously, while isoform-III is expressed predominantly in muscle tissues. In contrast to these isoforms, isoform-II is specific to late pupa and adult. In Drosophila, a single MLCK gene produces multiple isoforms whose regulatory regions and expression patterns are different. These differences suggest various cellular functions of MLCK in Drosophila.

  19. Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy.

    PubMed

    Schulz, Alexander; Baader, Stephan L; Niwa-Kawakita, Michiko; Jung, Marie Juliane; Bauer, Reinhard; Garcia, Cynthia; Zoch, Ansgar; Schacke, Stephan; Hagel, Christian; Mautner, Victor-Felix; Hanemann, C Oliver; Dun, Xin-Peng; Parkinson, David B; Weis, Joachim; Schröder, J Michael; Gutmann, David H; Giovannini, Marco; Morrison, Helen

    2013-04-01

    The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.

  20. Regulation of lipid droplets by metabolically controlled Ldo isoforms

    PubMed Central

    Johnsen, Lisa; Buxó, Laura; Idrissi, Fatima-Zahra; Ejsing, Christer S.

    2018-01-01

    Storage and consumption of neutral lipids in lipid droplets (LDs) are essential for energy homeostasis and tightly coupled to cellular metabolism. However, how metabolic cues are integrated in the life cycle of LDs is unclear. In this study, we characterize the function of Ldo16 and Ldo45, two splicing isoforms of the same protein in budding yeast. We show that Ldo proteins interact with the seipin complex, which regulates contacts between LDs and the endoplasmic reticulum (ER). Moreover, we show that the levels of Ldo16 and Ldo45 depend on the growth stage of cells and that deregulation of their relative abundance alters LD morphology, protein localization, and triglyceride content. Finally, we show that absence of Ldo proteins results in defects in LD morphology and consumption by lipophagy. Our findings support a model in which Ldo proteins modulate the activity of the seipin complex, thereby affecting LD properties. Moreover, we identify ER–LD contacts as regulatory targets coupling energy storage to cellular metabolism. PMID:29187528

  1. Isoliquiritigenin showed strong inhibitory effects towards multiple UDP-glucuronosyltransferase (UGT) isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation.

    PubMed

    Lu, Hang; Fang, Zhong-Ze; Cao, Yun-Feng; Hu, Cui-Min; Hong, Mo; Sun, Xiao-Yu; Li, Hua; Liu, Yan; Fu, Xiaoguang; Sun, Hongzhi

    2013-01-01

    Isoliquiritigenin, a herbal ingredient with chalcone structure, has been speculated to be able to inhibit one of the most drug-metabolizing enzymes (DMEs) UDP-glucuronosyltransferase (UGT). Therefore, the aim of the present study was to investigate the inhibition of isoliquiritigenin towards important UGT isoforms in the liver and intestine, including UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9 and 1A10. The recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as probe reactions. The results showed that 100μM of isoliquiritigenin inhibited the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 by 95.2%, 76.1%, 78.9%, 87.2%, 67.2%, 94.8%, and 91.7%, respectively. The data fitting using Dixon plot and Lineweaver-Burk plot showed that the inhibition of UGT1A1, UGT1A9 and UGT1A10 by isoliquiritigenin was all best fit to the competitive inhibition, and the second plot using the slopes from the Lineweaver-Burk plot versus isoliquiritigenin concentrations was used to calculate the inhibition kinetic parameter (K(i)) to be 0.7μM, 0.3μM, and 18.3μM for UGT1A1, UGT1A9, and UGT1A10, respectively. All these results indicated the risk of clinical application of isoliquiritigenin on the drug-drug interaction and other possible diseases induced by the inhibition of isoliquiritigenin towards these UGT isoforms. Copyright © 2012. Published by Elsevier B.V.

  2. Differential regulation of histamine- and bradykinin-stimulated phospholipase C in adrenal chromaffin cells: evidence for involvement of different protein kinase C isoforms.

    PubMed

    Sena, C M; Rosário, L M; Parker, P J; Patel, V; Boarder, M R

    1996-03-01

    In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin

  3. Identification of active retinaldehyde dehydrogenase isoforms in the postnatal human eye.

    PubMed

    Harper, Angelica R; Wiechmann, Allan F; Moiseyev, Gennadiy; Ma, Jian-Xing; Summers, Jody A

    2015-01-01

    Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined. Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye. In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels. Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.

  4. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  5. Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration

    NASA Astrophysics Data System (ADS)

    Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S.

    2016-11-01

    Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

  6. Distinct isoforms of the RFX transcription factor DAF-19 regulate ciliogenesis and maintenance of synaptic activity.

    PubMed

    Senti, Gabriele; Swoboda, Peter

    2008-12-01

    Neurons form elaborate subcellular structures such as dendrites, axons, cilia, and synapses to receive signals from their environment and to transmit them to the respective target cells. In the worm Caenorhabditis elegans, lack of the RFX transcription factor DAF-19 leads to the absence of cilia normally found on 60 sensory neurons. We now describe and functionally characterize three different isoforms of DAF-19. The short isoform DAF-19C is specifically expressed in ciliated sensory neurons and sufficient to rescue all cilia-related phenotypes of daf-19 mutants. In contrast, the long isoforms DAF-19A/B function in basically all nonciliated neurons. We discovered behavioral and cellular phenotypes in daf-19 mutants that depend on the isoforms daf-19a/b. These novel synaptic maintenance phenotypes are reminiscent of synaptic decline seen in many human neurodegenerative disorders. The C. elegans daf-19 mutant worms can thus serve as a molecular model for the mechanisms of functional neuronal decline.

  7. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

    PubMed

    Clausen, Michael V; Hilbers, Florian; Poulsen, Hanne

    2017-01-01

    The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  8. Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis.

    PubMed

    Wali, Vikram B; Gilmore-Hebert, Maureen; Mamillapalli, Ramanaiah; Haskins, Jonathan W; Kurppa, Kari J; Elenius, Klaus; Booth, Carmen J; Stern, David F

    2014-12-17

    Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer. We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed. Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this

  9. Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution

    PubMed Central

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian; Oturai, Peter; Meinild-Lundby, Anne-Kristine; Holstein-Rathlou, Niels-Henrik; Lundby, Carsten; Vidiendal Olsen, Niels

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p = 0.029); low-dose Epoetin beta: 73.1 (17.8)% (p = 0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal. PMID:25335123

  10. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    PubMed

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. Copyright © 2014 the American Physiological Society.

  11. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches

    PubMed Central

    Welch, Kenneth C.

    2014-01-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25–60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. PMID:24671242

  12. The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

    PubMed Central

    Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio

    2017-01-01

    Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

  13. Alternative relay and converter domains tune native muscle myosin isoform function in Drosophila.

    PubMed

    Kronert, William A; Melkani, Girish C; Melkani, Anju; Bernstein, Sanford I

    2012-03-02

    Myosin isoforms help define muscle-specific contractile and structural properties. Alternative splicing of myosin heavy chain gene transcripts in Drosophila melanogaster yields muscle-specific isoforms and highlights alternative domains that fine-tune myosin function. To gain insight into how native myosin is tuned, we expressed three embryonic myosin isoforms in indirect flight muscles lacking endogenous myosin. These isoforms differ in their relay and/or converter domains. We analyzed isoform-specific ATPase activities, in vitro actin motility and myofibril structure/stability. We find that dorsal acute body wall muscle myosin (EMB-9c11d) shows a significant increase in MgATPase V(max) and actin sliding velocity, as well as abnormal myofibril assembly compared to cardioblast myosin (EMB-11d). These properties differ as a result of alternative exon-9-encoded relay domains that are hypothesized to communicate signals among the ATP-binding pocket, actin-binding site and the converter domain. Further, EMB-11d shows significantly reduced levels of basal Ca- and MgATPase as well as MgATPase V(max) compared to embryonic body wall muscle isoform (EMB) (expressed in a multitude of body wall muscles). EMB-11d also induces increased actin sliding velocity and stabilizes myofibril structure compared to EMB. These differences arise from exon-11-encoded alternative converter domains that are proposed to reposition the lever arm during the power and recovery strokes. We conclude that relay and converter domains of native myosin isoforms fine-tune ATPase activity, actin motility and muscle ultrastructure. This verifies and extends previous studies with chimeric molecules and indicates that interactions of the relay and converter during the contractile cycle are key to myosin-isoform-specific kinetic and mechanical functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. The impact of tropomyosins on actin filament assembly is isoform specific.

    PubMed

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-03

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

  15. Effects of UV-B irradiation on isoforms of antioxidant enzymes and their activities in red alga Grateloupia filicina (Rhodophyta)

    NASA Astrophysics Data System (ADS)

    Zhao, Jiqiang; Li, Lixia

    2014-11-01

    Macroalgae in a littoral zone are inevitably exposed to UV-B irradiance. We analyzed the effects of UV-B on isoenzyme patterns and activities of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX) of red algae Grateloupia filicina (Lamour.) C. Agardh. The activities of SOD, CAT, and APX changed in response to UV-B in a time- and dose-dependent manner. POX activity increased significantly under all three UV-B treatments. The enzymatic assay showed three distinct bands of SODI (Mn-SOD), SODII (Fe-SOD), and SODIII (CuZn-SOD) under a low (Luv) and medium (Muv) dose of UV-B irradiation, while SODI and SODIII activities decreased significantly when exposed to a high dose of UV-B irradiation (Huv). The activity of POX isoenzymes increased significantly after exposure to UV-B, which is consistent with the total activity. In addition, a clear decrease in activity of CATIV was detected in response to all the three doses of UV treatments. Some bands of APX isoenzyme were also clearly influenced by UV-B irradiation. Correspondingly, the daily growth rate declined under all the three exposure doses, and was especially significant under Muv and Huv treatments. These data suggest that, although the protection mechanisms of antioxidant defense system are partly inducible by UV-B to prevent the damage, G. filicina has incomplete tolerance to higher UV-B irradiation stress.

  16. Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues.

    PubMed

    Reyes, Alejandro; Huber, Wolfgang

    2018-01-25

    Most human genes generate multiple transcript isoforms. The differential expression of these isoforms can help specify cell types. Diverse transcript isoforms arise from the use of alternative transcription start sites, polyadenylation sites and splice sites; however, the relative contribution of these processes to isoform diversity in normal human physiology is unclear. To address this question, we investigated cell type-dependent differences in exon usage of over 18 000 protein-coding genes in 23 cell types from 798 samples of the Genotype-Tissue Expression Project. We found that about half of the expressed genes displayed tissue-dependent transcript isoforms. Alternative transcription start and termination sites, rather than alternative splicing, accounted for the majority of tissue-dependent exon usage. We confirmed the widespread tissue-dependent use of alternative transcription start sites in a second, independent dataset, Cap Analysis of Gene Expression data from the FANTOM consortium. Moreover, our results indicate that most tissue-dependent splicing involves untranslated exons and therefore may not increase proteome complexity. Thus, alternative transcription start and termination sites are the principal drivers of transcript isoform diversity across tissues, and may underlie the majority of cell type specific proteomes and functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Isoforms of Vitamin E Have Opposing Immunoregulatory Functions during Inflammation by Regulating Leukocyte Recruitment1

    PubMed Central

    Berdnikovs, Sergejs; Abdala-Valencia, Hiam; McCary, Christine; Somand, Michelle; Cole, Rokeisha; Garcia, Alex; Bryce, Paul; Cook-Mills, Joan M.

    2009-01-01

    Reports indicate contradictory outcomes for anti-inflammatory functions of the α-tocopherol isoform of vitamin E in clinical studies of asthma and atherosclerosis. These seemingly disparate clinical results are consistent with novel unrecognized properties of isoforms of vitamin E reported in this study. We demonstrate that the isoform d-γ-tocopherol elevates inflammation in experimental asthma. Moreover, d-γ-tocopherol, at as little as 10% the concentration of d-α-tocopherol, ablates the anti-inflammatory benefit of the d-α-tocopherol isoform. A mechanism for these opposing immunoregulatory functions of purified tocopherols at physiological concentrations is not through modulation of expression of several cytokines, chemokines, or adhesion molecules, but is, at least in part, by regulation of endothelial cell signals during leukocyte recruitment. These opposing regulatory functions of vitamin E isoforms have impact on interpretations of vitamin E studies. In summary, our studies with purified tocopherol isoforms alter our understanding of vitamin E regulation of vascular function and asthma. PMID:19299740

  18. C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle.

    PubMed

    Hansberg-Pastor, Valeria; Piña-Medina, Ana Gabriela; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2015-01-01

    The CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP) with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

  19. C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle

    PubMed Central

    Hansberg-Pastor, Valeria; Piña-Medina, Ana Gabriela; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2015-01-01

    The CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP) with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle. PMID:26064112

  20. Decreased type I interferon receptor-soluble isoform in antiretroviral-treated HIV-positive children.

    PubMed

    Sottini, Alessandra; Ghidini, Claudia; Serana, Federico; Chiarini, Marco; Valotti, Monica; Badolato, Raffaele; Radeghieri, Annalisa; Caimi, Luigi; Imberti, Luisa

    2008-03-01

    We developed a real-time PCR assay to simultaneously measure the mRNA level of type I interferon (IFN) receptor (IFNAR) components in peripheral blood cells of children with chronic immune stimulation due to HIV infection. All patients were undergoing antiretroviral therapy and were divided into two groups on the basis of the induction of MxA mRNA, a marker of type I IFN bioactivity. We found that IFNAR-2 subunit mRNA was higher than that of the IFNAR-1 subunit, that the mRNA for the IFNAR-2.2 functional isoform was more expressed than that for the truncated IFNAR-2.1 isoform, and both were much more represented than that of the IFNAR-2.3 soluble isoform. We also demonstrated that soluble isoform mRNA was significantly diminished in the subgroup of patients with MxA mRNA below the cutoff value (determined as the 99th percentile of MxA measured in healthy controls). These results suggest that downregulation of the soluble receptor isoform, which would not compete with the functional isoform for binding to the target cytokine, would give type I IFN, eventually induced in these patients in the case of viral reactivation, the opportunity to promptly exert its antiviral activity.

  1. Isoforms of green fluorescent protein differ from each other in solvent molecules 'trapped' inside this protein.

    PubMed

    Glukhova, Kseniya F; Marchenkov, Victor V; Melnik, Tatiana N; Melnik, Bogdan S

    2017-05-01

    Green fluorescent protein (GFP) has been studied quite thoroughly, however, up to now some experimental data have not been explained explicitly. For example, under native conditions this protein can have two isoforms differing in their mobility in gel. In this case, no differences between the isoforms are revealed under denaturing conditions. In order to understand the difference in the isoforms of this protein, we have investigated GFP-cycle3 using mass spectrometry, gel electrophoresis, size exclusion chromatography, microcalorimetry, and spectroscopy methods under varying conditions. We have also designed and studied three mutant forms of this protein with substitutions of amino acid residues inside the GFP barrel. The mutations have allowed us to influence the formation of different GFP isoforms. Each of the mutant proteins has predominantly only one isoform. As a result of the performed research, it can be concluded that most likely the GFP isoforms differ in the solvent molecules 'trapped' inside the GFP barrel. In their turn, these molecules have an effect on the protein charge and consequently on its mobility at electrophoresis under native conditions.

  2. The two major glucokinase isoforms show conserved functionality in β-cells despite different subcellular distribution.

    PubMed

    Lu, Brian; Munoz-Gomez, Miguel; Ikeda, Yasuhiro

    2018-03-01

    Glucokinase (GCK) is crucial to regulating glucose metabolism in the liver and in pancreatic β-cells. There are two major GCK isoforms, hepatic and pancreatic GCKs, which differ only in exon 1. However, the functional differences between the two GCK isoforms remain poorly understood. Here, we used a β-cell-targeted gene transfer vector to determine the impact of isoform-specific GCK overexpression on β-cells in vitro and in vivo. We showed that pancreatic GCK had a nuclear localization signal unique to the pancreatic isoform, facilitating its nuclear distribution in β-cells. Despite the difference in subcellular distribution, overexpression of GCK isoforms similarly enhanced glucose uptake and β-cell proliferation in vitro. Overexpression of hepatic or pancreatic GCK also similarly enhanced β-cell proliferation in normal diet mice without affecting fasting glucose and intraperitoneal glucose tolerance tests. Our further study on human GCK sequences identified disproportional GCK amino acid variants in exon 1, while mutations linked to maturity onset diabetes of the young type 2 were disproportionally found in exons 2 through 10. Our results therefore indicate functional conservation between the two major GCK isoforms despite their distinct subcellular distribution.

  3. Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

  4. Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

    PubMed

    Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning

    2012-08-01

    Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

  5. Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

    PubMed Central

    Walker, Lauren Elizabeth; Frigerio, Federica; Ravizza, Teresa; Ricci, Emanuele; Tse, Karen; Jenkins, Rosalind E.; Sills, Graeme John; Jorgensen, Andrea; Porcu, Luca; Alapirtti, Tiina; Peltola, Jukka; Brodie, Martin J.; Park, Brian Kevin; Marson, Anthony Guy; Antoine, Daniel James

    2017-01-01

    Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies. PMID:28504645

  6. Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues

    PubMed Central

    2018-01-01

    Abstract Most human genes generate multiple transcript isoforms. The differential expression of these isoforms can help specify cell types. Diverse transcript isoforms arise from the use of alternative transcription start sites, polyadenylation sites and splice sites; however, the relative contribution of these processes to isoform diversity in normal human physiology is unclear. To address this question, we investigated cell type-dependent differences in exon usage of over 18 000 protein-coding genes in 23 cell types from 798 samples of the Genotype-Tissue Expression Project. We found that about half of the expressed genes displayed tissue-dependent transcript isoforms. Alternative transcription start and termination sites, rather than alternative splicing, accounted for the majority of tissue-dependent exon usage. We confirmed the widespread tissue-dependent use of alternative transcription start sites in a second, independent dataset, Cap Analysis of Gene Expression data from the FANTOM consortium. Moreover, our results indicate that most tissue-dependent splicing involves untranslated exons and therefore may not increase proteome complexity. Thus, alternative transcription start and termination sites are the principal drivers of transcript isoform diversity across tissues, and may underlie the majority of cell type specific proteomes and functions. PMID:29202200

  7. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    PubMed

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Adsorption Behavior of Charge Isoforms of Monoclonal Antibodies on Strong Cation Exchangers.

    PubMed

    Steinebach, Fabian; Wälchli, Ruben; Pfister, David; Morbidelli, Massimo

    2017-12-01

    In this work, the adsorption behavior of the different charge isoforms of the same monoclonal antibody (mAb) on strong cation-exchange resins is analyzed. While charge isoforms of the same antibody mainly differ in their effective charge, the similar structure and size allows developing a simplified model, which describes the adsorption behavior of mAb charge isoforms independently of the number of isoforms with only four parameters. In contrast to classical model-based descriptions of the adsorption isotherm, the proposed work enables retrieving some physical meaning in the definition of the model parameters. These model parameters are determined for several resin-antibody combinations. Thereby it is found that for mAbs on commercial cation exchangers an effective resin charge density of 0.22 ± 0.08 mmol mL -1 of solid phase is used for protein binding, which was found to be independent of the absolute resin charge density measured by titration. The presented results help to understand the adsorption behavior of mAbs on cation-exchangers, which is applicable both for the isolation of the main charge isoform or for preserving a certain charge isoform pattern during the polishing processes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Biochemical Characterization of Two Wheat Phosphoethanolamine N-Methyltransferase Isoforms with Different Sensitivities to Inhibition by Phosphatidic Acid*

    PubMed Central

    Jost, Ricarda; Berkowitz, Oliver; Shaw, John; Masle, Josette

    2009-01-01

    In plants the triple methylation of phosphoethanolamine to phosphocholine catalyzed by phosphoethanolamine N-methyltransferase (PEAMT) is considered a rate-limiting step in the de novo synthesis of phosphatidylcholine. Besides being a major membrane phospholipid, phosphatidylcholine can be hydrolyzed into choline and phosphatidic acid. Phosphatidic acid is widely recognized as a second messenger in stress signaling, and choline can be oxidized within the chloroplast to yield the putative osmoprotectant glycine betaine. Here we describe the cloning and biochemical characterization of a second wheat PEAMT isoform that has a four times higher specific activity than the previously described WPEAMT/TaPEAMT1 enzyme and is less sensitive to product inhibition by S-adenosyl homocysteine, but more sensitive to inhibition by phosphocholine. Both enzymes follow a sequential random Bi Bi mechanism and show mixed-type product inhibition patterns with partial inhibition for TaPEAMT1 and a strong non-competitive component for TaPEAMT2. An induction of TaPEAMT protein expression and activity is observed after cold exposure, ahead of an increase in gene expression. Our results demonstrate direct repression of in vitro enzymatic activities by phosphatidic acid for both enzymes, with TaPEAMT1 being more sensitive than TaPEAMT2 in the physiological concentration range. Other lipid ligands identified in protein-lipid overlays are phosphoinositide mono- as well as some di-phosphates and cardiolipin. These results provide new insights into the complex regulatory circuits of phospholipid biosynthesis in plants and underline the importance of head group biosynthesis in adaptive stress responses. PMID:19762471

  10. Indicators: Sediment Enzymes

    EPA Pesticide Factsheets

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  11. Enzymes in Analytical Chemistry.

    ERIC Educational Resources Information Center

    Fishman, Myer M.

    1980-01-01

    Presents tabular information concerning recent research in the field of enzymes in analytic chemistry, with methods, substrate or reaction catalyzed, assay, comments and references listed. The table refers to 128 references. Also listed are 13 general citations. (CS)

  12. Proteolytic enzymes from Bromelia antiacantha as tools for controlled tissue hydrolysis in entomology.

    PubMed

    Macció, Laura; Vallés, Diego; Cantera, Ana Maria

    2013-12-01

    A crude extract with high proteolytic activity (78.1 EU/mL), prepared from ripe fruit of Bromelia antiacantha was used to hydrolyze and remove soft tissues from the epigyne of Apopyllus iheringi. This enzymatic extract presented four actives isoforms which have a broad substrate specificity action. Enzyme action on samples was optimized after evaluation under different conditions of pH, enzyme-substrate ratio and time (parameters selected based on previous studies) of treatment (pH 4.0, 6.0 and 8.0 at 42°C with different amount of enzyme). Scanning electron microscopy was used to evaluate conditions resulting in complete digestion of epigyne soft tissues. Optimal conditions for soft tissue removal were 15.6 total enzyme units, pH 6.0 for 18 h at 42°C.

  13. Overproduction of ligninolytic enzymes

    SciTech Connect

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  14. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.

    1992-12-31

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid, polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for themore » oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.« less

  15. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.

    1993-07-13

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for themore » oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.« less

  16. Measurement of enzyme activity.

    PubMed

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  17. The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling

    PubMed Central

    Pamarthy, Sahithi; Mao, Liquin; Katara, Gajendra K; Fleetwood, Sara; Kulshreshta, Arpita; Gilman-Sachs, Alice; Beaman, Kenneth D

    2016-01-01

    Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of ‘a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker β-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-β (transforming growth factor-β) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-β pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-β are crucial for cell

  18. Alterations in mRNA 3' UTR Isoform Abundance Accompany Gene Expression Changes in Human Huntington's Disease Brains.

    PubMed

    Romo, Lindsay; Ashar-Patel, Ami; Pfister, Edith; Aronin, Neil

    2017-09-26

    The huntingtin gene has two mRNA isoforms that differ in their 3' UTR length. The relationship of these isoforms with Huntington's disease is not established. We provide evidence that the abundance of huntingtin 3' UTR isoforms differs between patient and control neural stem cells, fibroblasts, motor cortex, and cerebellum. Huntingtin 3' UTR isoforms, including a mid-3' UTR isoform, have different localizations, half-lives, polyA tail lengths, microRNA sites, and RNA-binding protein sites. Isoform shifts in Huntington's disease motor cortex are not limited to huntingtin; 11% of alternatively polyadenylated genes change the abundance of their 3' UTR isoforms. Altered expression of RNA-binding proteins may be associated with aberrant isoform abundance; knockdown of the RNA-binding protein CNOT6 in control fibroblasts leads to huntingtin isoform differences similar to those in disease fibroblasts. These findings demonstrate that mRNA 3' UTR isoform changes are a feature of molecular pathology in the Huntington's disease brain. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

    PubMed Central

    Hilbert, Lennart; Bates, Genevieve; Roman, Horia N.; Blumenthal, Jenna L.; Zitouni, Nedjma B.; Sobieszek, Apolinary; Mackey, Michael C.; Lauzon, Anne-Marie

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics. PMID:24204225

  20. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    PubMed Central

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation. PMID:23650515

  1. Contribution of the different UDP-glucuronosyltransferase (UGT) isoforms to buprenorphine and norbuprenorphine metabolism and relationship with the main UGT polymorphisms in a bank of human liver microsomes.

    PubMed

    Rouguieg, Koukeb; Picard, Nicolas; Sauvage, François-Ludovic; Gaulier, Jean-Michel; Marquet, Pierre

    2010-01-01

    The goal of this study was to evaluate the specific contribution of individual UDP-glucuronosyltransferase (UGT) isoforms in the metabolism of buprenorphine (BUP) and norbuprenorphine (Nor-BUP), as well as the impact of their genetic variations. The glucuronidation of BUP and Nor-BUP was examined using human liver microsomes (HLMs) and heterologously expressed UGTs. The individual contribution of UGT isoforms was estimated using enzyme kinetic experiments combined with the relative activity factor (RAF). Phenotype-genotype relationships were investigated in a bank of 52 HLMs. Among the six hepatic UGT isoforms tested, UGT1A1, UGT1A3, and UGT2B7 metabolized BUP and Nor-BUP. Using the RAF approach, we found that UGT1A1 and UGT2B7 accounted for approximately 10 and 41% of BUP glucuronidation, respectively. Nor-BUP glucuronidation involved predominantly UGT1A3 (approximately 63%) and UGT1A1 (34%), whereas UGT2B7 had only a minor role. The UGT1A1 promoter (TA)(6/7)TAA mutation (UGT1A1*28) resulted in a 28% decrease of BUP glucuronidation V(max) in pooled HLMs but was not statistically associated with glucuronidation rate in 52 individual HLMs. The presence of the UGT2B7 promoter (G-842A) mutation resulted in higher BUP glucuronidation V(max) in pooled HLMs (+80% on average) and in a significant higher glucuronidation rate in noncarriers (but not in carriers) of the UGT1A1*28 allele (P = 0.0352). This study represents a functional basis for further clinical pharmacogenetic studies.

  2. Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury

    PubMed Central

    Niyitegeka, Jean-Marie V.; Bastidas, Adam C.; Newman, Robert H.; Taylor, Susan S.

    2014-01-01

    Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways. PMID:25354939

  3. Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

    PubMed Central

    Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

    2015-01-01

    The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

  4. Isoform Evolution in Primates through Independent Combination of Alternative RNA Processing Events

    PubMed Central

    Zhang, Shi-Jian; Wang, Chenqu; Yan, Shouyu; Fu, Aisi; Luan, Xuke; Li, Yumei; Sunny Shen, Qing; Zhong, Xiaoming; Chen, Jia-Yu; Wang, Xiangfeng; Chin-Ming Tan, Bertrand; He, Aibin; Li, Chuan-Yun

    2017-01-01

    Abstract Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875 bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates. PMID:28957512

  5. Developmental expression of high molecular weight tropomyosin isoforms in Mesocestoides corti.

    PubMed

    Koziol, Uriel; Costábile, Alicia; Domínguez, María Fernanda; Iriarte, Andrés; Alvite, Gabriela; Kun, Alejandra; Castillo, Estela

    2011-02-01

    Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

    PubMed Central

    Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

    2012-01-01

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

  7. Identification of novel monocistronic HTLV-1 mRNAs encoding functional Rex isoforms.

    PubMed

    Rende, Francesca; Cavallari, Ilaria; Andresen, Vibeke; Valeri, Valerio W; D'Agostino, Donna M; Franchini, Genoveffa; Ciminale, Vincenzo

    2015-07-02

    Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently. Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms. The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections.

  8. Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii.

    PubMed

    Baines, I C; Corigliano-Murphy, A; Korn, E D

    1995-08-01

    The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.

  9. Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii

    PubMed Central

    1995-01-01

    The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70- 100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV. PMID:7622560

  10. NADPH-diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

    PubMed Central

    MORAES, NECI; ZAGO, DOUGLAS; GAGIOTI, SONIA; HOSHIDA, MARA SANDRA; BEVILACQUA, ESTELA

    2001-01-01

    The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface. PMID:11327206

  11. Human Mucin MUC1 RNA Undergoes Different Types of Alternative Splicing Resulting in Multiple Isoforms

    PubMed Central

    Zhang, Lixin; Vlad, Anda; Milcarek, Christine; Finn, Olivera J

    2012-01-01

    MUC1 is a transmembrane mucin with important functions in normal and transformed cells carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5′ splice site but variable 3′ splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3′ splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1+/−KrasG12D/+PtenloxP/loxP mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice. PMID:22941036

  12. Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis.

    PubMed

    Gavaldà-Navarro, Aleix; Domingo, Pere; Viñas, Octavi; Mampel, Teresa

    2015-07-01

    Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Role of molecular isoforms of acetylcholinesterase in learning and memory functions.

    PubMed

    Das, Amitava; Dikshit, Madhu; Nath, Chandishwar

    2005-05-01

    In the present study, activity of salt soluble (SS) G1 and detergent soluble (DS) G4 molecular isoforms of acetylcholinesterase (AChE) has been investigated in rat brain areas in trained (learned), scopolamine (amnesic) and Tacrine (anti-dementic) treated rats to find out their role in learning and memory functions. AChE was estimated spectrophotometrically at 412 nm in rat brain areas. Isolation and partial purification of molecular isoforms G1 and G4 of AChE was done by gel filtration chromatography. Passive avoidance was used to test learning and memory functions. AChE activity was altered in both the fractions SS and DS of different brain areas following passive avoidance in control, scopolamine treated, tacrine treated and tacrine treatment in scopolamine pretreated rats. The peak AChE activity obtained in the DS (fraction 9) and the SS (fraction 13) fraction following gel filtration chromatography. On the basis of molecular weight fraction 9 (DS) and 13 (SS) represent the G4 and G1, respectively. The pattern of changes in the AChE activity of G1 isoform (fraction 13 of SS) and G4 isoform (fraction 9 of DS) in brain areas were similar to those of SS and DS fraction, respectively. In hippocampus, AChE activity in the fraction G1 isoform (fraction 13 of SS) was decreased only in tacrine treated rats but AChE activity in the G4 isoform (fraction 9 of DS) was decreased in both trained and tacrine treated rats. Changes in activity of G4 isoform of AChE in hippocampus could be correlated with passive avoidance learning, scopolamine induced deficit in passive avoidance and reversal of scopolamine deficit by tacrine.

  14. Random-walk enzymes

    NASA Astrophysics Data System (ADS)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  15. Carboxylesterases: General detoxifying enzymes

    PubMed Central

    Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

    2016-01-01

    Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

  16. [Thrombin: a multifunctional enzyme].

    PubMed

    Polack, B

    2003-01-01

    Thrombin is the final enzyme of blood coagulation cascade. It belongs to the trypsin family of serine proteases. Its two primary actions are to cleave fibrinogen to release fibrin and to activate platelets through a limited proteolysis of a specific receptor. In addition, thrombin is the major regulator of blood coagulation. It is both a procoagulant enzyme in the activation of factors V and VIII, and an anticoagulant enzyme through the activation of protein C and TAFI. This multi-functionality of thrombin depends upon the conformation of its active site: depth for high specificity and shape for a finely tuned selection of substrates. Since new anticoagulant molecules, some with anti-thrombin activity, are emerging, it is important to understand the mechanisms allowing thrombin to be so specifically multifunctional.

  17. The pathogenomics of McArdle disease--genes, enzymes, models, and therapeutic implications.

    PubMed

    Nogales-Gadea, Gisela; Santalla, Alfredo; Brull, Astrid; de Luna, Noemi; Lucia, Alejandro; Pinós, Tomàs

    2015-03-01

    Numerous biomedical advances have been made since Carl and Gerty Cori discovered the enzyme phosphorylase in the 1940s and the Scottish physician Brian McArdle reported in 1951 a previously 'undescribed disorder characterized by a gross failure of the breakdown in muscle of glycogen'. Today we know that this disorder, commonly known as 'McArdle disease', is caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (GP). Here we review the main aspects of the 'pathogenomics' of this disease including, among others: the spectrum of mutations in the gene (PYGM) encoding muscle GP; the interplay between the different tissue GP isoforms in cellular cultures and in patients; what can we learn from naturally occurring and recently laboratory-generated animal models of the disease; and potential therapies.

  18. Chronic hypobaric hypoxia diminishes the expression of base excision repair OGG1 enzymes in spermatozoa.

    PubMed

    Farias, J G; Zepeda, A; Castillo, R; Figueroa, E; Ademoyero, O T; Pulgar, V M

    2018-03-01

    Hypobaric hypoxia induces DNA damage in rat testicular cells, the production of defective spermatozoids and decreased sperm count, associated with an increase in oxidative stress. 8-Oxoguanine glycosylase (OGG1) enzymes are main members of the base excision repair (BER) system, a DNA repair mechanism. We determined the expression levels of mitochondrial and nuclear OGG1 isoforms in spermatozoa collected from cauda epididymis in rats exposed to chronic hypobaric hypoxia (CHH) for 5, 15 and 30 days. CHH attenuates OGG1 expression in a time-dependent fashion, with a greater reduction in the mitochondrial isoform OGG1-2a (p < .05). Attenuation of the BER system may contribute to DNA damage under hypoxia exposure. © 2017 Blackwell Verlag GmbH.

  19. High-throughput screening of inhibitory effects of Bo-yang-hwan-o-tang on human cytochrome P450 isoforms in vitro using UPLC/MS/MS.

    PubMed

    Lee, Miran; Park, Jeonghyeon; Lim, Mi-sun; Seong, Sook Jin; Lee, Joomi; Seo, Jeong Ju; Park, Yong-Ki; Lee, Hae Won; Yoon, Young-Ran

    2012-01-01

    Bo-yang-hwan-o-tang (BHT) is an oriental herbal medicine for treating brain disorders such as cerebral ischemia. The objective of this study was to develop an economically feasible and time-saving high-throughput screening method to monitor the potential inhibitory effects of BHT on human cytochrome P450 (CYP) enzymes in vitro. Two cocktail sets were used for incubation of human liver microsomes: Cocktail A: 6 probe substrates for CYP1A2, CYP2A6, CYP2C8, CYP2C19, CYP2D6, CYP3A4; Cocktail B: 3 for CYP2B6, CYP2C9, CYP2E1. The concentrations of the substrate metabolites were simultaneously analyzed using UPLC/MS/MS. The BHT extract had almost negligible inhibitory effects on the nine human CYP isoforms tested, with the half-maximal inhibitory concentration value ranged from 3624.99 to 45412.44 μg/ml. The results suggest that BHT extract has no inhibitory effects on CYP isoforms within the clinically recommended dosage range. We conclude that BHT might be free of drug-herb interactions when co-administered with other medicines. However, more in vivo human studies are needed to confirm these results. The high-throughput screening method can be a useful tool for drug discovery and for understanding drug interactions.

  20. Activity-based protein profiling of hydrolytic enzymes induced by gibberellic acid in isolated aleurone layers of malting barley.

    PubMed

    Daneri-Castro, Sergio N; Chandrasekar, Balakumaran; Grosse-Holz, Friederike M; van der Hoorn, Renier A L; Roberts, Thomas H

    2016-09-01

    During barley germination, the aleurone layer secretes most of the enzymes required to degrade the endosperm, many of which are yet to be characterized. We used activity-based protein profiling (ABPP) to detect a range of active enzymes extracted from aleurone layers isolated from grains of a commercial malting barley variety incubated with or without gibberellic acid (GA). Enzymes found to be induced by GA were putative aleurains, cathepsin-B-like proteases and serine hydrolases. By using an inhibitory sugar panel, a specific active retaining β-glycosidase in the barley aleurone was identified as a putative xylanase. Our results show that ABPP can be used rapidly to identify a variety of active enzyme isoforms in cereal aleurone without the need for enzyme purification. © 2016 Federation of European Biochemical Societies.

  1. Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2Sshort isoform.

    PubMed

    Huang, Shar-Yin N; Dalla Rosa, Ilaria; Michaels, Stephanie A; Tulumello, David V; Agama, Keli; Khiati, Salim; Jean, Sae Rin; Baechler, Simone A; Factor, Valentina M; Varma, Sudhir; Murai, Junko; Miller Jenkins, Lisa M; Kelley, Shana O; Pommier, Yves

    2018-03-01

    Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2 S ) expressed from an alternative transcription start site. TDP2 S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type ( WT ) and TDP2 knockout ( TDP2 -/-/- ) DT40 cells shows that TDP2 -/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2 -/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2 -/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2 S isoform, we show that TDP2 S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2 S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria. © 2018 The Authors.

  2. Differential Cooperation between Heterochromatin Protein HP1 Isoforms and MyoD in Myoblasts*S⃞

    PubMed Central

    Yahi, Hakima; Fritsch, Lauriane; Philipot, Ophelie; Guasconi, Valentina; Souidi, Mouloud; Robin, Philippe; Polesskaya, Anna; Losson, Regine; Harel-Bellan, Annick; Ait-Si-Ali, Slimane

    2008-01-01

    Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1α, HP1β, and HP1γ play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1α and HP1β isoforms, but not HP1γ, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1α and HP1β, but not HP1γ, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner. PMID:18599480

  3. Characterizing functional differences in sea anemone Hsp70 isoforms using budding yeast.

    PubMed

    Waller, Shawn J; Knighton, Laura E; Crabtree, Lenora M; Perkins, Abigail L; Reitzel, Adam M; Truman, Andrew W

    2018-04-25

    Marine organisms experience abiotic stressors such as fluctuations in temperature, UV radiation, salinity, and oxygen concentration. Heat shock proteins (HSPs) assist in the response of cells to these stressors by refolding and maintaining the activity of damaged proteins. The well-conserved Hsp70 chaperone family is essential for cell viability as well as the response to stress. Organisms possess a variety of Hsp70 isoforms that differ slightly in amino acid sequence, yet very little is known about their functional relevance. In this study, we undertook analysis of three principal Hsp70 isoforms NvHsp70A, B, and D from the starlet sea anemone Nematostella vectensis. The functionality of Hsp70 isoforms in the starlet sea anemone was assessed through transcriptional analysis and by heterologous expression in budding yeast Saccharomyces cerevisiae. Interestingly, these isoforms were found to not only differ in expression under stress but also appear to have functional differences in their ability to mediate the cellular stress program. These results contribute to an understanding of Hsp70 isoform specificity, their shared and unique roles in response to acute and chronic environmental stress, and the potential basis of local adaptation in populations of N. vectensis.

  4. Elevated serum tartrate-resistant acid phosphatase isoform 5a levels in metabolic syndrome.

    PubMed

    Huang, Yi-Jhih; Huang, Tsai-Wang; Chao, Tsu-Yi; Sun, Yu-Shan; Chen, Shyi-Jou; Chu, Der-Ming; Chen, Wei-Liang; Wu, Li-Wei

    2017-09-29

    Tartrate-resistant phosphatase isoform 5a is expressed in tumor-associated macrophages and is a biomarker of chronic inflammation. Herein, we correlated serum tartrate-resistant phosphatase isoform 5a levels with metabolic syndrome status and made comparisons with traditional markers of inflammation, including c-reactive protein and interleukin-6. One hundred healthy volunteers were randomly selected, and cut-off points for metabolic syndrome related inflammatory biomarkers were determined using receiver operating characteristic curves. Linear and logistic regression models were subsequently used to correlate inflammatory markers with the risk of metabolic syndrome. Twenty-two participants met the criteria for metabolic syndrome, and serum tartrate-resistant phosphatase isoform 5a levels of >5.8 μg/L were associated with metabolic syndrome (c-statistics, 0.730; p = 0.001; 95% confidence interval, 0.618-0.842). In addition, 1 μg/L increases in tartrate-resistant phosphatase isoform 5a levels were indicative of a 1.860 fold increase in the risk of metabolic syndrome (p = 0.012). Elevated serum tartrate-resistant phosphatase isoform 5a levels are associated with the risk of metabolic syndrome, with a cut-off level of 5.8 μg/L.

  5. Ontogenic and sexually dimorphic expression of cyp19 isoforms in the rainbowfish, Melanotaenia fluviatilis (Castelnau 1878).

    PubMed

    Shanthanagouda, A H; Patil, J G; Nugegoda, D

    2012-02-01

    To investigate the role of cytochrome P450 aromatase, we isolated cyp19 isoforms in the Murray River rainbowfish, M. fluviatilis. The cloned cDNA for cyp19a1a and cyp19a1b had an open reading frame (ORF) of 492 and 499 amino acid residues, with shared identity of up to 83% and 87% with the corresponding homologues of other teleosts respectively. In contrast, the cyp19a1a and cyp19a1b of the Murray River rainbowfish had a shared identity of only 61%. Not surprisingly, the phylogenetic analysis clustered the M. fluviatilis cyp19 isoforms with the corresponding isoforms of other teleosts, suggesting a shared evolutionary ancestry of the respective isoforms. We also studied the expression of cyp19 isoforms during ontogeny and in adult fish using quantitative Real-Time PCR (qPCR). Results suggest that uniquely only cyp19a1b transcripts are maternally inherited, suggesting its role in early development and growth in the species. In contrast to reports in many teleosts, the cyp19a1a was exclusively expressed in the ovarian tissue and completely absent in other tissues examined, including testis. The cyp19a1b like in most teleosts was predominantly expressed in the brain of both males and females with low level of expression in other tissues including gonads of both sexes. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice[S

    PubMed Central

    Kim, Myung-Hee; de Beer, Maria C.; Wroblewski, Joanne M.; Charnigo, Richard J.; Ji, Ailing; Webb, Nancy R.; de Beer, Frederick C.; van der Westhuyzen, Deneys R.

    2016-01-01

    The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA’s rapid clearance. These studies enhance our understanding of SAA metabolism and SAA’s effects on AP-HDL composition and catabolism. PMID:27018443

  7. Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom.

    PubMed

    Machado, Leandro F; Laugesen, Sabrina; Botelho, Elvio D; Ricart, Carlos A O; Fontes, Wagner; Barbaro, Katia C; Roepstorff, Peter; Sousa, Marcelo V

    2005-05-01

    Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.

  8. Distinct Interactions of EBP1 Isoforms with FBXW7 Elicits Different Functions in Cancer

    DOE PAGES

    Wang, Yuli; Zhang, Pengju; Wang, Yunshan; ...

    2017-02-16

    The ErbB3 receptor–binding protein EBP1 encodes two alternatively spliced isoforms P48 and P42. While there is evidence of differential roles for these isoforms in tumorigenesis, little is known about their underlying mechanisms. In this paper, we demonstrate that EBP1 isoforms interact with the SCF-type ubiquitin ligase FBXW7 in distinct ways to exert opposing roles in tumorigenesis. EBP1 P48 bound to the WD domain of FBXW7 as an oncogenic substrate of FBXW7. EBP1 P48 binding sequestered FBXW7α to the cytosol, modulating its role in protein degradation and attenuating its tumor suppressor function. In contrast, EBP1 P42 bound to both the F-boxmore » domain of FBXW7 as well as FBXW7 substrates. This adapter function of EBP1 P42 stabilized the interaction of FBXW7 with its substrates and promoted FBXW7-mediated degradation of oncogenic targets, enhancing its overall tumor-suppressing function. Finally and overall, our results establish distinct physical and functional interactions between FBXW7 and EBP1 isoforms, which yield their mechanistically unique isoform-specific functions of EBP1 in cancer.« less

  9. Glucuronidation of the broad-spectrum antiviral drug arbidol by UGT isoforms.

    PubMed

    Song, Jin-Hui; Fang, Zhong-Ze; Zhu, Liang-Liang; Cao, Yun-Feng; Hu, Cui-Min; Ge, Guang-Bo; Zhao, De-Wei

    2013-04-01

    The aim of this work was to identify the uridine glucuronosyltransferase (UGT) isoforms involved in the metabolism of the broad-spectrum antiviral drug arbidol. A human liver microsome (HLM) incubation system was employed to catalyse the formation of arbidol glucuronide. The glucuronidation activity of commercially recombinant UGT isoforms towards arbidol was screened. A combination of kinetic analysis and chemical inhibition study was used to determine the UGT isoforms involved in arbidol's glucuronidation. The arbidol glucuronide was detected when arbidol was incubated with HLMs in the presence of UDP-glucuronic acid. The Eadie-Hofstee plot showed that glucuronidation of arbidol was best fit to the Michaelis-Menten kinetic model, and K(m) and apparent V(max) were calculated to be 8.0 ± 0.7 μm and 2.03 ± 0.05 nmol/min/mg protein, respectively. Assessment of a panel of recombinant UGT isoforms revealed that UGT1A1, UGT1A3 and UGT1A9 could catalyse the glucuronidation of arbidol. Kinetic analysis and chemical inhibition study demonstrated that UGT1A9 was the predominant UGT isoform involved in arbidol glucuronidation in HLMs. The major contribution of UGT1A9 towards arbidol glucuronidation was demonstrated in this study. © 2012 The Authors. JPP © 2012. Royal Pharmaceutical Society.

  10. Structure and Concentration Changes Affect Characterization of UGT Isoform-Specific Metabolism of Isoflavones

    PubMed Central

    Tang, Lan; Singh, Rashim; Liu, Zhongqiu; Hu, Ming

    2010-01-01

    We characterized the isoform specific glucuronidation of six isoflavones genistein, daidzein, glycitein, formononetin, biochanin A and prunetin using 12 expressed human UGTs and human intestinal and liver microsomes. The results indicated that these isoflavones are metabolized most rapidly at three different concentrations by one of these four UGT isoforms: UGT1A1, UGT1A8, UGT1A9 and UGT1A10. Furthermore, glycitein was usually metabolized the fastest whereas prunetin the slowest. Using the rates of metabolism by 12 UGT isoforms as a means to establish the metabolic “fingerprint”, we found that each isoflavone had distinctive concentration-dependent patterns. Determination of kinetic parameters of glucuronidation using genistein and prunetin indicated that the distinct concentration-dependent metabolic patterns were the result of differences in Km and Vmax values. We then measured how well metabolic “fingerprinting” predicted metabolism of these isoflavones by human intestinal and liver microsomes. We found that the prediction was rather successful for five isoflavones in the liver microsomes, but not successful in the intestinal microsomes. We propose that a newly discovered UGT3A1 isoform capable of metabolizing phenols and estrogens might be responsible for the metabolism of isoflavones such as formononetin in humans. In conclusion, the first systematic study of metabolic “fingerprinting” of six common isoflavones showed that each isoflavone has UGT isoform-specific metabolic patterns that are concentration-dependent and predictive of metabolism of the isoflavones in liver microsomes. PMID:19545173

  11. A molecular genetic approach to uncovering the differential functions of dopamine D2 receptor isoforms.

    PubMed

    Wang, Yanyan; Sasaoka, Toshikuni; Dang, Mai T

    2013-01-01

    Alterations in the activity of the dopamine D2 receptor (D2R) have been implicated in several neurological and psychiatric disorders, including schizophrenia, Parkinson's disease, Huntington's disease, Tourette syndrome, attention-deficit hyperactivity disorder (ADHD), and drug addiction. Two isoforms of D2R, long form (D2LR) and short form (D2SR), have been identified. The specific function of each D2R isoform is poorly understood, primarily because isoform-selective pharmacological agents are not available. Using homologous recombination, we have generated D2LR knockout (KO) mice. D2LR KO mice are completely deficient in D2LR, but still express functional D2SR at a level similar to the total D2R level in wild-type (WT) mice. D2LR is generally the predominant isoform expressed in WT mice. We showed that D2LR KO mice displayed a number of robust behavioral phenotypes distinct from WT mice, indicating that D2LR and D2SR have differential functions. In this chapter we describe the generation and characterization of the D2LR KO mouse. This genetic approach provides a valuable research tool to investigate the functional role of individual D2R isoforms in the mammalian central nervous system (CNS).

  12. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    SciTech Connect

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 ismore » not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.« less

  13. ROCK Isoform Regulation of Myosin Phosphatase and Contractility in Vascular Smooth Muscle Cells

    PubMed Central

    Wang, Yuepeng; Zheng, Xiaoyu Rayne; Riddick, Nadeene; Bryden, Meredith; Baur, Wendy; Zhang, Xin; Surks, Howard K.

    2009-01-01

    Abnormal VSMC contraction plays an important role in vascular diseases. The RhoA/ROCK signaling pathway is now well-recognized to mediate vascular smooth muscle contraction in response to vasoconstrictors by inhibiting myosin phosphatase (MLCP) activity and increasing myosin light chain (MLC) phosphorylation. Two ROCK isoforms, ROCK1 and ROCK2, are expressed in many tissues, yet the isoform specific roles of ROCK1 and ROCK2 in vascular smooth muscle (VSM) and the mechanism of ROCK-mediated regulation of MLCP are not well understood. In this study, ROCK2, but not ROCK1, bound directly to the myosin binding subunit (MBS) of MLCP, yet both ROCK isoforms regulated MLCP and MLC phosphorylation. Despite that both ROCK1 and ROCK2 regulated MLCP, the ROCK isoforms had distinct and opposing effects on VSMC morphology and ROCK2, but not ROCK1, had a predominant role in VSMC contractility. These data support that although the ROCK isoforms both regulate MLCP and MLC phosphorylation through different mechanisms, they have distinct roles in VSMC function. PMID:19131646

  14. Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

    PubMed Central

    Kurtz, Joachim; Schmucker, Dietmar; Chen, Wei

    2014-01-01

    The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens. PMID:25310676

  15. Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus.

    PubMed

    Westermeier, F; Sáez, T; Arroyo, P; Toledo, F; Gutiérrez, J; Sanhueza, C; Pardo, F; Leiva, A; Sobrevia, L

    2016-05-01

    The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Malic Enzymes of Higher Plants

    PubMed Central

    Wedding, Randolph T.

    1989-01-01

    The characteristics and distribution of the malic enzyme in plants is discussed as well as those features which appear to be limited to the plant NAD malic enzyme. Regulation of the malic enzyme as it relates to the physiological roles of this enzyme is also discussed. PMID:16666776

  17. Novel Isoform-specific Interfaces Revealed by PKA RIIβ Holoenzyme Structures

    PubMed Central

    Brown, Simon H.J.; Wu, Jian; Kim, Choel; Alberto, Kimberly; Taylor, Susan S.

    2012-01-01

    The PKA catalytic (C) subunit is inhibited by two classes of functionally non-redundant regulatory subunits, RI and RII. Unlike RI-subunits, RII-subunits are both substrates and inhibitors. Because RIIβ knockout mice have important disease phenotypes, the RIIβ holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain although this linker determines the unique globular architecture of the RIIβ holoenzyme. To understand how RIIβ functions as both an inhibitor and substrate and to elucidate the structural role of the linker, we engineered different RIIβ constructs. In the absence of nucleotide RIIβ(108–268) that contains a single cyclic nucleotide binding domain bound C-subunit poorly whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11nM. The RIIβ(108–268) holoenzyme structure (1.62Å) with AMP-PNP/Mn2+, showed that we trapped the RIIβ-subunit in an enzyme:substrate complex with the C-subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn2+ may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIβ(102–265) that contained six additional linker residues. The additional linker residues in RIIβ(102–265) increased the affinity to 1.6 nM suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the αF-αG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region

  18. The β1a Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

    PubMed Central

    Rebbeck, Robyn T.; Karunasekara, Yamuna; Gallant, Esther M.; Board, Philip G.; Beard, Nicole A.; Casarotto, Marco G.; Dulhunty, Angela F.

    2011-01-01

    Although it has been suggested that the C-terminal tail of the β1a subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca2+ release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β1a bound to RyR1 in affinity chromatography. The full-length β1a subunit and the C-terminal peptide increased [3H]ryanodine binding and RyR1 channel activity with an AC50 of 450–600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca2+, ATP, and Mg2+ concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg2+ inhibition or addition of 100 nM Ca2+ (without ATP). Maximum increases were seen with 1–10 μM Ca2+, in the absence of Mg2+ inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [3H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β1a subunit and RyR1 may support an in vivo function of β1a during voltage-activated Ca2+ release. PMID:21320436

  19. Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

    PubMed

    Szabó, C; Bryk, R; Zingarelli, B; Southan, G J; Gahman, T C; Bhat, V; Salzman, A L; Wolff, D J

    1996-08-01

    1. Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L-arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2. GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 microM, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L-arginine and non-competitive vs. tetrahydrobiopterin. 3. Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD-1 cells were stimulated with appropriate mixtures of pro-inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11, 9, 1 and 30 microM, respectively). This suggests that uptake of GED may be cell type and species-dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4. GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 microM). Part of this relaxation was endothelium-dependent, but was not blocked by methylene blue (100 microM), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine-induced, endothelium-dependent relaxations at 10 microM and caused a slight inhibition of the relaxations at 100 microM. The vascular studies

  20. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  1. Enzyme leaps fuel antichemotaxis

    PubMed Central

    Jee, Ah-Young; Dutta, Sandipan; Cho, Yoon-Kyoung

    2018-01-01

    There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration (“antichemotaxis”) by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot. PMID:29255047

  2. Implantable enzyme amperometric biosensors.

    PubMed

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony

    2012-05-15

    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

    SciTech Connect

    O'Donnell, Christopher D.; Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612; Tiwari, Vaibhav

    2006-03-15

    Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1more » gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.« less

  4. Isolation, characterization and comparison of antipeptide and antiprotein rabbit antibodies to the pi-isoform of glutathione S-transferase.

    PubMed

    Di Modugno, F; Rosano L; Castelli, M; Chersi, A

    1998-01-01

    The main linear epitopes of pi-glutathione transferase (pi-GST, EC 2.5.1.18), an enzyme related to cancer progression in a restricted number of tumours, were identified by testing in ELISA the reactivities of polyclonal anti-pi-GST rabbit sera against a panel of 51 overlapping decapeptides, covering the whole 216-residue sequence of the protein. Several major reactivity peaks were detected, each covering two or three adjacent peptides. The most active fragments were then reconstructed by conventional solid-phase synthesis, linked to Sepharose, and used as affinity ligands for isolating specific anti-pi-GST antibody subsets. A second group of antisera was then prepared in rabbits by using as immunogens some of the above described synthetic fragments, linked to a carrier protein, and antipeptide antibodies purified by affinity chromatography. An ELISA test was then performed, using as antigens a panel of peptides and different isoforms of GST, in order to establish whether antibodies isolated from total anti-pi-GST sera would display higher reactivity and specificity, as compared to traditional antipeptide antibodies. Binding data clearly confirm that the formers might be indeed better reagents for the detection and possibly quantitation of pi-GST.

  5. Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system.

    PubMed

    Ma, Yi; Miura, Eriko; Ham, Byung-Kook; Cheng, Hao-Wen; Lee, Young-Jin; Lucas, William J

    2010-11-01

    In yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post-translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel-filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

  6. Targeted Mutations in the Na,K-ATPase Alpha 2 Isoform Confer Ouabain Resistance and Result in Abnormal Behavior in Mice

    PubMed Central

    Schaefer, Tori L.; Lingrel, Jerry B; Moseley, Amy E.; Vorhees, Charles V.; Williams, Michael T.

    2011-01-01

    Sodium and potassium-activated adenosine triphosphatases (Na,K-ATPase) are ubiquitous, participate in osmotic balance and membrane potential, and are composed of α, β, and γ subunits. The α subunit is required for the catalytic and transport properties of the enzyme and contains binding sites for cations, ATP, and digitalis-like compounds including ouabain. There are four known α isoforms; three that are expressed in the CNS in a regional and cell-specific manner. The α2 isoform is most commonly found in astrocytes, pyramidal cells of the hippocampus in adults, and developmentally in several other neuronal types. Ouabain-like compounds are thought to be produced endogenously in mammals, bind the Na,K-ATPase, and function as a stress-related hormone, however, the impact of the Na,K-ATPase ouabain binding site on neurobehavioral function is largely unknown. To determine if the ouabain binding site of the α2 isoform plays a physiological role in CNS function, we examined knock-in mice in which the normally ouabain-sensitive α2 isoform was made resistant (α2R/R) while still retaining basal Na,K-ATPase enzymatic function. Egocentric learning (Cincinnati water maze) was impaired in adult α2R/R mice compared to wild type (WT) mice. They also exhibited decreased locomotor activity in a novel environment and increased responsiveness to a challenge with an indirect sympathomimetic agonist (methamphetamine) relative to WT mice. The α2R/R mice also demonstrated a blunted acoustic startle reflex and a failure to habituate to repeated acoustic stimuli. The α2R/R mice showed no evidence of altered anxiety (elevated zero maze) nor were they impaired in spatial learning or memory in the Morris water maze and neither group could learn in a large Morris maze. These results suggest that the ouabain binding site is involved in specific types of learning and the modulation of dopamine-mediated locomotor behavior. PMID:20936682

  7. The Enzyme Function Initiative†

    PubMed Central

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

    2011-01-01

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

  8. DJ-1 isoforms in whole blood as potential biomarkers of Parkinson disease

    NASA Astrophysics Data System (ADS)

    Lin, Xiangmin; Cook, Travis J.; Zabetian, Cyrus P.; Leverenz, James B.; Peskind, Elaine R.; Hu, Shu-Ching; Cain, Kevin C.; Pan, Catherine; Edgar, John Scott; Goodlett, David R.; Racette, Brad A.; Checkoway, Harvey; Montine, Thomas J.; Shi, Min; Zhang, Jing

    2012-12-01

    DJ-1 is a multifunctional protein that plays an important role in oxidative stress, cell death, and synucleinopathies, including Parkinson disease. Previous studies have demonstrated that total DJ-1 levels decrease in the cerebrospinal fluid, but do not change significantly in human plasma from patients with Parkinson disease when compared with controls. In this study, we measured total DJ-1 and its isoforms in whole blood of patients with Parkinson disease at various stages, Alzheimer disease, and healthy controls to identify potential peripheral biomarkers of PD. In an initial discovery study of 119 subjects, 7 DJ-1 isoforms were reliably detected, and blood levels of those with 4-hydroxy-2-nonenal modifications were discovered to be altered in late-stage Parkinson disease. This result was further confirmed in a validation study of another 114 participants, suggesting that, unlike total DJ-1 levels, post-translationally modified isoforms of DJ-1 from whole blood are candidate biomarkers of late-stage Parkinson disease.

  9. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism

    PubMed Central

    Corominas, Roser; Yang, Xinping; Lin, Guan Ning; Kang, Shuli; Shen, Yun; Ghamsari, Lila; Broly, Martin; Rodriguez, Maria; Tam, Stanley; Trigg, Shelly A.; Fan, Changyu; Yi, Song; Tasan, Murat; Lemmens, Irma; Kuang, Xingyan; Zhao, Nan; Malhotra, Dheeraj; Michaelson, Jacob J.; Vacic, Vladimir; Calderwood, Michael A.; Roth, Frederick P.; Tavernier, Jan; Horvath, Steve; Salehi-Ashtiani, Kourosh; Korkin, Dmitry; Sebat, Jonathan; Hill, David E.; Hao, Tong; Vidal, Marc; Iakoucheva, Lilia M.

    2014-01-01

    Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases. PMID:24722188

  10. The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells.

    PubMed

    Jerrell, Rachel J; Leih, Mitchell J; Parekh, Aron

    2017-06-26

    Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.

  11. Measurement of hepcidin isoforms in human serum by liquid chromatography with high resolution mass spectrometry.

    PubMed

    Handley, Simon; Couchman, Lewis; Sharp, Paul; Macdougall, Iain; Moniz, Cajetan

    2017-03-01

    Hepcidin-25 is the master regulator of iron homeostasis. N-truncated isoforms of hepcidin-25 have been identified (hepcidin-20, -22, -24), although data on the concentrations of these isoforms are sparse. Serum was mixed with aqueous formic acid, and the supernatant loaded onto a 96-well-SPE-plate. Eluted analytes were analyzed using LC-HR-MS. Forty-seven paired dipotassium-EDTA human plasma and serum samples were analyzed. The LLOQ was 1 μg/l (all analytes). Accuracy and precision were acceptable. There was a good correlation (R 2 >0.90, all analytes) between matrices. The median (range) serum hepcidin-20, -22, -24 and -25 concentrations measured were 4 (1-40), 8 (2-20), 8 (1-50) and 39 (1-334) μg/l, respectively. LC-HR-MS is widely applicable to the measurement of hepcidin-25, and truncated isoforms.

  12. Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl

    PubMed Central

    Paynel, Florence; Schaumann, Annick; Arkoun, Mustapha; Douchiche, Olfa; Morvan, Claudine

    2009-01-01

    Background and Aims In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling. Methods CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0·5 mm Cd(NO3)2. PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples. Key Results In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18. Conclusions The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling. PMID:19815572

  13. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

    PubMed Central

    Walsh, Martin J.; Brimacombe, Kyle R.; Veith, Henrike; Bougie, James M.; Daniel, Thomas; Leister, William; Cantley, Lewis C.; Israelsen, William J.; Vander Heiden, Matthew G.; Shen, Min; Auld, Douglas S.; Thomas, Craig J.; Boxer, Matthew B.

    2011-01-01

    Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described. PMID:21958545

  15. The dicotyledonous NAD malic enzyme C4 plant Cleome gynandra displays age-dependent plasticity of C4 decarboxylation biochemistry.

    PubMed

    Sommer, M; Bräutigam, A; Weber, A P M

    2012-07-01

    The C(4) photosynthetic pathway enriches carbon dioxide in the vicinity of Rubisco, thereby enabling plants to assimilate carbon more efficiently. Three canonical subtypes of C(4) exist, named after their main decarboxylating enzymes: NAD-dependent malic enzyme type, NADP-dependent malic enzyme type and phosphoenolpyruvate carboxykinase type. Cleome gynandra is known to perform NAD-ME type C(4) photosynthesis. To further assess the mode of C(4) in C. gynandra and its manifestation in leaves of different age, total enzyme activities of eight C(4) -related enzymes and the relative abundance of 31 metabolites were measured. C. spinosa was used as a C(3) control. C. gynandra was confirmed as an NAD-ME type C(4) plant in mid-aged leaves, whereas a mixed NAD-ME and PEPCK type was observed in older leaves. Young leaves showed a C(3) -C(4) intermediate state with respect to enzyme activities and metabolite abundances. Comparative transcriptome analysis of mid-aged leaves of C. gynandra and C. spinosa showed that the transcript of only one aspartate aminotransferase (AspAT) isoform is highly abundant in C. gynandra. However, the canonical model of the NAD-ME pathway requires two AspATs, a mitochondrial and a cytosolic isoform. Surprisingly, our results indicate the existence of only one highly abundant AspAT isoform. Using GFP-fusion, this isozyme was localised exclusively to mitochondria. We propose a revised model of NAD-ME type C(4) photosynthesis in C. gynandra, in which both AspAT catalysed reactions take place in mitochondria and PEPCK catalyses an alternative decarboxylating pathway. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

  16. Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression.

    PubMed

    Carlini, Federico; Picard, Christophe; Garulli, Céline; Piquemal, David; Roubertoux, Pierre; Chiaroni, Jacques; Chanez, Pascal; Gras, Delphine; Di Cristofaro, Julie

    2017-01-01

    Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab ® . New treatments will aim to target the bronchial epithelium-immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air-liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC ( p  < 0.05); in vitro HBEC re-differentiation from SA displays a particular isoform expression profile compared to MA and HC ( p  = 0.03); HLA-G*01:06 frequency in MA and SA was significantly higher than in the healthy population ( p  = 0.03 and p  < 0.001, respectively); and IL-13 exposure had no impact on HLA-G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs.

  17. Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression

    PubMed Central

    Carlini, Federico; Picard, Christophe; Garulli, Céline; Piquemal, David; Roubertoux, Pierre; Chiaroni, Jacques; Chanez, Pascal; Gras, Delphine; Di Cristofaro, Julie

    2017-01-01

    Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab®. New treatments will aim to target the bronchial epithelium–immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air–liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC (p < 0.05); in vitro HBEC re-differentiation from SA displays a particular isoform expression profile compared to MA and HC (p = 0.03); HLA-G*01:06 frequency in MA and SA was significantly higher than in the healthy population (p = 0.03 and p < 0.001, respectively); and IL-13 exposure had no impact on HLA-G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs. PMID:28303134

  18. Effect of different γ-subunit isoforms on the regulation of AMPK.

    PubMed

    Willows, Robin; Navaratnam, Naveenan; Lima, Ana; Read, Jon; Carling, David

    2017-05-09

    AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. AMPK activation results in a wide range of downstream responses, many of which are associated with improved metabolic outcome, making AMPK an attractive target for the treatment of metabolic diseases. AMPK is a heterotrimeric complex consisting of a catalytic subunit (α) and two regulatory subunits (β and γ). The γ-subunit harbours the nucleotide-binding sites and plays an important role in AMPK regulation in response to cellular energy levels. In mammals, there are three isoforms of the γ-subunit and these respond differently to regulation by nucleotides, but there is limited information regarding their role in activation by small molecules. Here, we determined the effect of different γ-isoforms on AMPK by a direct activator, 991. In cells, 991 led to a greater activation of γ2-containing AMPK complexes compared with either γ1 or γ3. This effect was dependent on the long N-terminal region of the γ2-isoform. We were able to rule out an effect of Ser 108 phosphorylation, since mutation of Ser 108 to alanine in the β2-isoform had no effect on activation of AMPK by 991 in either γ1- or γ2-complexes. The rate of dephosphorylation of Thr 172 was slower for γ2- compared with γ1-complexes, both in the absence and presence of 991. Our studies show that activation of AMPK by 991 depends on the nature of the γ-isoform. This finding may have implications for the design of isoform-selective AMPK activators. © 2017 The Author(s).

  19. miR-222 isoforms are differentially regulated by type-I interferon.

    PubMed

    Nejad, Charlotte; Pillman, Katherine A; Siddle, Katherine J; Pépin, Geneviève; Änkö, Minna-Liisa; McCoy, Claire E; Beilharz, Traude H; Quintana-Murci, Lluís; Goodall, Gregory J; Bracken, Cameron P; Gantier, Michael P

    2018-03-01

    Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) β stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with Salmonella Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation. © 2018 Nejad et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. Novel gb(3) isoforms detected in urine of fabry disease patients: a metabolomic study.

    PubMed

    Auray-Blais, C; Boutin, M

    2012-01-01

    Fabry disease is characterized by the accumulation of globotriaosylsphingosine (lyso-Gb(3)) and globotriaosylceramide (Gb(3)) in biological fluids and tissues. Metabolomic studies recently undertaken by our group, showed the presence of novel plasma and urine lyso-Gb(3)-related analogs in male and female Fabry patients. These analogs are distinguished by differences in structure of the sphingosine moiety. The principal aim of this study was to evaluate the possibility of detecting other Fabry disease biomarkers structurally related to Gb(3). A time-of-flight mass spectrometry metabolomic approach, focusing on mass-to-charge (m/z) ratios from 1000 to 1200 Da, was devised. This m/z window corresponds to the isoforms and potential analogs of Gb(3). Five different categories of Gb(3)- related isoforms/analogs were detected: Gb(3)-related isoforms with saturated fatty acids, methylated Gb(3)-related isoforms, Gb(3)-related isoforms/analogs with one double bond, Gb(3) analogs with hydrated sphingosine, and Gb(3)-related isoforms/analogs with two double bonds. A secondary objective was to elucidate the relationship between Gb(3) and lyso-Gb(3). The methylation observed on Gb(3)-related analogs was not detected on lyso-Gb(3). We speculate that the methylated Gb(3) may be an intermediate compound in the deacylation of Gb(3) to generate the lyso-Gb(3) molecule. We are in the process of devising a quantification methodology for these methylated Gb(3)-related analogs in Fabry patients to try to understand the underlying biochemical mechanisms involved in this complex disease.

  1. Isoform-level gene expression patterns in single-cell RNA-sequencing data.

    PubMed

    Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Pawitan, Yudi; Rantalainen, Mattias

    2018-02-27

    RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16,562 isoform-pairs from 4,929 genes. Among those, 26% of the discovered patterns were significant (p<0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. The effect of drop-out events, mean expression level, and properties of the expression distribution on the performances of ISOP were also investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoformlevel preference, commitment and heterogeneity in single-cell RNA-sequencing data. The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. mattias.rantalainen@ki.se. Supplementary data are available at Bioinformatics online.

  2. Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

    PubMed Central

    Schenk, Martijn F; Cordewener, Jan HG; America, Antoine HP; van't Westende, Wendy PC; Smulders, Marinus JM; Gilissen, Luud JWJ

    2009-01-01

    Background Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. Results All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives. Conclusion Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies. PMID:19257882

  3. Rapid plasmenylethanolamine-selective fusion of membrane bilayers catalyzed by an isoform of glyceraldehyde-3-phosphate dehydrogenase: discrimination between glycolytic and fusogenic roles of individual isoforms.

    PubMed

    Glaser, P E; Gross, R W

    1995-09-26

    Recently we demonstrated that the unique stereoelectronic relationships inherent in the structure of plasmenylethanolamine facilitate membrane fusion, and we postulated the existence of a membrane fusion protein which could exploit the propensity of plasmenylethanolamine molecular species to adapt an inverted hexagonal phase [Glaser & Gross (1994) Biochemistry 33, 5805-5812]. We now report a cryptic membrane fusion activity in rabbit brain cytosol, which requires separation from an endogenous inhibitor to express its activity, and demonstrate that vesicle fusion catalyzed by this protein is highly selective for membrane vesicles containing plasmenylethanolamine. The cytosolic protein catalyzing membrane fusion activity was purified to apparent homogeneity by sequential column chromatographies, revealing a single 38-kDa protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Automated Edman degradation demonstrated that the purified protein is an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was confirmed by Western blot analysis utilizing polyclonal antibodies and by solution-state inactivation of membrane fusion activity by a monoclonal antibody directed against GAPDH. Both GTP-affinity and Mono Q chromatographies resolved GAPDH isoforms that catalyzed dehydrogenase activity from the GAPDH isoform that catalyzed membrane fusion activity. The purified fusion protein was calcium-independent, resistant to treatment with N-ethylmaleimide, and possessed an obligatory requirement for plasmenylethanolamine and cholesterol. High-resolution stopped-flow kinetic analysis of plasmenylethanolamine-facilitated membrane fusion demonstrated that one tetramer of the GAPDH isoform catalyzed one fusion event between two vesicles containing plasmenylethanolamine every millisecond (on average). Collectively, these results constitute the first description of a protein which can catalyze the fusion of vesicles at a rate

  4. Cloning and characterization of cDNAs encoding two normal isoforms of bovine stem cell factor.

    PubMed

    Zhou, J H; Hikono, H; Ohtaki, M; Kubota, T; Sakurai, M

    1994-08-11

    The cDNA clones encoding two isoforms of bovine stem cell factor (bSCF) were obtained using reverse transcriptase-polymerase chain reaction, and their sequences were determined. The deduced amino acid sequences of the longer and shorter isoforms of bSCF consist, respectively, of 274 and 246 residues and show a high degree of identity to those of SCFs of different animal species. Northern blot analysis with the cDNA revealed the expression of a 5.8 kilobase bSCF RNA in fetal bovine tissues.

  5. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis.

    PubMed

    Myllyharju, Johanna

    2003-03-01

    The collagen prolyl 4-hydroxylases (P4Hs), enzymes residing within the endoplasmic reticulum, have a central role in the biosynthesis of collagens. In addition, cytoplasmic P4Hs play a critical role in the regulation of the hypoxia-inducible transcription factor HIFalpha. Collagen and HIF P4Hs constitute enzyme families as several isoenzymes have been identified. Two catalytic alpha subunit isoforms have been cloned and characterized for collagen P4Hs from vertebrates, both of them assembling into alpha(2)beta(2) P4H tetramers in which protein disulfide isomerase (PDI) acts as the beta subunit. The catalytic properties of the two isoenzymes are very similar, but distinct differences are found in the binding properties of peptide substrates and inhibitors, and major differences are seen in the expression patterns of the isoenzymes. The nematode Caenorhabditis elegans has five P4H alpha subunit isoforms, PHY1-PHY5. The C. elegans PHY1 and PHY2, together with PDI, are expressed in the collagen synthesizing hypodermal cells and three P4H forms are assembled from them, a PHY-1/PHY-2/PDI(2) mixed tetramer and PHY-1/PDI and PHY-2/PDI dimers. The mixed tetramer is the main P4H form in wild-type C. elegans. PHY-3 is much shorter than PHY-1 and PHY-2, has a unique expression pattern, and is most likely involved in the synthesis of collagens in early embryos. The genome of Drosophila melanogaster contains approximately 20 P4H alpha subunit-related genes, and that of Arabidopsis thaliana six. One A. thaliana P4H has been cloned and shown to be a soluble monomer with several unexpected properties. It effectively hydroxylates poly(L-proline), (Pro-Pro-Gly)(10) and many other proline-containing peptides.

  6. Structural and functional characterization of an arylamine N-acetyltransferase from the pathogen Mycobacterium abscessus: differences from other mycobacterial isoforms and implications for selective inhibition.

    PubMed

    Cocaign, Angélique; Kubiak, Xavier; Xu, Ximing; Garnier, Guillaume; Li de la Sierra-Gallay, Inès; Chi-Bui, Linh; Dairou, Julien; Busi, Florent; Abuhammad, Areej; Haouz, Ahmed; Dupret, Jean Marie; Herrmann, Jean Louis; Rodrigues-Lima, Fernando

    2014-11-01

    Mycobacterium abscessus is the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies of M. abscessus proteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and functional characterization of an arylamine N-acetyltransferase (NAT) from M. abscessus [(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significant N-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid and p-aminosalicylate. The enzyme is endogenously expressed and functional in both the rough and smooth M. abscessus morphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related to Nocardia farcinica NAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme from M. abscessus and provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance of M. abscessus.

  7. Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase.

    PubMed Central

    Foucault, G; Vacher, M; Merkulova, T; Keller, A; Arrio-Dupont, M

    1999-01-01

    Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres. PMID:9931306

  8. PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae.

    PubMed

    Brown, C E; Tarun, S Z; Boeck, R; Sachs, A B

    1996-10-01

    The Pab1p-dependent poly(A) nuclease (PAN) from Saccharomyces cerevisiae copurifies with polypeptides of approximately 127 and 76 kDa. Previously, it was demonstrated that the 127-kDa Pan2 protein is required for PAN activity (R. Boeck, S. Tarun, M. Reiger, J. Deardorff, S. Müller-Auer, and A.B. Sachs, J. Biol. Chem. 271:432-438, 1996). Here we demonstrate that the 76-kDa protein, encoded by the nonessential PAN3 gene, is also required for enzymatic activity. Deletion of PAN3 resulted in the loss of PAN activity in yeast extracts, and immunodepletion of Pan3p from purified PAN fractions abolished enzymatic activity. We show by coimmunoprecipitation and directed two-hybrid studies that the Pan2 and Pan3 proteins physically interact. In addition, we demonstrate that a deletion of PAN2, PAN3, or both resulted in similar increases in mRNA poly(A) tail lengths in vivo. These data strongly suggest that both Pan2p and Pan3p are required subunits of the PAN enzyme and that PAN functions in vivo to shorten mRNA poly(A) tails.

  9. PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae.

    PubMed Central

    Brown, C E; Tarun, S Z; Boeck, R; Sachs, A B

    1996-01-01

    The Pab1p-dependent poly(A) nuclease (PAN) from Saccharomyces cerevisiae copurifies with polypeptides of approximately 127 and 76 kDa. Previously, it was demonstrated that the 127-kDa Pan2 protein is required for PAN activity (R. Boeck, S. Tarun, M. Reiger, J. Deardorff, S. Müller-Auer, and A.B. Sachs, J. Biol. Chem. 271:432-438, 1996). Here we demonstrate that the 76-kDa protein, encoded by the nonessential PAN3 gene, is also required for enzymatic activity. Deletion of PAN3 resulted in the loss of PAN activity in yeast extracts, and immunodepletion of Pan3p from purified PAN fractions abolished enzymatic activity. We show by coimmunoprecipitation and directed two-hybrid studies that the Pan2 and Pan3 proteins physically interact. In addition, we demonstrate that a deletion of PAN2, PAN3, or both resulted in similar increases in mRNA poly(A) tail lengths in vivo. These data strongly suggest that both Pan2p and Pan3p are required subunits of the PAN enzyme and that PAN functions in vivo to shorten mRNA poly(A) tails. PMID:8816488

  10. Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

    SciTech Connect

    Geiger, Jim

    2013-11-30

    Starch is the major reserve polysaccharide in nature and accounts for the majority of the caloric intact of humans. It is also gaining importance as a renewable and biodegradable industrial material. There is burgeoning interest in increasing the amount and altering the properties of the plant starches by plant genetic modification. A rational approach to this effort will require a detailed, atomic-level understanding of the enzymatic processes that produce the starch granule. The starch granule is a complex particle made up of alternating layers of crystalline and amorphous lamellae. It consists of two types of polymer, amylose, a polymer ofmore » relatively long chains of α-1,4-linked glucans that contain virtually no branches, and amylopectin, which is highly branched and contains much shorter chains. This complex structure is synthesized by the coordinate activities of the starch synthases (SS), which elongate the polysaccharide chain by addition of glucose units via α-1,4 linkages using ADP- glucose as a donor, and branching enzymes (BE), which branch the polysaccharide chain by cleavage of α₋1,4 linkages and subsequent re-attachment via α₋1,6 linkages. Several isoforms of both starch synthase (SS) and branching enzyme (BE) are found in plants, including SSI, SSII, SSIII and granule- bound SS (GBSS), and SBEI, SBEIIa and SBEIIb. These isoforms have different activities and substrate and product specificities and play different roles in creating the granule and determining the properties of the resulting starch. The overarching goal of this proposal is to begin to understand the regulation and specificities of these enzymes at the atomic level. High-resolution X-ray structures of these enzymes bound to substrates and products will be determined to visualize the molecular interactions responsible for the properties of the enzymes. Hypotheses regarding these issues will then be tested using mutagenesis and enzyme assays. To date, we have determined the

  11. New insights on NOX enzymes in the central nervous system.

    PubMed

    Nayernia, Zeynab; Jaquet, Vincent; Krause, Karl-Heinz

    2014-06-10

    There is increasing evidence that the generation of reactive oxygen species (ROS) in the central nervous system (CNS) involves the NOX family of nicotinamide adenine dinucleotide phosphate oxidases. Controlled ROS generation appears necessary for optimal functioning of the CNS through fine-tuning of redox-sensitive signaling pathways, while overshooting ROS generation will lead to oxidative stress and CNS disease. NOX enzymes are not only restricted to microglia (i.e. brain phagocytes) but also expressed in neurons, astrocytes, and the neurovascular system. NOX enzymes are involved in CNS development, neural stem cell biology, and the function of mature neurons. While NOX2 appears to be a major source of pathological oxidative stress in the CNS, other NOX isoforms might also be of importance, for example, NOX4 in stroke. Globally speaking, there is now convincing evidence for a role of NOX enzymes in various neurodegenerative diseases, cerebrovascular diseases, and psychosis-related disorders. The relative importance of specific ROS sources (e.g., NOX enzymes vs. mitochondria; NOX2 vs. NOX4) in different pathological processes needs further investigation. The absence of specific inhibitors limits the possibility to investigate specific therapeutic strategies. The uncritical use of non-specific inhibitors (e.g., apocynin, diphenylene iodonium) and poorly validated antibodies may lead to misleading conclusions. Physiological and pathophysiological studies with cell-type-specific knock-out mice will be necessary to delineate the precise functions of NOX enzymes and their implications in pathomechanisms. The development of CNS-permeant, specific NOX inhibitors will be necessary to advance toward therapeutic applications.

  12. New Insights on NOX Enzymes in the Central Nervous System

    PubMed Central

    Nayernia, Zeynab; Jaquet, Vincent

    2014-01-01

    Abstract Significance: There is increasing evidence that the generation of reactive oxygen species (ROS) in the central nervous system (CNS) involves the NOX family of nicotinamide adenine dinucleotide phosphate oxidases. Controlled ROS generation appears necessary for optimal functioning of the CNS through fine-tuning of redox-sensitive signaling pathways, while overshooting ROS generation will lead to oxidative stress and CNS disease. Recent Advances: NOX enzymes are not only restricted to microglia (i.e. brain phagocytes) but also expressed in neurons, astrocytes, and the neurovascular system. NOX enzymes are involved in CNS development, neural stem cell biology, and the function of mature neurons. While NOX2 appears to be a major source of pathological oxidative stress in the CNS, other NOX isoforms might also be of importance, for example, NOX4 in stroke. Globally speaking, there is now convincing evidence for a role of NOX enzymes in various neurodegenerative diseases, cerebrovascular diseases, and psychosis-related disorders. Critical Issues: The relative importance of specific ROS sources (e.g., NOX enzymes vs. mitochondria; NOX2 vs. NOX4) in different pathological processes needs further investigation. The absence of specific inhibitors limits the possibility to investigate specific therapeutic strategies. The uncritical use of non-specific inhibitors (e.g., apocynin, diphenylene iodonium) and poorly validated antibodies may lead to misleading conclusions. Future Directions: Physiological and pathophysiological studies with cell-type-specific knock-out mice will be necessary to delineate the precise functions of NOX enzymes and their implications in pathomechanisms. The development of CNS-permeant, specific NOX inhibitors will be necessary to advance toward therapeutic applications. Antioxid. Redox Signal. 20: 2815–2837. PMID:24206089

  13. Tea consumption modulates hepatic drug metabolizing enzymes in Wistar rats.

    PubMed

    Maliakal, P P; Coville, P F; Wanwimolruk, S

    2001-04-01

    The antioxidant, antimutagenic and anticarcinogenic activities of green tea and its polyphenols have been reported. As bioactivation of the precarcinogens and detoxification of ultimate carcinogens are mainly carried out by hepatic metabolizing enzymes, we have investigated the modulation of these enzyme activities subsequent to tea consumption in rats. Female Wistar rats were divided into eight groups (n = 5). Six groups were given aqueous solutions (2%, w/v) of six different teas (New Zealand green tea, Australian green tea, Java green tea, Dragon green tea, Gunpowder green tea or English Breakfast black tea) as the sole source of fluid. One group was given a standard green tea extract (0.5%, w/v) while the control group had free access to water. At the end of four-weeks treatment, different cytochrome P450 (CYP) isoform and phase II enzyme activities were determined by incubation of the liver microsomes or cytosols with appropriate substrates. CYP 1A2 activity was markedly increased in all the tea treatment groups (P < 0.05). CYP 1A1 activity was increased significantly in most of the groups except for the Madura, Gunpowder, and Java green tea-treatment groups. Cytosolic glutathione-S-transferase activity was significantly increased (P< 0.05) in the New Zealand, Gunpowder, and Java green tea-treatment groups. The microsomal UDP-glucuronosyl transferase activity remained unchanged or was moderately increased in most of the groups. The balance between the phase I carcinogen-activating enzymes and the phase II detoxifying enzymes could be important in determining the risk of developing chemically-induced cancer.

  14. Isoform-specific changes in the Na,K-ATPase of rat soleus muscle during acute hindlinb suspension

    NASA Astrophysics Data System (ADS)

    Krivoi, Igor; Heiny, Judith; Bouzinova, Elena; Matchkov, Vladimir; Kravtsova, Violetta; Petrov, Aleksey; Zefirov, Andrey; Vasiliev, Alexander

    The largest pool of Na,K-ATPase (NKA) in a vertebrate's body is contained in the skeletal muscles where the alpha1 and alpha2 isoforms of NKA alpha subunit are expressed. The NKA is critically important for excitability, electrogenesis and contractility of skeletal muscle. Skeletal muscle use strongly regulates the content of NKA, and increased muscle activity differently regulates the alpha1 and alpha2 isoforms. However, whether skeletal muscle disuse affects NKA content and activity has not been investigated. This study examines for the first time the consequences of acute hindlinb suspension (HS) on the alpha1 and alpha2 NKA isozymes in rat soleus muscle. We subjected rats to HS for 6-12 hours and analyzed its effect on the resting membrane potential (RMP) in different sarcolemma regions of m.soleus fibers; the electrogenic transport activity, protein content and mRNA expression of the alpha1 and alpha2 NKA; the extracellular level of acetylcholine, and the plasma membrane localization of the alpha2 isozyme using confocal microscopy with cytochemistry. Our results show that 6 h HS specifically decreases the electrogenic activity of the NKA alpha2 isozyme and depolarizes m.soleus fibers. These effects are irreversible in the extrajunctional membrane region up to 12 h HS. The decreased alpha2 NKA activity is due to a decrease in enzyme activity rather than by altered protein content, mRNA expression, or localization in the sarcolemma. In addition, HS does not alter the alpha2 NKA electrogenic transport due to decreased extracellular acetylcholine level. However, adaptive mechanism(s) operate at the junctional membrane to restore alpha2 NKA electrogenic activities and the RMP after 12 h of HS. This mechanism operates specifically at the synaptic membrane region, presumably via increase in both alpha2 isozyme mRNA expression and protein content. This basic information on a protein as vital as the NKA is expected to advance our understanding of the cellular and

  15. Repertoire of malic enzymes in yeast and fungi: insight into their evolutionary functional and structural significance.

    PubMed

    Vorapreeda, Tayvich; Thammarongtham, Chinae; Cheevadhanarak, Supapon; Laoteng, Kobkul

    2013-12-01

    Malic enzyme (ME) is one of the important enzymes for furnishing the cofactor NAD(P)H for the biosynthesis of fatty acids and sterols. Due to the existence of multiple ME isoforms in a range of oleaginous microbes, a molecular basis for the evolutionary relationships amongst the enzymes in oleaginous fungi was investigated using sequence analysis and structural modelling. Evolutionary distance and structural characteristics were used to discriminate the MEs of yeasts and fungi into several groups. Interestingly, the NADP(+)-dependent MEs of Mucoromycotina had an unusual insertion region (FLxxPG) that was not found in other fungi. However, the subcellular compartment of the Mucoromycotina enzyme could not be clearly identified by an analysis of signal peptide sequences. A constructed structural model of the ME of Mucor circinelloides suggested that the insertion region is located at the N-terminus of the enzyme (aa 159-163). In addition, it is presumably part of the dimer interface region of the enzyme, which might provide a continuously positively charged pocket for the efficient binding of negatively charged effector molecules. The discovery of the unique structure of the Mucoromycotina ME suggests the insertion region could be involved in particular kinetics of this enzyme, which may indicate its involvement in the lipogenesis of industrially important oleaginous microbes.

  16. Quantitative variation in effector activity of ToxA isoforms from Stagonospora nodorum and Pyrenophora tritici-repentis

    USDA-ARS?s Scientific Manuscript database

    ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tritici-repentis. In this study, all eight mature isoforms of the ToxA protein were purified and compared. Circular dichroism spectra indicated that all isoforms were structurally intact and had indistingu...

  17. Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

    USDA-ARS?s Scientific Manuscript database

    The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) [Lp(a)], Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examinati...

  18. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    USDA-ARS?s Scientific Manuscript database

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  19. Complex tropomyosin and troponin T isoform expression patterns in orbital and global fibers of adult dog and rat extraocular muscles.

    PubMed

    Bicer, Sabahattin; Reiser, Peter J

    2013-08-01

    We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and β-Tm) and slow (γ-Tm and β-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, β and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

  20. Sulfite oxidizing enzymes

    PubMed Central

    Feng, Changjian; Tollin, Gordon; Enemark, John H.

    2007-01-01

    Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme

  1. Micellar Polymer Encapsulation of Enzymes.

    PubMed

    Besic, Sabina; Minteer, Shelley D

    2017-01-01

    Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between electrode and the biocatalyst. Another problem is inefficient and short term stability of catalytic activity within the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the non-uniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details encapsulation techniques where an enzyme will be immobilized within the pores/pockets of the hydrophobically modified micellar polymers such as

  2. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  3. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  4. Robust enzyme-silica composites made from enzyme nanocapsules.

    PubMed

    Li, Jie; Jin, Xin; Liu, Yang; Li, Fan; Zhang, Linlin; Zhu, Xianyuan; Lu, Yunfeng

    2015-06-14

    Novel enzyme composites are synthesized first by in situ polymerization around enzymes and a subsequent sol-gel process. Both the polymer shell and the silica shell with desired functional moieties provide not only great enzyme protection but also a favorable microenvironment, resulting in significantly enhanced activity and stability.

  5. Enzyme Molar Fractions: A Powerful Tool for Understanding Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Serra, Juan L.; And Others

    1986-01-01

    Deduces the relationship between reduced velocity and molar fractions for productive enzyme complexes; obtains the mathematical expression of molar fractions for an enzyme with two specific binding sites per molecule; and proposes a useful plot to follow the dependence of enzyme molar fractions with the concentration of one of its ligands. (JN)

  6. The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

    PubMed

    Cao, Yanyun; Zhao, Luping; Ying, Yusang; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-06-15

    Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions.

    PubMed

    Kjelgaard-Hansen, Mads; Christensen, Michelle B; Lee, Marcel H; Jensen, Asger L; Jacobsen, Stine

    2007-06-15

    Serum amyloid A (SAA) is a major acute phase protein in dogs. However, knowledge of qualitative properties of canine SAA and extent of its synthesis in extrahepatic tissues is limited. The aim of the study was to investigate expression of different SAA isoforms in serum and synovial fluid in samples obtained from dogs (n=16) suffering from different inflammatory or non-inflammatory conditions, which were either related or unrelated to joints. Expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. Serum amyloid A was present in serum from all dogs with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local production of these isoforms in the canine inflamed joint.

  8. Identification and functional analysis of peroxiredoxin isoforms in Euglena gracilis.

    PubMed

    Tamaki, Shun; Maruta, Takanori; Sawa, Yoshihiro; Shigeoka, Shigeru; Ishikawa, Takahiro

    2014-01-01

    Euglena gracilis lacks catalase and contains ascorbate peroxidase (APX) which is localized exclusively in the cytosol. Other enzymes that scavenge reactive oxygen species (ROS) in Euglena have not yet been identified; therefore, ROS metabolism, especially in organelles, remains unclear in Euglena. The full-length cDNAs of four Euglena peroxiredoxins (EgPrxs) were isolated in this study. EgPrx1 and -4 were predicted to be localized in the cytosol, and EgPrx2 and -3 in plastids and mitochondria, respectively. The catalytic efficiencies of recombinant EgPrxs were similar to those of plant thiol-peroxidases, but were markedly lower than those of APX from Euglena. However, transcript levels of EgPrx1, -2, and -3 were markedly higher than those of APX. The growth rate of Euglena cells, in which the expression of EgPrx1 and -4 was suppressed by gene silencing, was markedly reduced under normal conditions, indicating physiological significance of Prx proteins.

  9. Biochemical approaches to C4 photosynthesis evolution studies: the case of malic enzymes decarboxylases.

    PubMed

    Saigo, Mariana; Tronconi, Marcos A; Gerrard Wheeler, Mariel C; Alvarez, Clarisa E; Drincovich, María F; Andreo, Carlos S

    2013-11-01

    C4 photosynthesis enables the capture of atmospheric CO2 and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO2 and high atmospheric levels of O2 (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C3 ancestors which were already optimized for C4 function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C3 plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C4 mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C4 isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO2 for RuBisCO in engineering C3 species to perform C4 photosynthesis.

  10. Coordinated Changes in Antioxidative Enzymes Protect the Photosynthetic Machinery from Salinity Induced Oxidative Damage and Confer Salt Tolerance in an Extreme Halophyte Salvadora persica L.

    PubMed Central

    Rangani, Jaykumar; Parida, Asish K.; Panda, Ashok; Kumari, Asha

    2016-01-01

    Salinity-induced modulations in growth, photosynthetic pigments, relative water content (RWC), lipid peroxidation, photosynthesis, photosystem II efficiency, and changes in activity of various antioxidative enzymes were studied in the halophyte Salvadora persica treated with various levels of salinity (0, 250, 500, 750, and 1000 mM NaCl) to obtain an insight into the salt tolerance ability of this halophyte. Both fresh and dry biomass as well as leaf area (LA) declined at all levels of salinity whereas salinity caused an increase in leaf succulence. A gradual increase was observed in the Na+ content of leaf with increasing salt concentration up to 750 mM NaCl, but at higher salt concentration (1000 mM NaCl), the Na+ content surprisingly dropped down to the level of 250 mM NaCl. The chlorophyll and carotenoid contents of the leaf remained unaffected by salinity. The photosynthetic rate (PN), stomatal conductance (gs), the transpiration rate (E), quantum yield of PSII (ΦPSII), photochemical quenching (qP), and electron transport rate remained unchanged at low salinity (250 to 500 mM NaCl) whereas, significant reduction in these parameters were observed at high salinity (750 to 1000 mM NaCl). The RWC% and water use efficiency (WUE) of leaf remained unaffected by salinity. The salinity had no effect on maximum quantum efficiency of PS II (Fv/Fm) which indicates that PS II is not perturbed by salinity-induced oxidative damage. Analysis of the isoforms of antioxidative enzymes revealed that the leaves of S. persica have two isoforms each of Mn-SOD and Fe-SOD and one isoform of Cu-Zn SOD, three isoforms of POX, two isoforms of APX and one isoform of CAT. There was differential responses in activity and expression of different isoforms of various antioxidative enzymes. The malondialdehyde (MDA) content (a product of lipid peroxidation) of leaf remained unchanged in S. persica treated with various levels of salinity. Our results suggest that the absence of pigment

  11. Coordinated Changes in Antioxidative Enzymes Protect the Photosynthetic Machinery from Salinity Induced Oxidative Damage and Confer Salt Tolerance in an Extreme Halophyte Salvadora persica L.

    PubMed

    Rangani, Jaykumar; Parida, Asish K; Panda, Ashok; Kumari, Asha

    2016-01-01

    Salinity-induced modulations in growth, photosynthetic pigments, relative water content (RWC), lipid peroxidation, photosynthesis, photosystem II efficiency, and changes in activity of various antioxidative enzymes were studied in the halophyte Salvadora persica treated with various levels of salinity (0, 250, 500, 750, and 1000 mM NaCl) to obtain an insight into the salt tolerance ability of this halophyte. Both fresh and dry biomass as well as leaf area (LA) declined at all levels of salinity whereas salinity caused an increase in leaf succulence. A gradual increase was observed in the Na(+) content of leaf with increasing salt concentration up to 750 mM NaCl, but at higher salt concentration (1000 mM NaCl), the Na(+) content surprisingly dropped down to the level of 250 mM NaCl. The chlorophyll and carotenoid contents of the leaf remained unaffected by salinity. The photosynthetic rate (PN), stomatal conductance (gs), the transpiration rate (E), quantum yield of PSII (ΦPSII), photochemical quenching (qP), and electron transport rate remained unchanged at low salinity (250 to 500 mM NaCl) whereas, significant reduction in these parameters were observed at high salinity (750 to 1000 mM NaCl). The RWC% and water use efficiency (WUE) of leaf remained unaffected by salinity. The salinity had no effect on maximum quantum efficiency of PS II (Fv/Fm) which indicates that PS II is not perturbed by salinity-induced oxidative damage. Analysis of the isoforms of antioxidative enzymes revealed that the leaves of S. persica have two isoforms each of Mn-SOD and Fe-SOD and one isoform of Cu-Zn SOD, three isoforms of POX, two isoforms of APX and one isoform of CAT. There was differential responses in activity and expression of different isoforms of various antioxidative enzymes. The malondialdehyde (MDA) content (a product of lipid peroxidation) of leaf remained unchanged in S. persica treated with various levels of salinity. Our results suggest that the absence of pigment

  12. Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent

    PubMed Central

    Seutter von Loetzen, Christian; Jacob, Thessa; Hartl-Spiegelhauer, Olivia; Vogel, Lothar; Schiller, Dirk; Spörlein-Güttler, Cornelia; Schobert, Rainer; Vieths, Stefan; Hartl, Maximilian Johannes; Rösch, Paul

    2015-01-01

    Each spring millions of patients suffer from allergies when birch pollen is released into the air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms. Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms differing in their allergenic potential also show an individual, highly specific binding behaviour for the different ligands. This specificity is driven by the sugar moieties of the ligands rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1 isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus individual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective chemical sunscreen for pollen DNA, but may also play an important role in recognition processes during pollination. PMID:26042900

  13. Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice

    PubMed Central

    Diaz-Castroverde, Sabela; Gómez-Hernández, Almudena; Fernández, Silvia; García-Gómez, Gema; Di Scala, Marianna; González-Aseguinolaza, Gloria; Fernández-Millán, Elisa; González-Rodríguez, Águeda; García-Bravo, María; Chambon, Pierre; Álvarez, Carmen; Perdomo, Liliana; Beneit, Nuria; Benito, Manuel

    2016-01-01

    ABSTRACT Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte­-specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus. PMID:27562101

  14. HPLC separation of human serum albumin isoforms based on their isoelectric points

    PubMed Central

    Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA–SHg+), HSA with Cys34 oxidized to sulfenic acid (HSA–SOH) and HSA oxidized to sulfinate anion (HSA–SO2−) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3–585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA PMID:24316526

  15. Subcellular Targeting of Nine Calcium-Dependent Protein Kinase Isoforms from Arabidopsis1

    PubMed Central

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism. PMID:12913141

  16. ACA12 Is a Deregulated Isoform of Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana

    PubMed Central

    Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F.; De Michelis, Maria Ida

    2014-01-01

    Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues - highly conserved in other ACA isoforms - localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation. PMID:24101142

  17. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis.

    PubMed

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M; Hrabak, Estelle M; Harmon, Alice C; Pickard, Barbara G; Harper, Jeffrey F

    2003-08-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  18. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    PubMed

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Differential Regulation of Aromatase Isoforms and Tissue Responses to Environmental Chemicals in Fish

    EPA Science Inventory

    As in mammals, aromatase plays a basic role in fish reproduction. Unlike most mammals, with only one form of aromatase, fish have two distinct forms. One isoform, P450aromA, predominates in ovaries. Ovarian aromatase activity controls circulating levels of estrogens and is critic...

  20. Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles.

    PubMed

    Dai, Longjun; Nie, Zhiyi; Kang, Guijuan; Li, Yu; Zeng, Rizhong

    2017-02-01

    Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein). Copyright © 2016. Published by Elsevier Masson SAS.

  1. Functional distinctions of protein kinase B/Akt isoforms defined by their influence on cell migration.

    PubMed

    Stambolic, Vuk; Woodgett, James R

    2006-09-01

    The three mammalian members of the protein kinase B/Akt (PKB/Akt) family have been implicated in a plethora of cellular signaling processes with key functions in control of cellular metabolism, growth, proliferation and apoptosis. As a major target of phosphatidylinositol (PI) 3-kinase signaling, the PKB/Akt isoforms also have central roles in a variety of human cancers, with effects on tumor initiation, progression and metastasis. It has been shown that isoform-specific functions of PKB/Akt family members can contribute to tumorigenesis on multiple levels. A series of recent studies documents the isoform-specific functions of PKB/Akt family members in regulation of cellular motility and migration by influencing numerous cellular targets involved in organization of the actin cytoskeleton, cellular interaction with the extracellular matrix, expression of motility genes and establishment of cellular polarity. A thorough insight into the isoform-specific roles of PKB/Akt proteins is essential for a full understanding of the complex biological outcomes elicited by PI 3-kinase and PKB/Akt signaling.

  2. Distinct Isoforms of the RFX Transcription Factor DAF-19 Regulate Ciliogenesis and Maintenance of Synaptic Activity

    PubMed Central

    Senti, Gabriele

    2008-01-01

    Neurons form elaborate subcellular structures such as dendrites, axons, cilia, and synapses to receive signals from their environment and to transmit them to the respective target cells. In the worm Caenorhabditis elegans, lack of the RFX transcription factor DAF-19 leads to the absence of cilia normally found on 60 sensory neurons. We now describe and functionally characterize three different isoforms of DAF-19. The short isoform DAF-19C is specifically expressed in ciliated sensory neurons and sufficient to rescue all cilia-related phenotypes of daf-19 mutants. In contrast, the long isoforms DAF-19A/B function in basically all nonciliated neurons. We discovered behavioral and cellular phenotypes in daf-19 mutants that depend on the isoforms daf-19a/b. These novel synaptic maintenance phenotypes are reminiscent of synaptic decline seen in many human neurodegenerative disorders. The C. elegans daf-19 mutant worms can thus serve as a molecular model for the mechanisms of functional neuronal decline. PMID:18843046

  3. Role of APOE Isoforms in the Pathogenesis of TBI Induced Alzheimer’s Disease

    DTIC Science & Technology

    2015-10-01

    factor for sporadic Alzheimer disease (AD). Importantly, TBI is a risk factor for the subsequent development of AD particularly among APOE?4 carriers...APOE isoforms, ABCA1, Alzheimer disease, APPmice, amyloid beta, axonal injury, inflamma- tory reaction, trnascriptome, high-throughput massive

  4. NMR resonance assignments of a hypoallergenic isoform of the major birch pollen allergen Bet v 1.

    PubMed

    Ahammer, Linda; Grutsch, Sarina; Wallner, Michael; Ferreira, Fatima; Tollinger, Martin

    2017-10-01

    In Northern America and Europe a great number of people are suffering from birch pollen allergy and pollen related food allergies. The trigger for these immunological reactions is the 17.5 kDa major birch pollen allergen Bet v 1, which belongs to the family of PR-10 (pathogenesis-related) proteins. In nature, Bet v 1 occurs as a mixture of various isoforms that possess different immunological properties despite their high sequence identities. Bet v 1.0102 (Bet v 1d), which is investigated here, is a hypoallergenic isoform of Bet v 1 and a potential candidate for allergen-specific immunotherapy. We assigned the backbone and side chain 1 H, 13 C and 15 N resonances of this protein and predicted its secondary structure. The NMR-chemical shift data indicate that Bet v 1.0102 is composed of three α-helices and a seven stranded β-sheet, in agreement with the known structure of the hyperallergenic isoform Bet v 1.0101 (Bet v 1a). Our resonance assignments create the foundation for detailed characterization of the dynamic properties of Bet v 1 isoforms by NMR relaxation measurements.

  5. 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

  6. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  7. Streptozocin Diabetes Elevates all Isoforms of TGF-β in the Rat Kidney

    PubMed Central

    Snelling, Dustin M.; Langer, William J.

    2001-01-01

    Transforming growth factor beta (TGF-β) is a major promoter of diabetic nephropathy. While TGF-β1 is the most abundaft renal isoform, types 2 and 3 are present as well and have identical in vitro effects. Whole kidney extracts were studied 2 weeks after induction of streptozocin diabetes and in control rats. Mean glomerular area was 25% greater in the diabetic animals. TGF-β1 showed a 2-fold increase in message with a 3-fold increase in protein. TGF-β2 mRNA increased approximately 6% while its protein doubled. TGF-β-message increased by 25%, producing a 35% increase in its protein. TGF-β- inducible gene H3 mRNA was increased 35% in the diabetic animals, consistent with increased activity of this growth factor. All isoforms of TGF-β are increased in the diabetic rat kidney. Future studies need to address the specific role that each isoform plays in diabetic nephropathy as well as the impact of therapies on each isoform. PMID:12369727

  8. 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. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Treating Wastewater With Immobilized Enzymes

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  10. The Catalytic Function of Enzymes.

    ERIC Educational Resources Information Center

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  11. Identification of PIWIL1 Isoforms and Their Expression in Bovine Testes, Oocytes, and Early Embryos.

    PubMed

    Russell, Stewart J; Stalker, Leanne; Gilchrist, Graham; Backx, Alanna; Molledo, Gonzalo; Foster, Robert A; LaMarre, Jonathan

    2016-04-01

    PIWI proteins are members of the larger Argonaute family and bind to specific 24-32 nucleotide RNAs called PIWI-interacting RNAs (piRNAs). PIWI-interacting RNAs direct PIWI-mediated suppression of retrotransposon expression in the male germline in humans and mice, but their roles in bovine reproduction and embryogenesis are unknown. Although the majority of research in mammals has focused on the functions of PIWI proteins during spermatogenesis, this family of proteins and their associated piRNAs have recently been identified in early embryos. The goals of this study were to characterize the expression of PIWIL1 in bovine testis, oocytes, and early embryos. A full-lengthPIWIL1transcript and protein was found in the testis, specifically in the germs cells of mature seminiferous tubules. RNA-immunoprecipitation demonstrated the presence of putative piRNAs with a mean length of 30 nucleotides bound to PIWIL1 in testes. 3'-Rapid amplification of cDNA ends analysis ofPIWIL1transcripts in testes and oocytes revealed two shorter isoforms in addition to the full-length transcript that was only present in testes. TruncatedPIWIL1isoforms in oocytes and testes were confirmed through amplification of their unique intronic fragments. Expression profiling ofPIWIL1through early embryogenesis demonstrated peak mRNA expression at the 2-cell stage with decreasing levels through to the blastocyst. PIWIL1-YFP fusion plasmids were produced for each isoform and expressed in HEK 293 cells, demonstrating nuclear exclusion and size-specific banding of the different isoforms. These data represent the first comprehensive characterization of PIWIL1 in bovine, revealing functional similarities with PIWIL1 in other species and suggest tissue-specific expression of several isoforms. © 2016 by the Society for the Study of Reproduction, Inc.

  12. Characterization of sex hormone-binding globulin isoforms in hypothyroid women.

    PubMed

    Brenta, G; Bedecarras, P; Schnitman, M; Gurfinkiel, M; Damilano, S; Campo, S; Pisarev, M A

    2002-02-01

    Liver sex hormone-binding globulin (SHBG) biosynthesis is regulated by triiodothyronine (T3). This regulation is responsible for increased serum SHBG concentrations in hyperthyroid states. However, in hypothyroidism, normal SHBG levels are frequently found. To understand this we have characterized circulating SHBG isoforms according to their sialic acid content, which determines its half-life, in euthyroid and hypothyroid women. Six euthyroid (aged 56 +/- 8 years) and five hypothyroid women (51 +/- 13 years) were studied. Their body mass index (BMI) range was 20-25. Hypothyroidism diagnosis was based on clinical findings, elevated basal thyrotropin (TSH) and decreased T3 and thyroxine (T4) values. Total SHBG was measured by radioimmunoassay (RIA) and SHBG isoforms were isolated using preparative isoelectrofocusing. For comparisons, two-tailed t test was applied. No statistical difference was found between the total SHBG levels of hypothyroid and euthyroid postmenopausal women. Three groups of SHBG isoforms were isolated in the euthyroid group: S(I): pl: 5.0-5.2: 20% +/- 4%, S(II) : pl 5.2-5.4: 50% +/- 3% and S(III): pl 5.4-5.6: 29% +/- 4%. In hypothyroid patients, although the three groups of isoforms were isolated in the same pH range, S(I) and S(II) proportions were different (p < 0.001) when compared to normal women: S(I): 34% +/- 4%, S(II): 33% +/- 9.9% and S(III): 29% +/- 5.7%. These results show that hypothyroid patients have a higher proportion of more acidic SHBG isoforms. This variation may explain the normal levels of serum SHBG observed in hypothyroidism.