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Sample records for 1-aminocyclopropane-1-carboxylate isoforms regulates

  1. A Combinatorial Interplay Among the 1-Aminocyclopropane-1-carboxylate Isoforms Regulates Ethylene Biosynthesis in Arabidopsis thaliana

    Ethylene (C2H4) is a unique plant-signaling molecule that regulates numerous developmental processes. The key enzyme in the two-step biosynthetic pathway of ethylene is 1-aminocyclopropane-1-carboxylate synthase (ACS), which catalyzes the conversion of Sadenosyl-methionine (AdoMet) to ACC, the precu...

  2. Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress.

    Datta, Riddhi; Kumar, Deepak; Sultana, Asma; Hazra, Saptarshi; Bhattacharyya, Dipto; Chattopadhyay, Sharmila

    2015-12-01

    Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress. PMID:26463088

  3. Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

    Saito Koji

    2005-08-01

    Full Text Available Abstract Background In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1 is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS, in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits. Results Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type. Conclusion These results suggest that ETO1

  4. Isolation and Molecular Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase Genes in Hevea brasiliensis

    Jia-Hong Zhu

    2015-02-01

    Full Text Available Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1–7 of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment. These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production.

  5. Expression of 1-aminocyclopropane-1-carboxylate oxidase during leaf ontogeny in white clover.

    Hunter, D A; Yoo, S D; Butcher, S M; McManus, M T

    1999-05-01

    We examined the expression of three distinct 1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf ontogeny in white clover (Trifolium repens). Significant production of ethylene occurs at the apex, in newly initiated leaves, and in senescent leaf tissue. We used a combination of reverse transcriptase-polymerase chain reaction and 3'-rapid amplification of cDNA ends to identify three distinct DNA sequences designated TRACO1, TRACO2, and TRACO3, each with homology to 1-aminocyclopropane-1-carboxylic acid oxidase. Southern analysis confirmed that these sequences represent three distinct genes. Northern analysis revealed that TRACO1 is expressed specifically in the apex and TRACO2 is expressed in the apex and in developing and mature green leaves, with maximum expression in developing leaf tissue. The third gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were raised to each gene product expressed in Escherichia coli, and western analysis showed that the TRACO1 antibody recognizes a protein of approximately 205 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed preferentially in apical tissue. The TRACO2 antibody recognizes a protein of approximately 36.4 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed in the apex and in developing and mature green leaves, with maximum expression in mature green tissue. No protein recognition by the TRACO3 antibody could be detected in senescent tissue or at any other stage of leaf development. PMID:10318691

  6. RNA Interference of 1-Aminocyclopropane-1-carboxylic Acid Oxidase (ACO1 and ACO2 Genes Expression Prolongs the Shelf Life of Eksotika (Carica papaya L. Papaya Fruit

    Rogayah Sekeli

    2014-06-01

    Full Text Available The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6. Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  7. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[14C]Met. Peptide sequencing of both 3H- and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6

  8. Ethylene Production and 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase Gene Expression in Tomato(Lycopsicon esculentum Mill.) Leaves Under Enhanced UV-B Radiation

    Lizhe An; Xunling Wang; Xiaofeng Xu; Hongguan Tang; Manxiao Zhang; Zongdong Hou; Yanhong Liu; Zhiguang Zhao; Huyuan Feng; Shijian Xu

    2006-01-01

    Tomato (Lycopsicon esculentum Mill.) plants grown in a greenhouse were irradiated with two different levels of UV-B, namely 8.82 (T1) and 12.6 kJ/m2 per day (T2). Ethylene production, 1-aminocyclopropane-1carboxylate (ACC) content, 1-(malonylamino) cyclopvopane-1-carboxylic acid (MACC) content, gene expression of ACC synthase (EC 4.4.1.14), and ACC oxidase activity in tomato leaves were determined. The results indicated that ACC content, the activity of ACC synthase and ACC oxidase, and ethylene production increased continuously under low doses of UV-B radiation, whereas at high doses of radiation these parameters increased during the first 12 d and then started to decrease. The MACC content increased continuously over 18 d under both doses of UV-B irradiation. The changes in ACC content, ACC synthase activity,ACC oxidase activity, the transcriptional level of the ACC synthase gene, and ethylene production were consistent with each other, suggesting that ACC synthase was the key enzyme in ethylene biosynthesis and that ethylene production in tomato leaf tissues under UV-B radiation could be regulated by the expression of the ACC synthase gene. The results also indicate that the change in ethylene metabolism may be an adaptive mechanism to enhanced UV-B radiation.

  9. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants. PMID:25674802

  10. 1-Aminocyclopropane-1-carboxylic acid and abscisic acid during the germination of sugar beet (Beta vulgaris L.): a comparative study of fruits and seeds

    Hermann, K.; Meinhard, J.; Dobrev, Petre; Linkies, A.; Pešek, Bedřich; Heß, B.; Macháčková, Ivana; Fischer, U.; Leubner-Metzger, G.

    2007-01-01

    Roč. 58, č. 11 (2007), s. 3047-3060. ISSN 0022-0957 Institutional research plan: CEZ:AV0Z50380511 Keywords : abscisic acid (ABA) * ABA 8'-hydroxylase (CYP707A) * 1-aminocyclopropane-1-carboxylic acid (ACC) Subject RIV: EF - Botanics Impact factor: 3.917, year: 2007

  11. Isolation, characterization and colonization of 1-aminocyclopropane-1-carboxylate deaminase-producing bacteria XG32 and DP24.

    Wang, Mei-Xia; Liu, Jia; Chen, Shuang-Lin; Yan, Shu-Zhen

    2012-03-01

    Two 1-aminocyclopropane-1-carboxylate deaminase-producing bacterial strains (DP24 and XG32) were isolated from surface-sterilized tomato roots and rizhospere soil. The strains were identified as Pseudomonas fluorescens biovar. IV (XG2) and Erwinia herbicola (DP24) by physiological and biochemical tests, and 16S rRNA gene analysis. Both strains showed positive plant growth-promoting activity when inoculated into cucumber (Cucumis sativus), tomato (Lycopersicon esculentum), pepper (Capsicum annuum) and rapeseed (Brassica napus L.). Colonization ability and behavior of these two strains were determined by treating mutant strains with rifampicin and fluorescence in situ hybridization (FISH) assay with rRNA targeted probes, respectively. Both strains were endophytic colonizers of pepper plants. The behavior of the two strains was not identical. Strain XG32 only colonized the root and reached the max level of 27.7 × 10(7) c.f.u./g (fresh weight), after 12 days postinoculation, while strain DP24 was able to colonize the roots, stems and leaves. The max level was reached at 40.87 × 10(7) c.f.u./g (fresh weight) in the roots, 17 × 10(7) c.f.u./g in the stems after 7 days postinoculation and 44.84 × 10(7) c.f.u./g in the leaves after 12 days postinoculation. PMID:22805836

  12. Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway.

    Tsang, Dat L; Edmond, Clare; Harrington, Jennifer L; Nühse, Thomas S

    2011-06-01

    Cell expansion in plants requires cell wall biosynthesis and rearrangement. During periods of rapid elongation, such as during the growth of etiolated hypocotyls and primary root tips, cells respond dramatically to perturbation of either of these processes. There is growing evidence that this response is initiated by a cell wall integrity-sensing mechanism and dedicated signaling pathway rather than being an inevitable consequence of lost structural integrity. However, the existence of such a pathway in root tissue and its function in a broader developmental context have remained largely unknown. Here, we show that various types of cell wall stress rapidly reduce primary root elongation in Arabidopsis (Arabidopsis thaliana). This response depended on the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC). In agreement with the established ethylene signaling pathway in roots, auxin signaling and superoxide production are required downstream of ACC to reduce elongation. However, this cell wall stress response unexpectedly does not depend on the perception of ethylene. We show that the short-term effect of ACC on roots is partially independent of its conversion to ethylene or ethylene signaling and that this ACC-dependent pathway is also responsible for the rapid reduction of root elongation in response to pathogen-associated molecular patterns. This acute response to internal and external stress thus represents a novel, noncanonical signaling function of ACC. PMID:21508182

  13. Matriconditioning Improves Thermotolerance in Pepper Seeds through Increased in 1-Aminocyclopropane-1-Carboxylic Acid Synthesis and Utilization

    SATRIYAS ILYAS

    2006-03-01

    Full Text Available Matriconditioning using a solid carrier, Micro-Cel E, was applied on pepper seeds and its effects on the improvement of thermotolerance through the ethylene biosynthesis were studied. Untreated and matriconditioned seeds were soaked in 5 mM 1-aminocyclopropane-1-carboxylic acid (ACC for various time at 25 oC to studies the time course of ACC-derived ethylene production. To study the performance of the seeds at high temperature, they were planted at temperature regime of 35 oC, 12h light/27 oC, 12h dark. The ACC-oxidase activity of the seeds after incubated in ACC at 35 oC was also determined. The ACC contents in untreated and matriconditioned seeds during the 24h imbibition period at 35 oC were monitored. No ethylene was detected during soaking of pepper seeds in the absence of ACC. In 5 mM ACC detectable levels of ethylene were produced; the matriconditioned seeds producing 10-fold larger amounts than the untreated seeds at the time of germination. It is postulated that matriconditioning greatly increases the ACC-oxidase activity at the time of germination. Matriconditioned seeds imbibed at 35 oC produced larger amounts of ACC and greater ACC-oxidase activity than the untreated seeds. Thus, the basis for the thermotolerance by matriconditioned seeds may be increasing ability to synthesize ACC and to utilize it for ethylene production and stress alleviation.

  14. Isolation of a Tomato Protease that May Be Involved in Proteolysis of 1-Aminocyclopropane-1-Carboxylate Synthase

    Jian-Feng LI; Liang-Hu QU; Ning LI

    2005-01-01

    1-aminocyclopropane-1-carboxylate (ACC) synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase (LeACS2) in tomato (Lycopersicon esculentum Mill.) fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ℃. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthaseprocessing protease from plant tissues.

  15. Effect of 1-aminocyclopropane-1-carboxylic acid on the production of ethylene in senescing flowers of Ipomoea tricolor Cav

    Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to rib segments excised from flowers of Ipomoea tricolor Cav. resulted in the formation of C2H4 in greater quantities than produced under natural conditions. The ability of ACC to enhance C2H4 production was independent of the physiological age of the tissue and its capacity to synthesize C2H4 without applied ACC. When ACC was fed to rib segments that had been treated with [14C]methionine, incorporation of radioactivity into C2H4 was reduced by 80%. Aminoethoxyvinylglycine and aminooxyacetic acid inhibited C2H4 production in rib segments of I. tricolor but had no effect on ACC-enhanced C2H4 production. Protoplasts obtained from flower tissue of I. tricolor did not form C2H4, even when incubated with methionine or selenomethionine. They produced C2H4 upon incubation with ACC, however. ACC-dependent C2H4 production in protoplasts was inhibited by n-propyl gallate, AgCl, CoCl2, KCN, Na2S, and NaN3. ACC-dependent C2H4 synthesis in rib segments and protoplasts was dependent on O2, the K/sub m/ for O2 being 1.0 to 1.4% (v/v). These results confirm the following pathway for C2H4 biosynthesis in I. tricolor: methionine [selenomethionine] →S-adenosylmethionine [selenoadenosylmethionine] → ACC → C2H4

  16. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening. PMID:22419220

  17. Biosynthesis of 1-aminocyclopropane-1-carboxylic acid and ethylene from δ-aminolevulinic acid in ripening tomato fruits

    A new pathway for ethylene (C2H4) biosynthesis, which utilizes δ-aminolevulinic acid (ALA) as a precursor of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of C2H4, is presented. ALA enhanced ACC accumulation to 410% and C2H4 production to 232% of the control. The C2H4 production rate varied with the ALA concentration and the stage of tomato fruit development. As the ALA concentration increased from zero to 40 mM, the C2H4 production rate increased. Both treated and untreated pericarp discs from fruits at the pink stage of development yielded the largest C2H4 production rate. Radioactivity from (2,3-3H)ALA was detected in both ACC and C2H4, and radioactivity from (4-14C)ALA was detected in ACC and CO2 but not in C2H4. However, radioactivity from (5-14C)ALA was detected in CO2, and its amount was greater than that obtained from (4-14C)ALA. Neither ACC nor C2H4 showed any radioactivity when (5-14C)ALA was supplied to the fruit discs. In addition, when (2,3-3H)ALA or (4-14C)ALA was supplied to the fruit discs, radioactivity was detected in other metabolites such as fumarate, succinate, malate, glutamate, glutamine, α-ketoglutarate, and methionine, but the amount of radioactivity was insignificant as compared with the amount of radioactivity found in C2H4 and ACC

  18. ACC (1-Aminocyclopropane-1-Carboxylate) Deaminase Activity, a Widespread Trait in Burkholderia Species, and Its Growth-Promoting Effect on Tomato Plants▿

    Onofre-Lemus, Janette; Hernández-Lucas, Ismael; Girard, Lourdes; Caballero-Mellado, Jesús

    2009-01-01

    The genus Burkholderia includes pathogens of plants and animals and some human opportunistic pathogens, such as the Burkholderia cepacia complex (Bcc), but most species are nonpathogenic, plant associated, and rhizospheric or endophytic. Since rhizobacteria expressing ACC (1-aminocyclopropane-1-carboxylate) deaminase may enhance plant growth by lowering plant ethylene levels, in this work we investigated the presence of ACC deaminase activity and the acdS gene in 45 strains, most of which are...

  19. Study the effect of bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase on resistance to salt stress in tomato plant

    Maryam SADRNIA

    2011-11-01

    Full Text Available 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase produced by rhizobacteria could be remove theethylene precursor and stimulate plant growth. Aim of the work was investigation on effect of rhizosphere bacteria Pseudomonasmendocina containing plasmid carrying gene encoding ACC deaminase on resistance of tomato plant to salinity. Amplification ofacds gene in selected Pseudomonas was performed; the g e n e w a s c l o n e d i n Escherichia coli and was cloned subsequently in P.mendocina. Enzyme activity was determined in cloned Escherichia coli and cloned P. mendocina for confirmation of geneexpression. Effect of bacterial ACC deaminase on resistance of tomato plants to NaCl was studied in Pot and Greenhouse. In potexperiment, tomato plant treated by cloned P. mendocina was compared with plants treated by P. mendocina (without plasmid andcontrol group. Salinity were established by adding 172 and 207 mM of NaCl to irrigated water. Greenhouse experiments wereconducted in similar groups of bacteria in 207 mM of NaCl. Results obtained from pot experiment revealed that plants treated bycloned P. mendocina in 172 mM of NaCl was showed increasing content of growth than ones treated by P. mendocina and controlas 11%, 18.4% growth for the shoot, 16.6%, 3.7% for roots and 9.6%, 27.5% for wet weight after five weeks, respectively. In 207mM of NaCl, the results were as 14.9 %, 9.7% for shoot, 94.3%, 15.7% for roots and 96.4%, 50.6% for wet weight, respectively. Ingreenhouse experiment, results in same parameter in 207 mM of NaCl were revealed as 63.7%, 7 times for shoot, 2.8, 14 times forroots and 66.1%, 154 times for wet weight, respectively. We concluded that recombinant P. mendocina producing ACC deaminaseby reduction of ethylene content of tomato plant in high salt concentrations could result in improvement of plant resistance tosalinity.

  20. Immunopurification and characterization of a 40-kD 1-aminocyclopropane-1-carboxylic acid N-malonyltransferase from mung bean seedling hypocotyls

    Chick, WSH; Leung, PC

    1997-01-01

    1-Aminocyclopropane-1-carboxylic acid (ACC) N-malonyltransferase catalyzes the transfer of the malonyl group from malonyl coenzyme A to ACC to form malonyl ACC. Using partially purified ACC N-malonyltransferase from the hypocotyls of mung bean (Vigna radiata) seedlings, we produced two mouse monoclonal antibodies (1H5 and 2G3) to this enzyme. These antibodies bind to sites other than the active site of the enzyme because monoclonal antibody-bound ACC N-malonyltransferase still exhibits full c...

  1. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine

    Swarup Roy Choudhury; Sanjay Kumar Singh; Sujit Roy; Dibyendu N Sengupta

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of -adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana-ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5′-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP.

  2. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine.

    Choudhury, Swarup Roy; Singh, Sanjay Kumar; Roy, Sujit; Sengupta, Dibyendu N

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP. PMID:20689184

  3. The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Ⅰ on the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

    HU Zong-li; CHEN Xu-qing; CHEN Guo-ping; L(U) Li-juan; Grierson Donald

    2007-01-01

    The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase I on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato (Lycopersicon esculentum) were cloned, such as the 1-aminocyclopropane-1-carboxylic acid oxidase 1 gene (LeACO1), 1-aminocyclopropane-1-carboxylic acid oxidase 3 gene (LeAC03), EIN3-binding F-box 1 gene (LeEBF1), pathogenesisrelated protein 1 gene (LePR1), pathogenesis-related protein 5 gene (LePR5), and pathogenesis-related protein osmotin precursor gene (LeNP24) by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig (AC++) and the LeACO1 co-suppression tomatoes (V1187 and T4B), respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carried out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.

  4. Expression characteristics of CS-ACS1, CS-ACS2 and CS-ACS3, three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in cucumber (Cucumis sativus L.) fruit under carbon dioxide stress.

    Mathooko, F M; Mwaniki, M W; Nakatsuka, A; Shiomi, S; Kubo, Y; Inaba, A; Nakamura, R

    1999-02-01

    We investigated the expression pattern of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes, CS-ACS1, CS-ACS2 and CS-ACS3 in cucumber (Cucumis sativus L.) fruit under CO2 stress. CO2 stress-induced ethylene production paralleled the accumulation of only CS-ACS1 transcripts which disappeared upon withdrawal of CO2. Cycloheximide inhibited the CO2 stress-induced ethylene production but superinduced the accumulation of CS-ACS1 transcript. At higher concentrations, cycloheximide also induced the accumulation of CS-ACS2 and CS-ACS3 transcripts. In the presence of CO2 and cycloheximide, the accumulation of CS-ACS2 transcript occurred within 1 h, disappeared after 3 h and increased greatly upon withdrawal of CO2. Inhibitors of protein kinase and types 1 and 2A protein phosphatases which inhibited and stimulated, respectively, CO2 stress-induced ethylene production had little effect on the expression of these genes. The results presented here identify CS-ACS1 as the main ACC synthase gene responsible for the increased ethylene biosynthesis in cucumber fruit under CO2 stress and suggest that this gene is a primary response gene and its expression is under negative control since it is expressed by treatment with cycloheximide. The results further suggest that the regulation of CO2 stress-induced ethylene biosynthesis by reversible protein phosphorylation does not result from enhanced ACC synthase transcription. PMID:10202812

  5. Prospective Use of 1-Aminocyclopropane-1-Carboxylate Deaminase-Producing Bacteria for Plant Growth Promotion and Defense against Biotic and Abiotic Stresses in Peat-Soil-Agriculture

    EDI HUSEN

    2008-12-01

    Full Text Available The 1-aminocyclopropane-1-carboxylate (ACC deaminase (EC4.1.99.4 is an enzyme produced by some soil bacteria to degrade ACC (the immediate precursor of ethylene to reduce ethylene biosynthesis in higher plants. Increased concentrations of ethylene in plant tissues, which are triggered by various biotic and abiotic stresses, inhibits plant growth and weakens the plant defense against the stressors. Various findings on the successful use of ACC deaminase producing bacteria for plant growth under unfavorable soil conditions are inspiring their use in tropical peat-soil-agriculture, which possesses bio-physical constraints. It has been proven that inoculation of plants with ACC deaminase producing bacteria decreased ethylene inhibition generated by unfavorable environmental conditions, such as nutrient shortage, flooding, drought, high salts, and the presence of heavy metals and organic pollutants. Understanding the mechanisms by which ACC deaminase-producing bacteria act to reduce plant stress and the fitness of bacterial traits with the properties and constraints of peat-soils becomes a key to utilize these bacteria in improving crop productivity. The bacteria may ameliorate plant stress as well as promote plant growth under seasonal bio-physical changes of peat-soils that are usually encountered in the field.

  6. Characterization and optimization of 1-Aminocyclopropane-1-Carboxylate Deaminase (ACCD activity in different rhizospheric PGPR along with Microbacterium sp. strain ECI-12A

    Ashok Kumar

    2013-03-01

    Full Text Available A total of nine strains of plant growth promoting rhizobacteria were analyzed for ACC deaminase activity, where highest ACC deaminase activity was found in Klebsiella sp strain ECI-10A (539.1 nmol α-keto butyrate/ mg protein/ h and lowest in Microbacterium sp strain ECI-12A (122.0 nmol α-keto butyrate/ mg protein/ h. Although Microbacterium sp strain ECI-12A showed lowest level of ACC deaminase activity, but, the species of Microbacterium isolated from rhizosphere is the first report. Microbacterium sp strain ECI-12A was also analyzed under varying conditions of time, amount of 1-Aminocyclopropane-1- carboxylate (ACC, and temperature for optimization of the ACC deaminase activity. The optimum activity was recorded with the supplementation of 5mM ACC at 30oC temperature after 24h of culture growth. All the nine strains showed acdS gene in the PCR amplification of that gene. No any rhizospheric Microbacterium species showing ACC deaminase activity have been reported earlier, therefore, we report here ACC deaminase activity in Microbacterium sp ECI-12A isolated from rice rhizosphere is a novel finding.

  7. Structural analysis of the promoter of tomato 1-aminocyclopropane-1-carboxylate synthase 6 gene(Le-ACS6)

    LIN JingYu; FAN Rong; WAN XiaoRong; CHARNG Yeeyung; WANG NingNing

    2007-01-01

    Ethylene plays an important role in the regulation of many growth and developmental processes of higher plants. In tomato, Le-ACS6, a member of the ACC synthase multigene family involved in system 1 ethylene biosynthesis during fruit ripening, is subject to negative feedback regulation by ethylene. To identify the cis-elements that are responsible for the negative feedback control, we established an in vitro transient assay system employing particle bombardment on mature-green tomato fruit pericarp to examine the expression of a luciferase (LUC) reporter gene driven by a 5'-serially deleted Le-ACS6 promoter. The results localized putative cis-elements required for negative ethylene-response between -347 and -266 upstream from the translational start site ATG. Several lines of stable transformation of the Le-ACS6 promoter and GUS reporter fusion gene containing internal deletion from -347 to -266 were generated. The expression pattern of the GUS reporter showed that removal of the nucleotides from -347 to -266 completely eliminated the response of the Le-ACS6 promoter to exogenous ethylene.

  8. Characterization of cultivar differences in alcohol acyltransferase and 1-aminocyclopropane-1carboxylate synthase gene expression and volatile compound emission during apple fruit maturation and ripening

    Alcohol acyl transferase (AAT) catalyzes the last step of volatile ester biosynthesis, and ethylene purportedly regulates AAT gene expression. In this study, expession patterns of four apple AAT genes and two ethylene biosynthesis genes were investigated in two apple cultivars with relatively high ...

  9. Effects of aminoisobutyric acid on 1-aminocyclopropane-1-carboxylic acid uptake, ethylene production and content of ACC in water-stressed tomato plants

    The effect of water stress on the regulation of ethylene biosynthesis has not yet clearly been established. Both the formation and utilization of aminocyclopropane-1-carboxylic acid, ACC, are considered to be major regulatory points in ethylene biosynthesis. There is evidence that ACC synthase is the key control enzyme in response to various stimuli associated with the induction of ethylene biosynthesis. It has been reported that aminoisobutyric acid, AIB, inhibits ethylene production in some plants and AIB may inhibit the conversion of ACC to ethylene. For this reason, the possibility of inhibition of ACC uptake in the presence of AIB was examined. It was observed that the rate of 14C-ACC uptake decreased with an increase in the concentration of AIB in the solution. Calculating the percentage of ACC converted to ethylene on the basis of uptake shows that AIB inhibits the conversion of 14C-ACC to ethylene and that this inhibition is increased with an increase in the concentration of AIB in the solution. This suggests that a portion of the inhibition of the conversion of ACC to ethylene in the presence of AIB is partly due to the competition for absorption. However, the ability of AIB to inhibit ethylene production in leaf tissue without an exogenous supply of ACC clearly indicates that AIB inhibits ethylene production. The present study was undertaken to elucidate the regulation of ethylene biosynthesis in water-stressed plants and the results are discussed

  10. XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms,Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature

    Sofia D.Carvalho; Rita Saraiva; Teresa M.Maia; Isabel A.Abreu; Paula Duque

    2012-01-01

    The Arabidopsis XBAT35 is one of five structurally related ankyrin repeat-containing Really interesting New Gene (RING) E3 ligases involved in ubiquitin-mediated protein degradation,which plays key roles in a wide range of cellular processes.Here,we show that the XBAT35 gene undergoes alternative splicing,generating two transcripts that are constitutively expressed in all plant tissues.The two splice variants derive from an exon skipping event that excludes an in-frame segment from the XBAT35 precursor mRNA,giving rise to two protein isoforms that differ solely in the presence of a nuclear localization signal (NLS).Transient expression assays indicate that the isoform lacking the NLS localizes in the cytoplasm of plant cells,whereas the other is targeted to the nucleus,accumulating in nuclear speckles.Both isoforms are functional E3 ligases,as assessed by in vitro ubiquitination assays.Two insertion mutant alleles and RNA-interference (RNAi) silencing lines for XBAT35 display no evident phenotypes under normal growth conditions,but exhibit hypersensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) during apical hook exaggeration in the dark,which is rescued by an inhibitor of ethylene perception.Independent expression of each XBAT35 splice variant in the mutant background indicates that the two isoforms may differentially contribute to apical hook formation but are both functional in this ethylene-mediated response.Thus,XBAT35 defines a novel player in ethylene signaling involved in negatively regulating apical hook curvature,with alternative splicing controlling dual targeting of this E3 ubiquitin ligase to the nuclear and cytoplasmic compartments.

  11. Inibição da síntese da ACC (ácido 1-carboxílico-1-aminociclopropano oxidase em maçãs frigoconservadas em atmosfera controlada Inhibition of (1-aminocyclopropane-1-carboxylic acid oxydase synthesis in apple fruits by controlled atmosphere storage

    Paulo Dejalma ZIMMER

    1999-12-01

    Full Text Available Foi estudada a expressão da ACC oxidase em maçãs, cv. Jonagold, colhidas no estádio pré-climatérico e armazenadas sob refrigeração em atmosfera normal (0ºC, 95% UR - AN e controlada (0ºC, 95% UR, 1,5% O2 e 2,5% CO2 - AC, durante 180 dias. Na instalação do experimento, aos 90 e aos 120 dias, foram coletadas amostras para a determinação da firmeza de polpa, da acidez total titulável, dos sólidos solúveis totais, da produção de etileno, da atividade ACC oxidase e para a detecção imunoquímica das isoformas desta enzima. A dosagem da atividade ACC oxidase foi realizada por cromatografia gasosa a partir de extrato protéico solúvel acrescido de 250µM de ACC, 10µL de sulfato ferroso e 30µL de ascorbato de sódio. Para a detecção imunoquímica utilizou-se a técnica "western blot", com anticorpos policlonais anti-ACC oxidase de maçã, após separação das proteínas em eletroforese e isoeletrofocalização. Não foi detectada ACC oxidase em maçãs pré-climatéricas. Porém, após 120 horas em condições ambientais, houve a síntese dessa enzima e um incremento na produção de etileno. A refrigeração não exerceu controle na síntese da ACC oxidase e produção de etileno, resultando em significativas perdas físico-químicas nas frutas armazenadas em AN. Já a utilização de AC permitiu controlar a via de biossíntese do etileno, pela inibição da síntese da ACC oxidase, mantendo o material com boa qualidade para o consumo in natura. A adição de ACC e dos cofatores aumentou a atividade ACC oxidase e alterou o pI da ACC oxidase.This experiment was carried out to evaluate the inhibition of the 1-aminocyclopropane-1-carboxylic acid (ACC oxidase in apple fruits by controled atmosphere. Jonagold apples were stored in normal atmosphere (NA and controlled atmosphere (CA storage. The fruits were harvested at the preclimateric stage and stored for 120 days at 0ºC and 95±5% RH with O2 set to 1,5% and CO2 set to 2,5% (CA

  12. Southern blight disease of tomato control by 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Paenibacillus lentimorbus B-30488.

    Dixit, Ritu; Agrawal, Lalit; Gupta, Swati; Kumar, Manoj; Yadav, Sumit; Chauhan, Puneet Singh; Nautiyal, Chandra Shekhar

    2016-02-01

    Tomato cultivation is highly susceptible for soil born diseases and among them southern blight disease caused by Scelerotium rolfsii is very common. For its management use of chemical fungicides is not very successful as their spores are able to survive for many years in the soil. As an alternative eco-friendly approach to control the disease antagonistic microbes are being characterized.Among them plant growth promoting rhizobacteria Paenibacillus lentimorbus B-30488 (B-30488) with antagonistic properties, multiple PGP attributes stress tolerance and ACC deaminase enzyme activity is characterized to decipher its mode of action against S. rolfsii under in vitro and in vivo conditions. In vitro results obtained from this study clearly demonstrate that B-30488 has ability to show antagonistic properties under different abiotic stresses against S. rolfsii. Similar results were also obtained from in vivo experiments where B-30488 inoculation has efficiently controlled the disease caused by S. rolfsii and improve the plant growth. Deleterious enhanced ethylene level in S. rolfsii infected plants was also ameliorated by inoculation of ACC deaminase producing B-30488. The ACC accumulation, ACO and ACS activities were also modulated in S. rolfsii infected plants. Results from defense enzymes and other biochemical attributes were also support the role of B-30488 inoculation in ameliorating the biotic stress caused by S. rolfsii in tomato plants. These results were further validated by pathogen related gene expression analysis by real time PCR. Overall results from the present study may be concluded that ACC deaminase producing B-30488 has ability to control the southern blight disease caused by S. rolfsii and commercial bioinoculant package may be developed. PMID:26825539

  13. Differential water permeability and regulation of three aquaporin 4 isoforms

    Fenton, Robert A.; Moeller, Hanne B; Zelenina, Marina;

    2010-01-01

    Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed...... in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected...... by changes in external K(+) concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms...

  14. Molecular regulation of skeletal muscle myosin heavy chain isoforms

    Brown, David M.

    2015-01-01

    Research investigating the regulation of muscle fibre type has traditionally been conducted in vivo, analyzing global changes at a whole muscle level. Broadly, this thesis aimed to explore more “molecular” approaches, utilizing molecular and cell biology to understand the expression and regulation of myosin heavy chain (MyHC) isoforms as an indicator of muscle fibre composition. The mRNA expression profile of six MyHC isoform genes during C2C12 myogenesis was elucidated to reveal that the...

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

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

    1995-01-01

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

  16. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stress can increase susceptibility to developing psychiatric disorders, including depression. Understanding the neurobiological mechanisms underlying stress resilience and susceptibility is key to identifying novel targets for the development of more effective treatments for stress-related psychiatric disorders. Here we show that specific isoforms of GABAB receptor subunits differentially regulate stress resilience. Specifically, GABAB(1a)−/− mice are more susceptible whereas GABAB(1b)−/− mic...

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

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

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam ...

  18. Apoplastic Alkalinization Is Instrumental for the Inhibition of Cell Elongation in the Arabidopsis Root by the Ethylene Precursor 1-Aminocyclopropane-1-Carboxylic Acid

    Staal, Marten; De Cnodder, Tinne; Simon, Damien; Vandenbussche, Filip; Van Der Straeten, Dominique; Verbelen, Jean-Pierre; Elzenga, Theo; Vissenberg, Kris

    2011-01-01

    In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200-450 mu m proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the

  19. Characterization of Alcohol Acyl Transferase and 1-Aminocyclopropane-1-Carboxylate Synthase Gene Expression and Volatile Compound Emission during Apple Fruit Development and Ripening

    Alcohol acyl transferase (AAT) catalyzes the last step of volatile ester biosynthesis, and in this study, expression of four apple AAT genes was investigated in the peel of two apple cultivars with relatively high (‘Golden Delicious’) or low (‘Granny Smith’) volatile ester production. All four AAT ...

  20. Purification of 1-aminocyclopropane-1-carboxylate synthase from apple fruits using s-adenosyl [3,414C]-methionine (SAM) as a probe

    Tomato ACC synthase is inactivated by its substrate SAM, with the moiety of aminobutyrate being covalently linked to ACC synthase during the catalytic reactions. A partial purified ACC synthase (the catalytic activity 100 μmol/h·mg protein) from pellets of apple extract was incubated with [3,414C] SAM. Only one radioactive peak was revealed in a C-4 reverse phase HPLC and one radioactive band on SDS-PAGE with an M.W. of 48 kDa. Apple ACC synthase in native form is resistant to V8, α-chromtrypsin and carboxylpeptidase A digestion, but effectively inactivated by trypsin and ficin, as demonstrated by both the activity assay and SAM labeling. The radioactive protein cut from the SDS-PAGE was injected to three mice, two of the mice showed responses to the protein in western blot analysis. The antibodies from mice is currently under characterization

  1. Isoform-specific phosphorylation-dependent regulation of connexin hemichannels

    Alstrøm, Jette Skov; Hansen, Daniel Bloch; Nielsen, Morten Schak;

    2015-01-01

    Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of...... fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30...... hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC...

  2. Differential regulation of macropinocytosis by Abi1/Hssh3bp1 isoforms.

    Patrycja M Dubielecka

    Full Text Available BACKGROUND: Macropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown. PRINCIPAL FINDINGS: Here, using flow cytometric assay analysis for Alexa Fluor 647, we demonstrate that Abi1 isoforms 2 and 3 differentially regulate macropinocytosis. LNCaP cells expressing isoform 3 had increased macropinocytic uptake that correlated with enhanced cell spreading and higher Rac1 activation in comparison to cells expressing isoform 2. Isoform 2 expressing cells had decreased macropinocytic uptake, but demonstrated greater sensitivity to Rac1 activation. Moreover, more isoform 2 was localized within the cytoplasm in comparison to isoform 3, which was more associated with the plasma membrane. Activated Rac1 was found to specifically bind to a site in exon 10 of isoform 2 in vitro. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably. CONCLUSION: Based on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions.

  3. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  4. Inter-isoform-dependent Regulation of the Drosophila Master Transcriptional Regulator SIN3.

    Chaubal, Ashlesha; Todi, Sokol V; Pile, Lori A

    2016-05-27

    SIN3 is a transcriptional corepressor that acts as a scaffold for a histone deacetylase (HDAC) complex. The SIN3 complex regulates various biological processes, including organ development, cell proliferation, and energy metabolism. Little is known, however, about the regulation of SIN3 itself. There are two major isoforms of Drosophila SIN3, 187 and 220, which are differentially expressed. Intrigued by the developmentally timed exchange of SIN3 isoforms, we examined whether SIN3 187 controls the fate of the 220 counterpart. Here, we show that in developing tissue, there is interplay between SIN3 isoforms: when SIN3 187 protein levels increase, SIN3 220 protein decreases concomitantly. SIN3 187 has a dual effect on SIN3 220. Expression of 187 leads to reduced 220 transcript, while also increasing the turnover of SIN3 220 protein by the proteasome. These data support the presence of a novel, inter-isoform-dependent mechanism that regulates the amount of SIN3 protein, and potentially the level of specific SIN3 complexes, during distinct developmental stages. PMID:27129248

  5. Specific regulation of NRG1 isoform expression by neuronal activity

    Liu, Xihui; Bates, Ryan; Wang, Fay; Su, Nan; Kirov, Sergei A.; Luo, Yuling; Wang, Jian-Zhi; Xiong, Wen-Cheng; Mei, Lin

    2011-01-01

    Neuregulin 1 (NRG1) is a trophic factor that has been implicated in neural development, neurotransmission and synaptic plasticity. NRG1 has multiple isoforms that are generated by usage of different promoters and alternative splicing of a single gene. However, little is known about NRG1 isoform composition profile, whether it changes during development or the underlying mechanisms. We found that each of the six types of NRG1 has a distinct expression pattern in the brain at different ages, re...

  6. Regulation of three isoforms of SOD gene by environmental stresses in citrus red mite, Panonychus citri.

    Feng, Ying-Cai; Liao, Chong-Yu; Xia, Wen-Kai; Jiang, Xuan-Zhao; Shang, Feng; Yuan, Guo-Rui; Wang, Jin-Jun

    2015-09-01

    Superoxide dismutase (SOD) is a family of enzymes with multiple isoforms that possess antioxidative abilities in response to environmental stresses. Panonychus citri is one of the most important pest mites and has a global distribution. In this study, three distinct isoforms of SOD were cloned from P. citri and identified as cytoplasmic Cu-ZnSOD (PcSOD1), extracellular Cu-ZnSOD (PcSOD2), and mitochondrial MnSOD (PcSOD3). mRNA expression level analysis showed that all three isoforms were up-regulated significantly after exposure to the acaricide abamectin and to UV-B ultraviolet irradiation. In particular, PcSOD3 was up-regulated under almost all environmental stresses tested. The fold change of PcSOD3 expression was significantly higher than those of the two Cu-ZnSOD isoforms. Taken together, the results indicate that abamectin and UV-B can induce transcripts of all three SOD isoforms in P. citri. Furthermore, PcSOD3 seems to play a more important role in P. citri tolerance to oxidative stress. PMID:26063404

  7. Kiwifruit EIL and ERF Genes Involved in Regulating Fruit Ripening1[W

    Yin, Xue-ren; Allan, Andrew C.; Chen, Kun-song; Ferguson, Ian B.

    2010-01-01

    Kiwifruit (Actinidia deliciosa) is a climacteric fruit sensitive to low concentrations of ethylene. To investigate the transcriptional mechanisms underlying kiwifruit ethylene response, transcription factors encoding four EIN3-Like (EILs) and 14 Ethylene Response Factors (ERFs) were cloned from kiwifruit. Expression of these transcription factors was examined during fruit development. The expression of transcripts of most AdERFs was higher during early fruit development, with the exception of AdERF3, which increased with maturity. Several AdERFs were apparently down-regulated by ethylene, as they were affected by the ethylene inhibitor 1-methylcyclopropene and by antisense suppression of ACO (for 1-aminocyclopropane-1-carboxylic acid oxidase) in the fruit. In contrast, AdEILs were constitutively expressed during fruit development and ripening. The transcription factors AdEIL2 and AdEIL3 activated transcription of the ripening-related genes AdACO1 and AdXET5 (xyloglucan endotransglycosylase gene) and, when overexpressed in Arabidopsis (Arabidopsis thaliana), stimulated ethylene production. The potential repressor AdERF9 suppressed this promoter activity. These results support a role for kiwifruit EILs and ERFs in transcriptional regulation of ripening-related genes and in the regulation of kiwifruit fruit-ripening processes. PMID:20457803

  8. Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ETHYLENE RESPONSE FACTOR 057.

    Sun, Xiaoming; Zhao, Tingting; Gan, Shuheng; Ren, Xiaodie; Fang, Linchuan; Karungo, Sospeter Karanja; Wang, Yi; Chen, Liang; Li, Shaohua; Xin, Haiping

    2016-01-01

    Ethylene (ET) is a gaseous plant hormone that plays essential roles in biotic and abiotic stress responses in plants. However, the role of ET in cold tolerance varies in different species. This study revealed that low temperature promotes the release of ET in grapevine. The treatment of exogenous 1-aminocyclopropane-1-carboxylate increased the cold tolerance of grapevine. By contrast, the application of the ET biosynthesis inhibitor aminoethoxyvinylglycine reduced the cold tolerance of grapevine. This finding suggested that ET positively affected cold stress responses in grapevine. The expression of VaERF057, an ET signaling downstream gene, was strongly induced by low temperature. The overexpression of VaERF057 also enhanced the cold tolerance of Arabidopsis. Under cold treatment, malondialdehyde content was lower and superoxide dismutase, peroxidase, and catalase activities were higher in transgenic lines than in wild-type plants. RNA-Seq results showed that 32 stress-related genes, such as CBF1-3, were upregulated in VaERF057-overexpressing transgenic line. Yeast one-hybrid results further demonstrated that VaERF057 specifically binds to GCC-box and DRE motifs. Thus, VaERF057 may directly regulate the expression of its target stress-responsive genes by interacting with a GCC-box or a DRE element. Our work confirmed that ET positively regulates cold tolerance in grapevine by modulating the expression of VaERF057. PMID:27039848

  9. Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of banana fruit.

    Inaba, Akitsugu; Liu, Xuejun; Yokotani, Naoki; Yamane, Miki; Lu, Wang-Jin; Nakano, Ryohei; Kubo, Yasutaka

    2007-01-01

    The feedback regulation of ethylene biosynthesis in banana [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit was investigated in an attempt to clarify the opposite effect of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, before and after the onset of ripening. 1-MCP pre-treatment completely prevented the ripening-induced effect of propylene in pre-climacteric banana fruit, whereas treatment after the onset of ripening stimulated ethylene production. In pre-climacteric fruit, higher concentrations of propylene suppressed ethylene production more strongly, despite their earlier ethylene-inducing effect. Exposure of the fruit ripened by propylene to 1-MCP increased ethylene production concomitantly with an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC content, and prevented a transient decrease in MA-ACS1 transcripts in the pulp tissues. In contrast, in the peel of ripening fruit, 1-MCP prevented the increase in ethylene production and subsequently the ripening process by reduction of the increase in MA-ACS1 and MA-ACO1 transcripts and of ACC synthase and ACC oxidase activities. These results suggest that ethylene biosynthesis in ripening banana fruit may be controlled negatively in the pulp tissue and positively in the peel tissue. This differential regulation by ethylene in pulp and peel tissues was also observed for MA-PL, MA-Exp, and MA-MADS genes. PMID:17185740

  10. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

  11. Transgenic analysis reveals LeACS-1 as a positive regulator of ethylene-induced shikonin biosynthesis in Lithospermum erythrorhizon hairy roots.

    Fang, Rongjun; Wu, Fengyao; Zou, Ailan; Zhu, Yu; Zhao, Hua; Zhao, Hu; Liao, Yonghui; Tang, Ren-Jie; Yang, Tongyi; Pang, Yanjun; Wang, Xiaoming; Yang, Rongwu; Qi, Jinliang; Lu, Guihua; Yang, Yonghua

    2016-03-01

    The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon. PMID:26780904

  12. Functional studies and expression regulation of two leptin isoforms in grass carp

    Chen, Ting; 陈廷

    2012-01-01

    Leptin, the protein product of obese gene, is a 16-kD adipokine with regulatory functions on food intake and energy metabolism. At present, limited information is available on leptin functions and regulation in lower vertebrates mainly due to the fact that the primary structure of leptin is highly diversified from fish to mammals. Leptin in teleost fish is even more complicated as leptin isoforms have been reported presumably as a result of whole-genome duplication that occurred during the ev...

  13. Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis

    Lei, Mingguang

    2010-11-30

    With the exception of root hair development, the role of the phytohormone ethylene is not clear in other aspects of plant responses to inorganic phosphate (Pi) starvation. The induction of AtPT2 was used as a marker to find novel signalling components involved in plant responses to Pi starvation. Using genetic and chemical approaches, we examined the role of ethylene in the regulation of plant responses to Pi starvation. hps2, an Arabidopsis mutant with enhanced sensitivity to Pi starvation, was identified and found to be a new allele of CTR1 that is a key negative regulator of ethylene responses. 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, increases plant sensitivity to Pi starvation, whereas the ethylene perception inhibitor Ag+ suppresses this response. The Pi starvation-induced gene expression and acid phosphatase activity are also enhanced in the hps2 mutant, but suppressed in the ethylene-insensitive mutant ein2-5. By contrast, we found that ethylene signalling plays a negative role in Pi starvation-induced anthocyanin production. These findings extend the roles of ethylene in the regulation of plant responses to Pi starvation and will help us to gain a better understanding of the molecular mechanism underlying these responses. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  14. Down regulation of ethylene biosynthesis in apples. Cloning and sequencing of the partial ACC synthase gene in the McIntosh cultivar

    Ethylene is a plant hormone that regulates many aspects of plant growth, development and senescence. 1-aminocyclopropane-1-carboxylate (ACC) synthase has been identified as the key enzyme in the biosynthesis of ethylene. Down regulation of ethylene biosynthesis via transformation with the antisense ACC synthase gene (as has already been proved with tomato) might lengthen the storability of apple. To produce the antisense gene of the apple, RNA was isolated from the McIntosh cultivar and the cDNA synthesized. The cDNA template was amplified by polymerase chain reaction (PCR) using ACC specific primers that have been found to be highly conserved in several plant species. PCR resulted in a 1.1 kb DNA band, which was cloned and sequenced. Sequenced analysis of the McIntosh clones (K1, G1, G2, G3, G4) showed a similarity of 63.5-71.6% with the apple ACC synthase known so far and found comparable homology with the ACC syntheses of other plant species in the databank. 8 refs, 2 tabs

  15. Yin-Yang Regulation of Adiponectin Signaling by APPL Isoforms in Muscle Cells*

    Wang, Changhua; Xin, Xiaoban; Xiang, Ruihua; Ramos, Fresnida J.; Liu, Meilian; Lee, Hak Joo; Chen, Hongzhi; Mao, Xuming; Kikani, Chintan K.; Liu, Feng; Dong, Lily Q.

    2009-01-01

    APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 ...

  16. Computational identification of transcriptionally co-regulated genes, validation with the four ANT isoform genes

    Dupont Pierre-Yves

    2012-09-01

    Full Text Available Abstract Background The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. Results From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases. Conclusions Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.

  17. Pear 14-3-3a gene (Pp14-3-3a) is regulated during fruit ripening and senescense, and involved in response to salicylic acid and ethylene signalling

    Haiyan Shi; Yuxing Zhang

    2014-12-01

    14-3-3 proteins play important roles in regulating plant development and phytohormone (abscisic acid, gibberellin and brassinosteroids) signalling. However, their regulation in fruit ripening and senescense, and response to salicylic acid and ethylene signalling are yet to be illustrated. One cDNA encoding putative 14-3-3 protein was isolated from pear (Pyrus pyrifolia) and designated Pp14-3-3a. Phylogenetic analysis clearly demonstrated that Pp14-3-3a belonged to -like group of 14-3-3 super-families. Real-time quantitative PCR analysis indicated that the expression of Pp14-3-3a gene was developmentally regulated in the fruit. Further study demonstrated that Pp14-3-3a expression was inhibited by salicylic acid and induced by ethylene precursor 1-aminocyclopropane-1-carboxylic acid in pear fruit. These data suggested that Pp14-3-3a might be involved in response to salicylic acid and ethylene signalling during fruit ripening and senescence of pear.

  18. RAS and RHO Families of GTPases Directly Regulate Distinct Phosphoinositide 3-Kinase Isoforms

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S.; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-01-01

    Summary RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind an...

  19. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-05-23

    RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs. PMID:23706742

  20. Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms.

    Weeks, Kate L; Avkiran, Metin

    2015-04-15

    Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms. PMID:25362149

  1. Mechanistic insights into isoform-dependent and species-specific regulation of bile salt export pump by farnesoid X receptor

    Song, Xiulong; Chen, Yuan; Valanejad, Leila; Kaimal, Rajani; Yan, Bingfang; Stoner, Matthew; Deng, Ruitang

    2013-01-01

    Expression of bile salt export pump (BSEP) is regulated by the bile acid/farnesoid X receptor (FXR) signaling pathway. Two FXR isoforms, FXRα1 and FXRα2, are predominantly expressed in human liver. We previously showed that human BSEP was isoform-dependently regulated by FXR and diminished with altered expression of FXRα1 and FXRα2 in patients with hepatocellular carcinoma. In this study, we demonstrate that FXRα1 and FXRα2 regulate human BSEP through two distinct FXR responsive elements (FXR...

  2. AMPK regulates circadian rhythms in a tissue- and isoform-specific manner.

    Jee-Hyun Um

    Full Text Available BACKGROUND: AMP protein kinase (AMPK plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo. METHODOLOGY/PRINCIPAL FINDING: THE CATALYTIC SUBUNIT OF AMPK HAS TWO ISOFORMS: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1-/- and AMPKα2-/- mice. We found that both α1-/- and α2-/- mice are able to maintain a circadian rhythm of activity in dark-dark (DD cycle, but α1-/- mice have a shorter circadian period whereas α2-/- mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1-/- mice, but not in α2-/- mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1-/- mice, but it was severely disrupted in the heart and skeletal muscle of α2-/- mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1-/- and α2-/- mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT activity, which converts nicotinamide (NAM to NAD+, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells. CONCLUSION/SIGNIFICANCE: This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.

  3. Concordant and discordant regulation of target genes by miR-31 and its isoforms.

    Yu-Tzu Chan

    Full Text Available It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA variants designated as "isomiR". Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M, which differ only slightly in their 5'- and/or 3'-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3'-untranslated region (UTR of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.

  4. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  5. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  6. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  7. LPA is a novel lipid regulator of mesangial cell hexokinase activity and HKII isoform expression.

    Coy, Platina E; Taneja, Navin; Lee, Iris; Hecquet, Claudie; Bryson, Jane M; Robey, R Brooks

    2002-08-01

    The prototypical extracellular phospholipid mediator, lysophosphatidic acid (LPA), exhibits growth factor-like properties and represents an important survival factor in serum. This potent mesangial cell mitogen is increased in conditions associated with glomerular injury. It is also a known activator of the classic mitogen-activated protein kinase (MAPK) pathway, which plays an important role in the regulation of mesangial cell hexokinase (HK) activity. To better understand the mechanisms coupling metabolism to injury, we examined the ability of LPA to regulate HK activity and expression in cultured murine mesangial cells. LPA increased total HK activity in a concentration- and time-dependent manner, with maximal increases of >50% observed within 12 h of exposure to LPA concentrations > or =25 microM (apparent ED(50) 2 microM). These effects were associated with increased extracellular signal-regulated kinase (ERK) activity and were prevented by the pharmacological inhibition of either MAPK/ERK kinase or protein kinase C (PKC). Increased HK activity was also associated with increased glucose (Glc) utilization and lactate accumulation, as well as selectively increased HKII isoform abundance. The ability of exogenous LPA to increase HK activity was both Ca2+ independent and pertussis toxin insensitive and was mimicked by LPA-generating phospholipase A2. We conclude that LPA constitutes a novel lipid regulator of mesangial cell HK activity and Glc metabolism. This regulation requires sequential activation of both Ca2+-independent PKC and the classic MAPK pathway and culminates in increased HKII abundance. These previously unrecognized metabolic consequences of LPA stimulation have both physiological and pathophysiological implications. They also suggest a novel mechanism whereby metabolism may be coupled to cellular injury via extracellular lipid mediators. PMID:12110510

  8. N-Myc Differentially Regulates Expression of MXI1 Isoforms in Neuroblastoma

    Michael B. Armstrong

    2013-12-01

    Full Text Available Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB. MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation.

  9. Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function.

    Hubert, Nadia; Hentze, Matthias W

    2002-09-17

    Divalent metal transporter 1 (DMT1) mediates apical iron uptake into duodenal enterocytes and also transfers iron from the endosome into the cytosol after cellular uptake via the transferrin receptor. Hence, mutations in DMT1 cause systemic iron deficiency and anemia. DMT1 mRNA levels are increased in the duodenum of iron-deficient animals. This regulation has been observed for DMT1 mRNA harboring an iron-responsive element (IRE) in its 3' UTR, but not for a processing variant lacking a 3'UTR IRE, suggesting that the IRE regulates the expression of DMT1 mRNA in response to iron levels. Here, we show that iron regulation of DMT1 involves the expression of a previously unrecognized upstream 5' exon (exon 1A) of the human and murine DMT1 gene. The expression of this previously uncharacterized 5' exon is tissue-specific and particularly prevalent in the duodenum and kidney. It adds an in-frame AUG translation initiation codon extending the DMT1 ORF by a conserved sequence of 29-31 amino acids. In combination with the IRE- and non-IRE variants in the 3'UTR, our results reveal the existence of four DMT1 mRNA isoforms predicting the synthesis of four different DMT1 proteins. We show that two regulatory regions, the 5' promoter/exon 1A region and the IRE-containing terminal exon participate in iron regulation of DMT1 expression, which operate in a tissue-specific way. These results uncover an unexpected complexity of DMT1 expression and regulation, with implications for understanding the physiology, cell biology, and pathophysiology of mammalian iron metabolism. PMID:12209011

  10. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    Fabian V Filipp

    2013-01-01

    Full Text Available Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2 is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor activation-induced tumorigenesis. These biochemical activities are controlled by a shift in the oligomeric state of PKM2 that includes acetylation, oxidation, phosphorylation, prolyl hydroxylation and sumoylation. Metabolically active PKM2 tetramer is allosterically regulated and responds to nutritional and stress signals. Metabolically inactive PKM2 dimer is imported into the nucleus and can function as protein kinase stimulating transcription. A systems biology approach to PKM2 at the genome, transcriptome, proteome, metabolome and fluxome level reveals how differences in biomolecular structure translate into a global rewiring of cancer metabolism. Cancer systems biology takes us beyond the Warburg effect, opening unprecedented therapeutic opportunities.

  11. Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

    Vázquez-Martínez, Edgar Ricardo; Camacho-Arroyo, Ignacio; Zarain-Herzberg, Angel; Rodríguez, María Carmen; Mendoza-Garcés, Luciano; Ostrosky-Wegman, Patricia; Cerbón, Marco

    2016-06-01

    Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells. PMID:26676302

  12. Fruit ripening regulation of α-mannosidase expression by the MADS Box transcription factor RIPENING INHIBITOR and ethylene

    Mohammad eIrfan

    2016-01-01

    Full Text Available α-Mannosidase (α-Man, a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN, a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site that contained three CArG boxes (binding sites for RIN was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC. Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression.

  13. Fruit Ripening Regulation of α-Mannosidase Expression by the MADS Box Transcription Factor RIPENING INHIBITOR and Ethylene.

    Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-01-01

    α-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression. PMID:26834776

  14. Epimorphin is a novel regulator of the progesterone receptor isoform-a.

    Bascom, Jamie L; Radisky, Derek C; Koh, Eileen; Fata, Jimmie E; Lo, Alvin; Mori, Hidetoshi; Roosta, Neda; Hirai, Yohei; Bissell, Mina J

    2013-09-15

    Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy. PMID:23867473

  15. Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes

    Müller, Margit S; Pedersen, Sofie E; Walls, Anne B;

    2015-01-01

    Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely...... understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each...... determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with...

  16. Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

    Guil-Luna, Silvia; Stenvang, Jan; Brünner, Nils; De Andrés, Francisco Javier; Rollón, Eva; Domingo, Víctor; Sánchez-Céspedes, Raquel; Millán, Yolanda; Mulas, Juana Martín de las

    2014-01-01

    Background Progesterone receptors play a key role in the development of canine mammary tumours, and recent research has focussed on their possible value as therapeutic targets using antiprogestins. Cloning and sequencing of the progesterone receptor gene has shown that the receptor has two isoforms, A and B, transcribed from a single gene. Experimental studies in human breast cancer suggest that the differential expression of progesterone receptor isoforms has implications for hormone therapy...

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

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

    2008-02-01

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

  18. Red light regulation of ethylene biosynthesis and gravitropism in etiolated pea stems

    Steed, C. L.; Taylor, L. K.; Harrison, M. A.

    2004-01-01

    During gravitropism, the accumulation of auxin in the lower side of the stem causes increased growth and the subsequent curvature, while the gaseous hormone ethylene plays a modulating role in regulating the kinetics of growth asymmetries. Light also contributes to the control of gravitropic curvature, potentially through its interaction with ethylene biosynthesis. In this study, red-light pulse treatment of etiolated pea epicotyls was evaluated for its effect on ethylene biosynthesis during gravitropic curvature. Ethylene biosynthesis analysis included measurements of ethylene; the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC); malonyl-conjugated ACC (MACC); and expression levels of pea ACC oxidase (Ps-ACO1) and ACC synthase (Ps-ACS1, Ps-ACS2) genes by reverse transcriptase-polymerase chain reaction analysis. Red-pulsed seedlings were given a 6 min pulse of 11 micromoles m-2 s-1 red-light 15 h prior to horizontal reorientation for consistency with the timeline of red-light inhibition of ethylene production. Red-pulse treatment significantly reduced ethylene production and MACC levels in epicotyl tissue. However, there was no effect of red-pulse treatment on ACC level, or expression of ACS or ACO genes. During gravitropic curvature, ethylene production increased from 60 to 120 min after horizontal placement in both control and red-pulsed epicotyls. In red-pulsed tissues, ACC levels increased by 120 min after horizontal reorientation, accompanied by decreased MACC levels in the lower portion of the epicotyl. Overall, our results demonstrate that ethylene production in etiolated epicotyls increases after the initiation of curvature. This ethylene increase may inhibit cell growth in the lower portion of the epicotyl and contribute to tip straightening and reduced overall curvature observed after the initial 60 min of curvature in etiolated pea epicotyls.

  19. Expression and beta-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP)

    Sooriyaarachchi, Sanjeewani; Jaber, Emad; Covarrubias, Adrian Suárez;

    2011-01-01

    . Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The c...

  20. The formation of ACC and competition between polyamines and ethylene for SAM

    Ethylene biosynthesis involves the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). ACC is then converted to ethylene. The genes that encode enzymes in this pathway all belong to a family of genes. Differential transcriptional regulation ...

  1. Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms

    Tabuchi, Mitsuaki; Tanaka, Naotaka; Nishida-Kitayama, Junko; Ohno, Hiroshi; Kishi, Fumio

    2002-01-01

    Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3′ exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element ...

  2. Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

    Guil-Luna, Silvia; Stenvang, Jan; Brünner, Nils;

    2014-01-01

    and mRNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n¿=¿22) or vehicle (n¿=¿5) twice before surgery.ResultsFormalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total......BackgroundProgesterone receptors play a key role in the development of canine mammary tumours, and recent research has focussed on their possible value as therapeutic targets using antiprogestins. Cloning and sequencing of the progesterone receptor gene has shown that the receptor has two isoforms...

  3. A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends.

    Kern, David M; Nicholls, Peter K; Page, David C; Cheeseman, Iain M

    2016-05-01

    The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. PMID:27138257

  4. Foliar abscisic acid-to-ethylene accumulation and response regulate shoot growth sensitivity to mild drought in wheat

    Ravi eValluru

    2016-04-01

    Full Text Available Although plant hormones play an important role in adjusting growth in response to environmental perturbation, the relative contributions of abscisic acid (ABA and ethylene remain elusive. Using six spring wheat genotypes differing for stress tolerance, we show that young seedlings of the drought-tolerant (DT group maintained or increased shoot dry weight (SDW while the drought-susceptible (DS group decreased SDW in response to mild drought. Both the DT and DS groups increased endogenous ABA and ethylene concentrations under mild drought compared to control. The DT and DS groups exhibited different SDW response trends, whereby the DS group decreased while the DT group increased SDW, to increased concentrations of ABA and ethylene under mild drought, although both groups decreased ABA/ethylene ratio under mild drought albeit at different levels. We concluded that SDW of the DT and DS groups might be distinctly regulated by specific ABA:ethylene ratio. Further, a foliar-spray of low concentrations (0.1 μM of ABA increased shoot relative growth rate (RGR in the DS group while ACC (1-aminocyclopropane-1-carboxylic acid, ethylene precursor spray increased RGR in both groups compared to control. Furthermore, the DT group accumulated a significantly higher galactose while a significantly lower maltose in the shoot compared to the DS group. Taken all together, these results suggest an impact of ABA, ethylene and ABA:ethylene ratio on SDW of wheat seedlings that may partly underlie a genotypic variability of different shoot growth sensitivities to drought among crop species under field conditions. We propose that phenotyping based on hormone accumulation, response and hormonal ratio would be a viable, rapid, and an early–stage selection tool aiding genotype selection for stress tolerance.

  5. Foliar Abscisic Acid-To-Ethylene Accumulation and Response Regulate Shoot Growth Sensitivity to Mild Drought in Wheat.

    Valluru, Ravi; Davies, William J; Reynolds, Matthew P; Dodd, Ian C

    2016-01-01

    Although, plant hormones play an important role in adjusting growth in response to environmental perturbation, the relative contributions of abscisic acid (ABA) and ethylene remain elusive. Using six spring wheat genotypes differing for stress tolerance, we show that young seedlings of the drought-tolerant (DT) group maintained or increased shoot dry weight (SDW) while the drought-susceptible (DS) group decreased SDW in response to mild drought. Both the DT and DS groups increased endogenous ABA and ethylene concentrations under mild drought compared to control. The DT and DS groups exhibited different SDW response trends, whereby the DS group decreased while the DT group increased SDW, to increased concentrations of ABA and ethylene under mild drought, although both groups decreased ABA/ethylene ratio under mild drought albeit at different levels. We concluded that SDW of the DT and DS groups might be distinctly regulated by specific ABA:ethylene ratio. Further, a foliar-spray of low concentrations (0.1 μM) of ABA increased shoot relative growth rate (RGR) in the DS group while ACC (1-aminocyclopropane-1-carboxylic acid, ethylene precursor) spray increased RGR in both groups compared to control. Furthermore, the DT group accumulated a significantly higher galactose while a significantly lower maltose in the shoot compared to the DS group. Taken all together, these results suggest an impact of ABA, ethylene, and ABA:ethylene ratio on SDW of wheat seedlings that may partly underlie a genotypic variability of different shoot growth sensitivities to drought among crop species under field conditions. We propose that phenotyping based on hormone accumulation, response and hormonal ratio would be a viable, rapid, and an early-stage selection tool aiding genotype selection for stress tolerance. PMID:27148292

  6. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  7. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn, E-mail: LoneB.Madsen@agrsci.dk

    2013-08-23

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  8. PPARγ isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation

    Strand, D.W.; Jiang, M; Murphy, T A; Yi, Y.; Konvinse, K C; Franco, O E; Wang, Y.; Young, J D; Hayward, S.W.

    2012-01-01

    Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithel...

  9. Regulation of carnitine palmitoyltransferase I (CPT-Iα) gene expression by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) isoforms

    Sadana, Prabodh; Zhang, Yi; Song, Shulan; Cook, George A.; Elam, Marshall B.; Park, Edwards A.

    2006-01-01

    The peroxisome proliferator-activated receptor gamma coactivators (PGC-1) have important roles in mitochondrial biogenesis and metabolic control in a variety of tissues. There are multiple isoforms of PGC-1 including PGC-1α and PGC-1β. Both the PGC-1α and β isoforms promote mitochondrial biogenesis and fatty acid oxidation, but only PGC-1α stimulates gluconeogenesis in the liver. Carnitine palmitoyltransferase I (CPT-I) is a key enzyme regulating mitochondrial fatty acid oxidation. In these s...

  10. Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions

    Lee, Gloria; Spring, Frances A.; Parons, Stephen F.; Mankelow, Tosti J.; Peters, Luanne L.; Koury, Mark J.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

    2003-02-18

    ICAM-4, a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding a4b1 and aV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express a4b1 and ICAM-4 and macrophages exhibit aV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68 percent overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic a4b1-expressing HEL cells and non-hematopoietic aV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with GFP constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.

  11. DNA signals at isoform promoters.

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

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

  12. Tissue- and isoform-specific phytochrome regulation of light-dependent anthocyanin accumulation in Arabidopsis thaliana

    Warnasooriya, Sankalpi N.; Porter, Katie J.; Montgomery, Beronda L

    2011-01-01

    Phytochromes regulate light- and sucrose-dependent anthocyanin synthesis and accumulation in many plants. Mesophyll-specific phyA alone has been linked to the regulation of anthocyanin accumulation in response to far-red light in Arabidopsis thaliana. However, multiple mesophyll-localized phytochromes were implicated in the photoregulation of anthocyanin accumulation in red-light conditions. Here, we report a role for mesophyll-specific phyA in blue-light-dependent regulation of anthocyanin l...

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

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Justine R. Stehn; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.; Fath, Thomas; Schevzov, Galina; Gunning, Peter W.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to...

  14. Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of β1 integrin-null cells

    The presence of many laminin receptors of the β1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin α6β4 and dystroglycan. We therefore tested the binding of a β1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin α6Aβ4A variant. GD25 β1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin α6 antibody, but not by a dystroglycan antibody. Hence, integrin α6Aβ4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin α6Aβ4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin α6Aβ4A

  15. A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism

    Herman, Mark Andrew; Peroni, Odile Daniele; Villoria, Jorge; Schön, Michael R; Abumrad, Nada A.; Blüher, Matthias; Klein, Samuel; Kahn, Barbara

    2012-01-01

    Summary The prevalence of obesity and type 2-diabetes is increasing worldwide and threatens to shorten lifespan. Impaired insulin action in peripheral tissues is a major pathogenic factor. Insulin stimulates glucose uptake in adipose tissue through the Glut4-glucose transporter and alterations in adipose-Glut4 expression or function regulate systemic insulin sensitivity. Downregulation of adipose tissue-Glut4 occurs early in diabetes development. Here we report that adipose tissue-Glut4 regul...

  16. Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblastoma tumour suppressor protein (pRB) and the pRB-related proteins p107 and p130, key regulators of the cell-cycle, differentiation and apoptosis. The interaction between pRB and DGKzeta is regulated ...

  17. Calponin isoforms CNN1, CNN2 and CNN3: Regulators for actin cytoskeleton functions in smooth muscle and non-muscle cells.

    Liu, Rong; Jin, J-P

    2016-07-01

    Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and many types of non-muscle cells. Three homologous genes, CNN1, CNN2 and CNN3, encoding calponin isoforms 1, 2, and 3, respectively, are present in vertebrate species. All three calponin isoforms are actin-binding proteins with functions in inhibiting actin-activated myosin ATPase and stabilizing the actin cytoskeleton, while each isoform executes different physiological roles based on their cell type-specific expressions. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Calponin 2 is expressed in both smooth muscle and non-muscle cells and regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development and myogenesis. Phosphorylation has been extensively studied for the regulation of calponin functions. Cytoskeleton tension regulates the transcription of CNN2 gene and the degradation of calponin 2 protein. This review summarizes our knowledge learned from studies over the past three decades, focusing on the evolutionary lineage of calponin isoform genes, their tissue- and cell type-specific expressions, structure-function relationships, and mechanoregulation. PMID:26970176

  18. Nkx factors specifically regulate expression of Hedgehog receptor isoforms in early embryonic development

    BACKGROUND: NK homeobox family members are tissue-specific transcription factors that regulate developmental genes. Homozygous disruption of Nkx3.2 produces severe developmental defects of the axial skeleton, skull, spleen, and stomach. Murine mutation of Nkx2.5 results in death at E9 with defects i...

  19. Nuclear Factor I isoforms regulate gene expression during the differentiation of human neural progenitors to astrocytes

    Wilczynska, Katarzyna M.; Singh, Sandeep K.; Adams, Bret; Bryan, Lauren; Rao, Raj R.; Valerie, Kristoffer; Wright, Sarah; Griswold-Prenner, Irene; Kordula, Tomasz

    2009-01-01

    Even though astrocytes are critical for both normal brain functions and the development and progression of neuropathological states, including neuroinflammation associated with neurodegenerative diseases, the mechanisms controlling gene expression during astrocyte differentiation are poorly understood. Thus far, several signaling pathways were shown to regulate astrocyte differentiation, including JAK-STAT, BMP-2/Smads, and Notch. More recently, a family of Nuclear Factor-1 (NFI-A, -B, -C, an...

  20. Muscle Lim Protein isoform negatively regulates striated muscle actin dynamics and differentiation

    Vafiadaki, Elizabeth; Arvanitis, Demetrios A.; Papalouka, Vasiliki; Terzis, Gerasimos; ROUMELIOTIS, Theodoros I.; Spengos, Konstantinos; Garbis, Spiros D.; Manta, Panagiota; Kranias, Evangelia G.; Sanoudou, Despina

    2014-01-01

    Muscle Lim Protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, while aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum ...

  1. Opposition between PKC Isoforms Regulates Histone Deimination and Neutrophil Extracellular Chromatin Release

    Marko eRadic; Indira eNeeli

    2013-01-01

    In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase IV (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol ...

  2. Both liver-X receptor (LXR) isoforms control energy expenditure by regulating Brown Adipose Tissue activity

    Korach-André, Marion; Archer, Amena; Barros, Rodrigo P.; Parini, Paolo; Gustafsson, Jan-Åke

    2010-01-01

    Brown adipocytes are multilocular lipid storage cells that play a crucial role in nonshivering thermogenesis. Uncoupling protein 1 (UCP1) is a unique feature of brown fat cells that allows heat generation on sympathetic nervous system stimulation. As conventional transcriptional factors that are activated in various signaling pathways, liver-X receptors (LXRs) play important roles in many physiological processes. The role of LXRs in the regulation of energy homeostasis remains unclear, howeve...

  3. The novel CXCL12gamma isoform encodes an unstructured cationic domain which regulates bioactivity and interaction with both glycosaminoglycans and CXCR4.

    Cédric Laguri

    Full Text Available BACKGROUND: CXCL12alpha, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4 and heparan sulfate (HS. The alternative splicing of the cxcl12 gene generates a recently identified isoform, CXCL12gamma, which structure/function relationships remain unexplored. The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS. METHODOLOGY/PRINCIPAL FINDINGS: Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12gamma first 68 amino acids adopt a structure closely related to the well described alpha isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60% of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM, and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine. CONCLUSIONS/SIGNIFICANCE: Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the gamma isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS

  4. Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: Implications for regulation and cellular function

    Hubert, Nadia; Hentze, Matthias W.

    2002-01-01

    Divalent metal transporter 1 (DMT1) mediates apical iron uptake into duodenal enterocytes and also transfers iron from the endosome into the cytosol after cellular uptake via the transferrin receptor. Hence, mutations in DMT1 cause systemic iron deficiency and anemia. DMT1 mRNA levels are increased in the duodenum of iron-deficient animals. This regulation has been observed for DMT1 mRNA harboring an iron–responsive element (IRE) in its 3′ UTR, but not for a processing variant lacking a 3′UTR...

  5. A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy

    Ruas, Jorge L.; White, James P.; Rao, Rajesh R.; Kleiner, Sandra; Brannan, Kevin T.; Harrison, Brooke C.; Greene, Nicholas P.; Wu, Jun; Estall, Jennifer L.; Irving, Brian A.; Lanza, Ian R.; Rasbach, Kyle A.; Okutsu, Mitsuharu; Nair, K. Sreekumaran; Yan, Zhen

    2012-01-01

    PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise, but has no effects on muscle strength or hypertrophy. We have identified a novel form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induc...

  6. SUMO5, a Novel Poly-SUMO Isoform, Regulates PML Nuclear Bodies.

    Liang, Ya-Chen; Lee, Chia-Chin; Yao, Ya-Li; Lai, Chien-Chen; Schmitz, M Lienhard; Yang, Wen-Ming

    2016-01-01

    Promyelocytic leukemia nuclear bodies (PML-NBs) are PML-based nuclear structures that regulate various cellular processes. SUMOylation, the process of covalently conjugating small ubiquitin-like modifiers (SUMOs), is required for both the formation and the disruption of PML-NBs. However, detailed mechanisms of how SUMOylation regulates these processes remain unknown. Here we report that SUMO5, a novel SUMO variant, mediates the growth and disruption of PML-NBs. PolySUMO5 conjugation of PML at lysine 160 facilitates recruitment of PML-NB components, which enlarges PML-NBs. SUMO5 also increases polySUMO2/3 conjugation of PML, resulting in RNF4-mediated disruption of PML-NBs. The acute promyelocytic leukemia oncoprotein PML-RARα blocks SUMO5 conjugation of PML, causing cytoplasmic displacement of PML and disruption of PML-NBs. Our work not only identifies a new member of the SUMO family but also reveals the mechanistic basis of the PML-NB life cycle in human cells. PMID:27211601

  7. Down-regulated expression of atypical PKC-binding domain deleted asip isoforms in human hepatocellular carcinomas

    2001-01-01

    Asip is a mammalian homologue of polarity protein Par-3 of Caenorhabditis elegans and Bazooka of Drosophila melanogaster. Asip/Par-3/Bazooka are PDZ-motif containing proteins that localize asymmetrically to the cell periphery and play a pivotal role in cell polarity and asymmetric cell division. In the present study, we have cloned human asip cDNA and its splicing variants by 5'-RACE and RT-PCR using candidate human EST clones which have a high homology to rat asip cDNA. The full-length cDNA of human asip encodes a 1,353 aa protein exhibiting 88% similarity to the rat one. Human asip is a single copy gene consisting of at least 26 exons and localizing in human chromosome 10, band p11.2, with some extraordinarily long introns. All exon/intron boundary nucleotides conform to the “gt-ag” rule. Three main transcripts were detected by Northern blot analysis, and at least five variants, from alternative splicing and polyadenylation, have been identified by RT-PCR and liver cDNA library screening. Exon 17b deleted asip mRNAs expressed ubiquitously in normal human tissues, including liver, on RT-PCR analysis. However, they were absent from most human liver cancer cell lines examined. More interestingly, the expression of exon 17b deleted variants was down regulated in 52.6% (10/19) clinic specimens of human hepatocellular carcinomas (HCCs), compared with the surrounding nontumorous liver tissues from the same patients. The presence of various splicing transcripts, the variation of their distribution among different tissues and cells, and their differential expressions in human HCCs suggest that human Asip isoforms may function in different context.

  8. Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites

    Lindgren, Niklas; Usiello, Alessandro; Goiny, Michel; Haycock, John; Erbs, Eric; Greengard, Paul; Hökfelt, Tomas; Borrelli, Emiliana; Fisone, Gilberto

    2003-01-01

    Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knock...

  9. Regulation of carnitine palmitoyltransferase I (CPT-Iα) gene expression by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) isoforms

    Sadana, Prabodh; Zhang, Yi; Song, Shulan; Cook, George A.; Elam, Marshall B.; Park, Edwards A.

    2007-01-01

    Summary The peroxisome proliferator-activated receptor gamma coactivators (PGC-1) have important roles in mitochondrial biogenesis and metabolic control in a variety of tissues. There are multiple isoforms of PGC-1 including PGC-1α and PGC-1β. Both the PGC-1α and β isoforms promote mitochondrial biogenesis and fatty acid oxidation, but only PGC-1α stimulates gluconeogenesis in the liver. Carnitine palmitoyltransferase I (CPT-I) is a key enzyme regulating mitochondrial fatty acid oxidation. In these studies, we determined that PGC-1β stimulated expression of the “liver” isoform of CPT-I (CPT-Iα) but that PGC-1β did not induce pyruvate dehydrogenase kinase 4 (PDK4) which is a regulator of pyruvate metabolism. The CPT-Iα gene is induced by thyroid hormone. We found that T3 increased the expression of PGC-1β and that PGC-1β enhanced the T3 induction of CPT-Iα. The thyroid hormone receptor interacts with PGC-1β in a ligand dependent manner. Unlike PGC-1α, the interaction of PGC-1β and the T3 receptor does not occur exclusively through the leucine-X-X-leucine-leucine motif in PGC-1β. We have found that PGC-1β is associated with the CPT-Iα gene in vivo. Overall, our results demonstrate that PGC-1β is a coactivator in the T3 induction of CPT-Iα and that PGC-1β has similarities and differences with the PGC-1α isoform. PMID:17239528

  10. Ethylene evolution and endo-beta-mannanase activity during lettuce seed germination at high temperature Evolução de etileno e atividade da enzima endo-beta-mananase durante a germinação de sementes de alface sob altas temperaturas

    Warley Marcos Nascimento; Daniel James Cantliffe; Donald John Huber

    2004-01-01

    High temperatures during lettuce seed imbibition can delay or completely inhibit germination and the endosperm layer appears to restrict the radicle protrusion. The role of endo-beta-mannanase during lettuce seed germination at 35°C and the influence of ethylene in endo-beta-mannanase regulation were investigated. Seeds of 'Dark Green Boston' (DGB) and 'Everglades' (EVE) were germinated in water, or 10 mmol L-1 of 1-aminocyclopropane-1-carboxylic acid (ACC), or 10 mmol L-1 of aminoethoxyvinyl...

  11. The amyloid precursor protein (APP) intracellular domain regulates translation of p44, a short isoform of p53, through an IRES-dependent mechanism.

    Li, Mi; Pehar, Mariana; Liu, Yan; Bhattacharyya, Anita; Zhang, Su-Chun; O'Riordan, Kenneth J; Burger, Corinna; D'Adamio, Luciano; Puglielli, Luigi

    2015-10-01

    p44 is a short isoform of the tumor suppressor protein p53 that is regulated in an age-dependent manner. When overexpressed in the mouse, it causes a progeroid phenotype that includes premature cognitive decline, synaptic defects, and hyperphosphorylation of tau. The hyperphosphorylation of tau has recently been linked to the ability of p44 to regulate transcription of relevant tau kinases. Here, we report that the amyloid precursor protein (APP) intracellular domain (AICD), which results from the processing of the APP, regulates translation of p44 through a cap-independent mechanism that requires direct binding to the second internal ribosome entry site (IRES) of the p53 mRNA. We also report that AICD associates with nucleolin, an already known IRES-specific trans-acting factor that binds with p53 IRES elements and regulates translation of p53 isoforms. The potential biological impact of our findings was assessed in a mouse model of Alzheimer's disease. In conclusion, our study reveals a novel aspect of AICD and p53/p44 biology and provides a possible molecular link between APP, p44, and tau. PMID:26174856

  12. Inibição da síntese da ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em maçãs frigoconservadas em atmosfera controlada Inhibition of (1-aminocyclopropane-1-carboxylic acid) oxydase synthesis in apple fruits by controlled atmosphere storage

    Zimmer, Paulo Dejalma; Jaqueline Dettmann BIERHALS; SILVA Jorge Adolfo; ROMBALDI Cesar Valmor

    1999-01-01

    Foi estudada a expressão da ACC oxidase em maçãs, cv. Jonagold, colhidas no estádio pré-climatérico e armazenadas sob refrigeração em atmosfera normal (0ºC, 95% UR - AN) e controlada (0ºC, 95% UR, 1,5% O2 e 2,5% CO2 - AC), durante 180 dias. Na instalação do experimento, aos 90 e aos 120 dias, foram coletadas amostras para a determinação da firmeza de polpa, da acidez total titulável, dos sólidos solúveis totais, da produção de etileno, da atividade ACC oxidase e para a detecção imunoquímica d...

  13. Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

    Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.;

    2013-01-01

    When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2......-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from...... turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site...

  14. CKI isoforms α and ε regulate Star–PAP target messages by controlling Star–PAP poly(A) polymerase activity and phosphoinositide stimulation

    Laishram, Rakesh S.; Barlow, Christy A.; Richard A. Anderson

    2011-01-01

    Star–PAP is a non-canonical, nuclear poly(A) polymerase (PAP) that is regulated by the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PI4,5P2), and is required for the expression of a select set of mRNAs. It was previously reported that a PI4,5P2 sensitive CKI isoform, CKIα associates with and phosphorylates Star–PAP in its catalytic domain. Here, we show that the oxidative stress-induced by tBHQ treatment stimulates the CKI mediated phosphorylation of Star–PAP, which is crit...

  15. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth. - Highlights: • Glutamate induced neuronal progenitor cells in the mature rat retina. • Proteomic analysis revealed the up-regulation of DRP-3, DRP-2 and STIP1. • mRNA expression of DRP-3, especially, its long isoform, robustly increased

  16. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina

    Tokuda, Kazuhiro, E-mail: r502um@yamaguchi-u.ac.jp [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Kuramitsu, Yasuhiro; Byron, Baron; Kitagawa, Takao [Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Tokuda, Nobuko [Faculty of Health Sciences, Yamaguchi University Graduate School of Medicine, Ube (Japan); Kobayashi, Daiki; Nagayama, Megumi; Araki, Norie [Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sonoda, Koh-Hei [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Nakamura, Kazuyuki [Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan)

    2015-08-07

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth. - Highlights: • Glutamate induced neuronal progenitor cells in the mature rat retina. • Proteomic analysis revealed the up-regulation of DRP-3, DRP-2 and STIP1. • mRNA expression of DRP-3, especially, its long isoform, robustly increased.

  17. p53 isoforms, Δ133p53 and p53β, are endogenous regulators of replicative cellular senescence

    Fujita, Kaori; Mondal, Abdul M.; Horikawa, Izumi; Nguyen, Giang H.; Kumamoto, Kensuke; Sohn, Jane J.; Bowman, Elise D.; Mathe, Ewy A.; Schetter, Aaron J.; Pine, Sharon R.; Ji, Helen; Vojtesek, Borivoj; Bourdon, Jean-Christophe; Lane, David P; Harris, Curtis C.

    2009-01-01

    The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo1–3. We show that human p53 isoforms (Δ133p53 and p53β)4 constitute an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expre...

  18. Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5

    Fushimi, Kazuo; Ray, Payal; Kar, Amar; Wang, Lei; Sutherland, Leslie C.; Jane Y Wu

    2008-01-01

    Similar to many genes involved in programmed cell death (PCD), the caspase 2 (casp-2) gene generates both proapoptotic and antiapoptotic isoforms by alternative splicing. Using a yeast RNA–protein interaction assay, we identified RBM5 (also known as LUCA-15) as a protein that binds to casp-2 pre-mRNA. In both transfected cells and in vitro splicing assay, RBM5 enhances the formation of proapoptotic Casp-2L. RBM5 binds to a U/C-rich sequence immediately upstream of the previously identified In...

  19. Human CD72 splicing isoform responsible for resistance to systemic lupus erythematosus regulates serum immunoglobulin level and is localized in endoplasmic reticulum

    Hitomi Yuki

    2012-12-01

    Full Text Available Abstract Background CD72 is an inhibitory co-receptor expressed on B cells. We previously demonstrated significant association of the polymorphism of the CD72 gene with susceptibility to human systemic lupus erythematosus (SLE in individuals carrying a SLE-susceptible FCGR2B genotype (FCGR2B-232Thr/Thr. The human CD72 locus generates a splicing isoform that lacks exon 8 (CD72Δex8 as well as full-length CD72 (CD72fl, and the CD72 polymorphism regulates exon 8 skipping. Results Here we demonstrated that individuals carrying the disease-protective CD72 genotype exhibit significantly lower serum immunoglobulin levels than do individuals carrying other CD72 genotypes (P CD72 genotype, the protein level of CD72Δex8 was increased in individuals carrying the disease-protective CD72 genotype, suggesting a crucial role of CD72Δex8 in regulation of antibody production. By expressing these human CD72 isoforms in mouse cell lines, we further demonstrated that CD72Δex8 is accumulated in endoplasmic reticulum (ER and fails to regulate BCR signaling whereas human CD72fl is efficiently transported to the cell surface and inhibits signaling through the B cell antigen receptor (BCR, as is the case for mouse CD72. Conclusion Human CD72 polymorphism appears to regulate antibody production as well as susceptibility to SLE by regulating expression of ER-localizing CD72Δex8.

  20. Antioxidant diet and sex interact to regulate NOS isoform expression and glomerular mesangium proliferation in Zucker diabetic rat kidney.

    Slyvka, Yuriy; Malgor, Ramiro; Inman, Sharon R; Ding, Julia; Heh, Victor; Nowak, Felicia V

    2016-03-01

    Oxidative stress contributes substantially to the pathophysiology of diabetic nephropathy (DN). Consumption of an antioxidant-fortified (AO) diet from an early age prevents or delays later development of DN in the Zucker rat female with type 2 diabetes. We hypothesize this is due to effects on mesangial matrix and renal nitric oxide synthase (NOS) distribution and to sex-specific differences in NOS responses in the diabetic kidney. Total glomerular tuft area (GTA) and PAS-positive tuft area (PTA), endothelial (e), neuronal (n) and inducible (i) NOS were quantified in males and females on AO or regular (REG) diet at 6 and 20 weeks of age. eNOS was observed in glomeruli and tubules. nNOS predominantly localized to tubular epithelium in both cortex and medulla. iNOS was expressed in proximal and distal tubules and collecting ducts. Sex, diabetes duration and AO diet affected the distribution of the three isoforms. GTA and PTA increased with duration of hyperglycemia and showed a negative correlation with renal levels of all NOS isoforms. AO diet in both genders was associated with less PAS-positive staining and less mesangial expansion than the REG diet, an early increase in cortical iNOS in males, and sex-specific changes in cortical eNOS at 20 weeks. These effects of AO diet may contribute to sex-specific preservation of renal function in females. PMID:26797190

  1. Urocortins and CRF type 2 receptor isoforms expression in the rat stomach are regulated by endotoxin: role in the modulation of delayed gastric emptying.

    Yuan, Pu-Qing; Wu, S Vincent; Taché, Yvette

    2012-07-01

    Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF(2) isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100 μg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 μg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P gastric motor alterations to endotoxemia. PMID:22517775

  2. The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

    Halsey Christina

    2012-08-01

    Full Text Available Abstract Background Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL where GATA1FL mutations are an essential driver for disease pathogenesis. Methods Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. Results We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. Conclusions These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.

  3. Analysis of opa1 isoforms expression and apoptosis regulation in autosomal dominant optic atrophy (ADOA) patients with mutations in the opa1 gene.

    Formichi, Patrizia; Radi, Elena; Giorgi, Eleonora; Gallus, Gian Nicola; Brunetti, Jlenia; Battisti, Carla; Rufa, Alessandra; Dotti, Maria Teresa; Franceschini, Rossella; Bracci, Luisa; Federico, Antonio

    2015-04-15

    Autosomal dominant optic atrophy (ADOA) is a hereditary optic neuropathy characterized by bilateral symmetrical visual loss, decrease in retinal ganglion cells and a loss of myelin within the optic nerve. ADOA is associated to mutations in Optic atrophy 1 gene (OPA1), which encodes a mitochondrial protein involved in cristae remodeling, maintenance of mitochondrial membrane integrity, mitochondrial fusion and apoptosis regulation. We thus evaluated the rate of apoptosis and the expression levels of OPA1 isoforms in ADOA and control cells. Peripheral blood lymphocytes from eight patients with OPA1 mutation and age matched controls were cultivated both in basal conditions or with 2-deoxy-D-ribose, a reducing sugar that induces apoptosis through oxidative stress. Apoptosis was analyzed by flow cytometry, phosphatidylserine translocation, mitochondrial membrane depolarization and caspase 3 activation. We also analyzed the expression levels of OPA1 isoforms in ADOA and control cells cultured with and without 2-deoxy-D-ribose. We showed an increased percentage of apoptotic cells in ADOA patients compared to controls, both in basal culture conditions and after 2-deoxy-D-ribose treatment. This suggested a great susceptibility of ADOA cells to oxidative stress and a strong correlation between OPA1 protein dysfunctions and morphological-functional alterations to mitochondria. Moreover OPA1 protein expression was significantly decreased in lymphocytes from the ADOA patients after 2-deoxy-D-ribose treatment, implying a great sensitivity of the mutated protein to free radical damage. Concluding, we could confirm that oxidative stress-induced apoptosis may play a key role in the pathophysiological process bringing to retinal ganglion cells degeneration in ADOA. PMID:25796301

  4. Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

    Wang, Jiajing; Hmadcha, Abdelkrim; Zakarian, Vaagn; Song, Fei; Loeb, Jeffrey A

    2015-09-01

    The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions. PMID:25913151

  5. Differential Expression and Regulation of Brain-Derived Neurotrophic Factor (BDNF) mRNA Isoforms in Brain Cells from Mecp2(308/y) Mouse Model.

    Rousseaud, Audrey; Delépine, Chloé; Nectoux, Juliette; Billuart, Pierre; Bienvenu, Thierry

    2015-08-01

    Rett syndrome (RTT) is a severe neurodevelopmental disease caused by mutations in methyl-CpG-binding protein 2 (MECP2), which encodes a transcriptional modulator of many genes including BDNF. BDNF comprises nine distinct promoter regions, each triggering the expression of a specific transcript. The role of this diversity of transcripts remains unknown. MeCP2 being highly expressed in neurons, RTT was initially considered as a neuronal disease. However, recent studies have shown that MeCP2 was also expressed in astrocytes. Though several studies explored Bdnf IV expression in Mecp2-deficient mice, the differential expression of Bdnf isoforms in Mecp2-deficient neurons and astrocytes was never studied. By using TaqMan technology and a mouse model expressing a truncated Mecp2 (Mecp2(308/y)), we firstly showed in neurons that Bdnf transcripts containing exon I, IIb, IIc, IV, and VI are prominently expressed, whereas in astrocytes, Bdnf transcript containing exon VI is preferentially expressed, suggesting a specific regulation of Bdnf expression at the cellular level. Secondly, we confirmed the repressive role of Mecp2 only on the expression of Bdnf VI in neurons. Our data suggested that the truncated Mecp2 protein maintains its function on Bdnf expression regulation in neurons and in astrocytes. Interestingly, we observed that Bdnf transcripts (I and IXA), regulated by neural activity induced by bicuculline in Mecp2(308/y) neurons, were not affected by histone deacetylase inhibition. In contrast, Bdnf transcripts (IIb, IIc, and VI), regulated by histone deacetylation, were not affected by bicuculline treatment in wild-type and Mecp2(308/y) neurons. All these results reflect the complexity of regulation of Bdnf gene. PMID:25634725

  6. Isoforms of elongation factor eEF1A may be differently regulated at post-transcriptional level in breast cancer progression

    Vislovukh A. A.

    2013-01-01

    Full Text Available Eukaryotic translation elongation factor 1A exists as two 98 % homologous isoforms: eEF1A1 (A1 and eEF1A2 (A2 which are tissue and development specific. Despite high homology in an open reading frame (ORF region, mRNAs coding for eEF1A1 and eEF1A2 are different in their untranslated regions (UTR, suggesting a possibility of their dissimilar post-transcriptional regulation. Aim. To analyze the existence of cis-acting motifs in the UTRs of EEF1A1/A2 mRNAs, to confirm the possibility of post-transcriptional control of eEF1A1 and eEF1A2 expression. Methods. An ensemble of bioinformatic methods was applied to predict regulatory motifs in the UTRs of EEF1A1/A2 mRNAs. Dual-luciferase reporter assay was employed to detect post-transcriptional regulation of eEF1A1/A2 expression. Results. Numerous regulatory motifs in the UTR of EEF1A1/A2 mRNAs were found bioinformatically. The experimental evidence was obtained for the existence of negative regulation of EEF1A1 and positive regulation of EEF1A2 mRNA in the model of breast cancer development. Conclusions. EEF1A1 and EEF1A2 mRNAs contain distinct motifs in the UTRs and are differently regulated in cancer suggesting the possibility of their control by different cellular signals.

  7. Transcription regulation of AAC3 gene encoding hypoxic isoform of ADP/ATP carrier in Saccharomyces cerevisiae

    Two repressoric regions are present in the AAC3 promoter, termed URS1 and URS2. URS1 region is responsible for a carbon source-dependent regulation and plays a role under both, aerobic and anaerobic conditions. By deletion analysis URS1 was localized into the -322/-244 region and was found that the regulation is likely exerted by the repression by non-fermentable or non-repressing fermentable carbon sources than by the activation by repressing carbon source. By computer analysis cis sequences for two potential transcription factors, Rap1 and ERA, were identified within URS1. Rap1 binding into its consensus sequence was proved, effort to find the protein binding to the ERA cis regulatory sequences has failed. By the means of mutational analysis we revealed that the regulation pathway mediating the carbon source-dependent regulation via URS1 differs according to the presence or absence of oxygen in the growth medium. Under aerobic conditions the carbon source-dependent repression is mediated by the ERA factor and the role of Rap1 is only marginal. On the contrary, under anaerobic conditions, the repression is mediated solely by Rap1. AAC1 gene product might be involved in the regulation of the AAC3 gene, the regulation pathway has not been characterized yet. (author)

  8. p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter.

    Marcel, V; Vijayakumar, V; Fernández-Cuesta, L; Hafsi, H; Sagne, C; Hautefeuille, A; Olivier, M; Hainaut, P

    2010-05-01

    The tumor suppressor p53 protein is activated by genotoxic stress and regulates genes involved in senescence, apoptosis and cell-cycle arrest. Nine p53 isoforms have been described that may modulate suppressive functions of the canonical p53 protein. Among them, Delta133p53 lacks the 132 proximal residues and has been shown to modulate p53-induced apoptosis and cell-cycle arrest. Delta133p53 is expressed from a specific mRNA, p53I4, driven by an alternative promoter P2 located between intron 1 and exon 5 of TP53 gene. Here, we report that the P2 promoter is regulated in a p53-dependent manner. Delta133p53 expression is increased in response to DNA damage by doxorubicin in p53 wild-type cell lines, but not in p53-mutated cells. Chromatin immunoprecipitation and luciferase assays using P2 promoter deletion constructs indicate that p53 binds functional response elements located within the P2 promoter. We also show that Delta133p53 does not bind specifically to p53 consensus DNA sequence in vitro, but competes with wild-type p53 in specific DNA-binding assays. Finally, we report that Delta133p53 counteracts p53-dependent growth suppression in clonogenic assays. These observations indicate that Delta133p53 is a novel target of p53 that may participate in a negative feedback loop controlling p53 function. PMID:20190805

  9. Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-κB in human aortic smooth muscle cells

    Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-κB (NF-κB) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-κB signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-α (TNFα), a potent inducer of both Nox and NF-κB, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-κB pathway reduced significantly the TNFα-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-κB elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-κB significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-κB proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-κB is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-κB and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

  10. Differentiation-induced cleavage of Cutl1/CDP generates a novel dominant-negative isoform that regulates mammary gene expression.

    Maitra, Urmila; Seo, Jin; Lozano, Mary M; Dudley, Jaquelin P

    2006-10-01

    Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland. PMID:17015474

  11. The Short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction

    Chen, Lanfen; Chen, Zhangguo; Baker, Kristi; Halvorsen, E lizabeth M.; da Cunha, Andre Pires; Flak, Magdalena B.; Gerber, Georg; Huang, Yu-Hwa; Hosomi, Shuhei; Arthur, J anelle C.; Dery, Ken J.; Nagaishi, Takashi; Beauchemin, Nicole; Kathryn V Holmes; Joshua W K Ho

    2012-01-01

    Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in ti...

  12. CCAAT/enhancer binding protein β (C/EBPβ isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer cells.

    André Albergaria

    Full Text Available P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

  13. Morphometric evaluation of nitric oxide synthase isoforms and their cytokine regulators predict pulmonary dysfunction and survival in systemic sclerosis

    E.R. Parra

    2013-01-01

    Full Text Available Because histopathological changes in the lungs of patients with systemic sclerosis (SSc are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO and plasminogen activator inhibitor-1 (PAI-1 synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP. We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS related significantly to staining of septal cells for interleukin (IL-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04, septal IL-13 (P=0.03, and septal basic fibroblast growth factor (bFGF; P=0.02. Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.

  14. Endoplasmic Reticulum-resident Heat Shock Protein 90 (HSP90) Isoform Glucose-regulated Protein 94 (GRP94) Regulates Cell Polarity and Cancer Cell Migration by Affecting Intracellular Transport.

    Ghosh, Suman; Shinogle, Heather E; Galeva, Nadezhda A; Dobrowsky, Rick T; Blagg, Brian S J

    2016-04-15

    Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion. PMID:26872972

  15. A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

    Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi

    2016-01-01

    Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression. PMID:26153462

  16. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    Ivanov, Sergey V., E-mail: Sergey.Ivanov@med.nyu.edu [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States); Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I. [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States)

    2009-05-08

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

  17. Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena;

    2014-01-01

    Plasma membrane Ca2+-ATPase (PMCA) by extruding Ca2+ outside the cell, actively participates in the regulation of intracellular Ca2+ concentration. Acting as Ca2+/H+ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four...... isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca2+ overload evoked by 59 m......+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient...

  18. The Zebrafish Equivalent of Alzheimer's Disease-Associated PRESENILIN Isoform PS2V Regulates Inflammatory and Other Responses to Hypoxic Stress.

    Ebrahimie, Esmaeil; Moussavi Nik, Seyyed Hani; Newman, Morgan; Van Der Hoek, Mark; Lardelli, Michael

    2016-03-31

    Dominant mutations in the PRESENILIN genes PSEN1 and PSEN2 cause familial Alzheimer's disease (fAD) that usually shows onset before 65 years of age. In contrast, genetic variation at the PSEN1 and PSEN2 loci does not appear to contribute to risk for the sporadic, late onset form of the disease (sAD), leading to doubts that these genes play a role in the majority of AD cases. However, a truncated isoform of PSEN2, PS2V, is upregulated in sAD brains and is induced by hypoxia and high cholesterol intake. PS2V can increase γ-secretase activity and suppress the unfolded protein response (UPR), but detailed analysis of its function has been hindered by lack of a suitable, genetically manipulable animal model since mice and rats lack this PRESENILIN isoform. We recently showed that zebrafish possess an isoform, PS1IV, that is cognate to human PS2V. Using an antisense morpholino oligonucleotide, we can block specifically the induction of PS1IV that normally occurs under hypoxia. Here, we exploit this ability to identify gene regulatory networks that are modulated by PS1IV. When PS1IV is absent under hypoxia-like conditions, we observe changes in expression of genes controlling inflammation (particularly sAD-associated IL1B and CCR5), vascular development, the UPR, protein synthesis, calcium homeostasis, catecholamine biosynthesis, TOR signaling, and cell proliferation. Our results imply an important role for PS2V in sAD as a component of a pathological mechanism that includes hypoxia/oxidative stress and support investigation of the role of PS2V in other diseases, including schizophrenia, when these are implicated in the pathology. PMID:27031468

  19. Apolipoprotein E Regulates the Integrity of Tight Junctions in an Isoform-dependent Manner in an in Vitro Blood-Brain Barrier Model*

    Nishitsuji, Kazuchika; Hosono, Takashi; Nakamura, Toshiyuki; Bu, Guojun; Michikawa, Makoto

    2011-01-01

    Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ϵ4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3- and apoE4-knock-in mice, we show that the barri...

  20. Sex-Steroid Regulation of Relaxin Receptor Isoforms (RXFP1 & RXFP2) Expression in the Patellar Tendon and Lateral Collateral Ligament of Female WKY Rats

    Dehghan, Firouzeh; Muniandy, Sekaran; Yusof, Ashril; Salleh, Naguib

    2014-01-01

    The incidence of non-contact knee injury was found higher in female than in male and is related to the phases of the menstrual cycle. This raised the possibility that female sex-steroids are involved in the mechanism underlying this injury via affecting the expression of the receptors for relaxin, a peptide hormone known to modulate ligament laxity. Therefore, this study aims to investigate the effect of sex-steroids on relaxin receptor isoforms (RXFP1 & RXFP2) expression in the ligaments and...

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

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

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

  2. The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

    Fabiana Salm

    Full Text Available Medulloblastoma (MB is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

  3. Ikaros isoforms:The saga continues

    Laura; A; Perez-Casellas; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    Through alternate splicing,the Ikaros gene produces multiple proteins.Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity.Ikaros isoforms interact to form dimers and potentially multimeric complexes.Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions.Small dominantnegative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros.Here,we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H.Although IK-1 is described as full-length Ikaros,IK-H is the longest Ikaros isoform.IK-H,which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4),is abundant in human but not murine hematopoietic cells.Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1.Moreover,IK-H can potentiate or inhibit the ability of IK-1 to bind DNA.IK-H binds to the regulatory regions of genes that are upregulated by Ikaros,but not genes that are repressed by Ikaros.Although IK-1 localizes to pericentromeric heterochromatin,IK-H can be found in both pericentromeric and non-pericentromeric locations.Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H,but not other Ikaros isoforms.The unique features of IK-H,its influence on Ikaros activity,and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

  4. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  5. DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans.

    Jennifer M A Tullet

    2014-02-01

    Full Text Available The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK, which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be

  6. DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans.

    Tullet, Jennifer M A; Araiz, Caroline; Sanders, Matthew J; Au, Catherine; Benedetto, Alexandre; Papatheodorou, Irene; Clark, Emily; Schmeisser, Kathrin; Jones, Daniel; Schuster, Eugene F; Thornton, Janet M; Gems, David

    2014-02-01

    The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved

  7. p53 Family: Role of Protein Isoforms in Human Cancer

    Jinxiong Wei

    2012-01-01

    Full Text Available TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.

  8. EASI—enrichment of alternatively spliced isoforms

    Julian P Venables; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This ...

  9. β-Adrenergic regulation of a novel isoform of NCX: sequence and expression of shark heart NCX in human kidney cells

    Janowski, Einsley; Day, Regina; Kraev, Alexander; Roder, John C.; Cleemann, Lars; Morad, Martin

    2009-01-01

    The function, regulation, and molecular structure of the cardiac Na+/Ca2+ exchangers (NCXs) vary significantly among vertebrates. We previously reported that β-adrenergic suppression of amphibian cardiac NCX1.1 is associated with specific molecular motifs. Here we investigated the bimodal, cAMP-dependent regulation of spiny dogfish shark (Squalus acanthias) cardiac NCX, exploring the effects of molecular structure, host cell environment, and ionic milieu. The shark cardiac NCX sequence (GenBa...

  10. Two conventional PKC isoforms, α and βI, are involved in the ATP-induced regulation of VRAC and glutamate release in cultured astrocytes

    Rudkouskaya, Alena; Chernoguz, Artur; Haskew-Layton, Renée E.; Mongin, Alexander A.

    2008-01-01

    Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompte...

  11. Ethylene is involved in brassinosteroids induced alternative respiratory pathway in cucumber (Cucumis sativus L. seedlings response to abiotic stress

    Lijie eWei

    2015-11-01

    Full Text Available Effects of brassinosteroids (BRs on cucumber (Cucumis sativus L. abiotic stresses resistance to salt, polyethylene glycol (PEG, cold and the potential mechanisms were investigated in this work. Previous reports have indicated that BRs can induce ethylene production and enhance alternative oxidase (AOX pathway. The mechanisms whether ethylene is involved as a signal molecule which connected BR with AOX in regulating stress tolerance are still unknown. Here, we found that pretreatment with 1 µM brassinolide (BL, the most active BRs relieved stress-caused oxidative damage in cucumber seedlings and clearly enhanced the capacity of AOX and the ethylene biosynthesis. Furthermore, transcription level of ethylene signaling biosynthesis genes including ripening-related ACC synthase1 (CSACS1, ripening-related ACC synthase2 (CSACS2, ripening-related ACC synthase3 (CSACS3, 1-aminocyclopropane-1-carboxylate oxidase1 (CSACO1, 1-aminocyclopropane-1-carboxylate oxidase2 (CSACO2 and CSAOX were increased after BL treatment. Importantly, the application of the salicylhydroxamic acid (SHAM, AOX inhibitor and ethylene biosynthesis inhibitor aminooxyacetic acid (AOA decreased plant resistance to environmental stress by blocking BRs-induced alternative respiration. Taken together, our results demonstrated that ethylene was involved in BRs-induced AOX activity which played important roles in abiotic stresses tolerance in cucumber seedlings.

  12. Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets.

    Lever, Robert A; Hussain, Azhar; Sun, Benjamin B; Sage, Stewart O; Harper, Alan G S

    2015-12-01

    -selective cation permeability of the plasma membrane as assessed by Mn(2+) quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca(2+)]cyt following SERCA inhibition and either removal of extracellular Na(+) or inhibition of Na(+)/K(+)-ATPase activity by removal of extracellular K(+) or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca(2+)]cyt by acceleration of SERCA activity, whilst rises in [Ca(2+)]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na(+)/K(+)-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na(+)]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca(2+) signalling. PMID:26434503

  13. CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

    Miura, Yuka; Hagiwara, Natsumi [Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, 2-1 Gakuen, Sanda 669-1337 Japan (Japan); Radisky, Derek C. [Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32225 (United States); Hirai, Yohei, E-mail: y-hirai@kwansei.ac.jp [Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, 2-1 Gakuen, Sanda 669-1337 Japan (Japan)

    2014-09-10

    Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.

  14. CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

    Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination

  15. Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure*S⃞

    Qiu, Hongyu; Dai, Huacheng; Jain, Komal; Shah, Rina; Hong, Chull; Pain, Jayashree; Tian, Bin; Vatner, Dorothy E.; Vatner, Stephen F.; Depre, Christophe

    2008-01-01

    Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p < 0.05), which was confirmed by quan...

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

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A., E-mail: roberto.perego@unimib.it

    2013-08-01

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

  17. A novel functional rabbit IL- 7 isoform

    Siewe, Basile T.; Kalis, Susan L.; Esteves, Pedro J; Zhou, Tong; Knight, Katherine L.

    2010-01-01

    IL-7 is required for B cell development in mouse and is a key regulator of T cell development and peripheral T cell homeostasis in mouse and human. Recently, we found that IL-7 is expressed in rabbit bone marrow and in vitro, is required for differentiation of lymphoid progenitors to B and T lineage cells. Herein, we report the identification of a novel rabbit IL-7 isoform, IL-7II. Recombinant IL-7II (rIL-7II) binds lymphocytes via the IL-7R and induces phosphorylation of STAT5. Further, rIL-...

  18. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  19. Altered Alpha-Synuclein, Parkin, and Synphilin Isoform Levels in Multiple System Atrophy Brains

    Brudek, Tomasz; Winge, Kristian; Bredo Rasmussen, Nadja;

    2016-01-01

    -1 isoforms. In MSA brains, alpha-synuclein140 and alpha-synuclein112 isoform levels were significantly increased,whereas levels of the alpha-synuclein126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus vs. CONTROLS: Moreover, in MSA cases, we showed...... increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphiln-1A isoform that causes neuronal toxicity in MSA. In PD brains, Parkin transcript variant 3, 7 and 11 were significantly and specifically overexpressed in the striatum and cerebellar cortex......, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of alpha-synuclein in the...

  20. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T; Clausen, Henrik; Linstedt, Adam D

    2014-01-01

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms...

  1. Differential roles of PML isoforms

    MouniraKChelbi-Alix

    2013-05-01

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

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

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

    2014-01-01

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

  3. Vitamin E Isoforms as Modulators of Lung Inflammation

    Hiam Abdala-Valencia

    2013-10-01

    Full Text Available Asthma and allergic diseases are complex conditions caused by a combination of genetic and environmental factors. Clinical studies suggest a number of protective dietary factors for asthma, including vitamin E. However, studies of vitamin E in allergy commonly result in seemingly conflicting outcomes. Recent work indicates that allergic inflammation is inhibited by supplementation with the purified natural vitamin E isoform α-tocopherol but elevated by the isoform γ-tocopherol when administered at physiological tissue concentrations. In this review, we discuss opposing regulatory effects of α-tocopherol and γ-tocopherol on allergic lung inflammation in clinical trials and in animal studies. A better understanding of the differential regulation of inflammation by isoforms of vitamin E provides a basis towards the design of clinical studies and diets that would effectively modulate inflammatory pathways in lung disease.

  4. Apolipoprotein E isoform-specific effects on lipoprotein receptor processing.

    Bachmeier, Corbin; Shackleton, Ben; Ojo, Joseph; Paris, Daniel; Mullan, Michael; Crawford, Fiona

    2014-12-01

    Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood–brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low-density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained increased. Likewise, intracranial administration of Aβ to apoE-targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4 > apoE3 > apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain. PMID:25015123

  5. Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains

    Dalton, Justin E.; Fear, Justin M.; Knott, Simon; Baker, Bruce S.; McIntyre, Lauren M.; Arbeitman, Michelle N.

    2013-01-01

    Background Drosophila melanogaster adult males perform an elaborate courtship ritual to entice females to mate. fruitless (fru), a gene that is one of the key regulators of male courtship behavior, encodes multiple male-specific isoforms (FruM). These isoforms vary in their carboxy-terminal zinc finger domains, which are predicted to facilitate DNA binding. Results By over-expressing individual FruM isoforms in fru-expressing neurons in either males or females and assaying the global transcri...

  6. Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

    Yousaf, N; Low, W Y; Onipinla, A; Mein, C; Caulfield, M; Munroe, P B; Chernajovsky, Y

    2015-05-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme. PMID:25545008

  7. Perspectives of bacterial ACC deaminase in phytoremediation.

    Arshad, Muhammad; Saleem, Muhammad; Hussain, Sarfraz

    2007-08-01

    Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics. PMID:17573137

  8. Activation of antithrombin III isoforms by heparan sulphate glycosaminoglycans and other sulphated polysaccharides.

    Carlson, T H; Kolman, M R; Piepkorn, M

    1995-07-01

    Antithrombin III occurs naturally as two functionally distinct molecular species that differ in glycosylation at Asn135. Whereas the predominant, glycosylated isoform has high affinity for heparin, a quantitatively minor isoform lacking glycosylation at that site displays relatively higher affinity for both heparins and heparinoids. We characterized the ability of various sulphated polysaccharides to potentiate the rates of thrombin inhibition by the isoforms. High-molecular-weight dextran sulphate was the most effective of those studied, increasing thrombin inhibition by the higher-affinity antithrombin III isoform up to five-fold more efficiently than did heparin fractions with low-affinity for antithrombin III. In addition, dextran sulphate activated the higher-affinity isoform as much as twelve times more effectively than it did the lower-affinity isoform. Pentosan polysulphate was up to three-fold, and some heparan sulphate fractions up to two-fold, more effective with the higher, compared with the lower affinity, isoform. Heparan sulphate preparations less effectively increased the rate of thrombin inhibition than did the other low-affinity polysaccharides. Structure-function studies indicated positive correlations between activity and both polymer length and anionic group density of low-affinity sulphated polysaccharides. The observed effects of the heparan sulphates on this anticoagulant pathway, although of low potency, are consistent with the hypotheses that these substances naturally regulate blood homeostasis in vascular tissues and that much of this function may be mediated by the higher-affinity antithrombin III isoform. PMID:8589216

  9. Inference of Isoforms from Short Sequence Reads

    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.

  10. Role of p53 isoforms and aggregations in cancer.

    Kim, SeJin; An, Seong Soo A

    2016-06-01

    p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers.Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways.Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects.As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  11. Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

    Lopez-Mejia, Isabel C; de Toledo, Marion; Chavey, Carine; Lapasset, Laure; Cavelier, Patricia; Lopez-Herrera, Celia; Chebli, Karim; Fort, Philippe; Beranger, Guillaume; Fajas, Lluis; Amri, Ez Z; Casas, Francois; Tazi, Jamal

    2014-05-01

    Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals. PMID:24639560

  12. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities. PMID:27091317

  13. SchA-p85-FAK complex dictates isoform-specific activation of Akt2 and subsequent PCBP1-mediated post-transcriptional regulation of TGFβ-mediated epithelial to mesenchymal transition in human lung cancer cell line A549.

    Xue, Xinying; Wang, Xin; Liu, Yuxia; Teng, Guigen; Wang, Yong; Zang, Xuefeng; Wang, Kaifei; Zhang, Jinghui; Xu, Yali; Wang, Jianxin; Pan, Lei

    2014-08-01

    A post-transcriptional pathway by which TGF-β modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-β for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-β induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-β treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-β-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma. PMID:24819169

  14. Expression of Two Novel Alternatively Spliced COL2A1 Isoforms During Chondrocyte Differentiation

    McAlinden, Audrey; Johnstone, Brian; Kollar, John; Kazmi, Najam; Hering, Thomas M.

    2007-01-01

    Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 n...

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

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

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week ...

  16. Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

    Hisayo Sadamoto

    2010-05-01

    Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

  17. Optimatization of transient transformation methods to study gene expression in Musa acuminata (AAA group) cultivar Ambon Lumut

    Prayuni, Kinasih; Dwivany, Fenny M.

    2015-09-01

    Banana is classified as a climateric fruit, whose ripening is regulated by ethylene. Ethylene is synthesized from ACC (1-aminocyclopropane-1-carboxylic acid) by ACC oxidase enzyme which is encoded by ACO gene. Controling an important gene expression in ethylene biosynthesis pathway has became a target to delay the ripening process. Therefore in the previous study we have designed a MaACO-RNAi construct to control MaACO gene expression. In this research, we study the effectiveness of different transient transformation methods to deliver the construct. Direct injection, with or no vaccum infiltration methods were used to deliver MaACO-RNAi construct. All of the methods succesfully deliver the construct into banana fruits based on RT-PCR result.

  18. Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria.

    Vurukonda, Sai Shiva Krishna Prasad; Vardharajula, Sandhya; Shrivastava, Manjari; SkZ, Ali

    2016-03-01

    Drought is one of the major constraints on agricultural productivity worldwide and is likely to further increase. Several adaptations and mitigation strategies are required to cope with drought stress. Plant growth promoting rhizobacteria (PGPR) could play a significant role in alleviation of drought stress in plants. These beneficial microorganisms colonize the rhizosphere/endo-rhizosphere of plants and impart drought tolerance by producing exopolysaccharides (EPS), phytohormones, 1-aminocyclopropane- 1-carboxylate (ACC) deaminase, volatile compounds, inducing accumulation of osmolytes, antioxidants, upregulation or down regulation of stress responsive genes and alteration in root morphology in acquisition of drought tolerance. The term Induced Systemic Tolerance (IST) was coined for physical and chemical changes induced by microorganisms in plants which results in enhanced tolerance to drought stresses. In the present review we elaborate on the role of PGPR in helping plants to cope with drought stress. PMID:26856449

  19. Co-inoculation with Rhizobium and plant growth promoting rhizobacteria (PGPR for inducing salinity tolerance in mung bean under field condition of semi arid climate

    Muhammad Aamir

    2013-04-01

    Full Text Available Salinity stress severely affects the growth, nodulation and yield of mung bean (Vigna radiata L.. However, its growth can be improved under salinity stress by inoculation/co-inoculation with rhizobia and plant growth promoting rhizobacteria (PGPR containing 1-Aminocyclopropane-1-carboxylic acid (ACC deaminase enzyme. ACC-deaminase containing bacteria regulate the stress induced ethylene production by hydrolyzing the ACC (immediate precursor of ethylene into ammonia and ketobutyric acid, thus improve plant growth by lowering the ethylene level. A study was conducted under salt affected field conditions where pre-isolated strains of Rhizobium and PGPR were used alone as well as in combination for mitigating the salinity stress on growth, nodulation and yield of mung bean by following the randomized complete block design (RCBD. The data were recorded and analyzed statistically to see the difference among treatments.

  20. Dysregulation of miRNA isoform level at 5' end in Alzheimer's disease.

    Wang, Shengqin; Xu, Yuming; Li, Musheng; Tu, Jing; Lu, Zuhong

    2016-06-15

    Alzheimer's disease (AD) is the most common form of dementia, whose mechanism is still not yet fully understood. A miRNA-based signature method, commonly according to the changes of expression levels, is widely used for AD analysis in previous studies. Recently, miRNA isoforms called as isomiR variants, which is considered to play important biological roles, have been demonstrated as the applications of high throughput sequencing platforms. Here, we presented an entropy-based model to detect the miRNA isoform level at the 5' end, and found many miRNAs with significant changes of isoform levels between the early stage and the late stage of AD by the application of this model to the public data. The statistical significance of the overlap between isoform-level changed miRNAs and AD related miRNAs extracted from HMDD2 supports that these miRNA isoforms are not degradation products. Based on the most common isomiR seed analysis of isoform-level changed AD related miRNAs, the predicted targets are also found to be enriched for genes involved in transcriptional regulation and the nervous system. After comparing with the expression level based method, we detected that changes of 5' isoform levels are more stable than those of expression levels for AD related miRNA detecting. PMID:26899870

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

    Gareth W. Fearnley

    2016-05-01

    Full Text Available 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.

  2. FSH isoform pattern in classic galactosemia

    Gubbels, Cynthia S.; Thomas, Chris M.G.; Wodzig, Will K. W. H.; Olthaar, André J.; Jaeken, Jaak; Sweep, Fred C. G. J.; Rubio-Gozalbo, M. Estela

    2010-01-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns...

  3. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    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…

  4. PKC Isoform Expression in Modeled Microgravity

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

    1999-01-01

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

  5. DAF-16/FoxO Directly Regulates an Atypical AMP-Activated Protein Kinase Gamma Isoform to Mediate the Effects of Insulin/IGF-1 Signaling on Aging in Caenorhabditis elegans

    Tullet, J. M.; Araiz, C.; Sanders, M J; Au, C.; Benedetto, A.; Papatheodorou, I.; Clark, E.; Schmeisser, K.; Jones, D.; Schuster, E F; Thornton, J M; Gems, D.

    2014-01-01

    The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, ...

  6. DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans.

    Tullet, Jennifer M. A.; Caroline Araiz; Sanders, Matthew J.; Catherine Au; Alexandre Benedetto; Irene Papatheodorou; Emily Clark; Kathrin Schmeisser; Daniel Jones; Eugene F Schuster; Thornton, Janet M.; David Gems

    2014-01-01

    The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, ...

  7. Molecular modeling study on tunnel behavior in different histone deacetylase isoforms.

    Sundarapandian Thangapandian

    Full Text Available Histone deacetylases (HDACs have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E in HDAC10 and leucine (L in HDAC 11 based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.

  8. Bacteria in combination with fertilizers promote root and shoot growth of maize in saline-sodic soil

    Muhammad Zafar-ul-Hye; Hafiz Muhammad Farooq; Mubshar Hussain

    2015-01-01

    Salinity is the leading abiotic stress hampering maize ( Zea mays L.) growth throughout the world, especially in Pakistan. During salinity stress, the endogenous ethylene level in plants increases, which retards proper root growth and consequent shoot growth of the plants. However, certain bacteria contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which converts 1-aminocyclopropane-1-carboxylic acid (an immediate precursor of ethylene biosynthesis in higher plants) into am...

  9. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  10. Influenza A Viruses Control Expression of Proviral Human p53 Isoforms p53β and Δ133p53α

    Terrier, Olivier; Marcel, Virginie; Cartet, Gaëlle; Lane, David P; Lina, Bruno; Rosa-Calatrava, Manuel; Bourdon, Jean-Christophe

    2012-01-01

    Previous studies have described the role of p53 isoforms, including p53β and Δ133p53α, in the modulation of the activity of full-length p53, which regulates cell fate. In the context of influenza virus infection, an interplay between influenza viruses and p53 has been described, with p53 being involved in the antiviral response. However, the role of physiological p53 isoforms has never been explored in this context. Here, we demonstrate that p53 isoforms play a role in influenza A virus infec...

  11. The FU gene and its possible protein isoforms

    Nöthen Markus M

    2004-07-01

    Full Text Available Abstract Background FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci. Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. Results The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1 maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. Conclusions The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing

  12. Absolute Quantification of Endogenous Ras Isoform Abundance.

    Craig J Mageean

    Full Text Available Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.

  13. Telomerase Regulation

    Cifuentes-Rojas, Catherine; Dorothy E Shippen

    2011-01-01

    The intimate connection between telomerase regulation and human disease is now well established. The molecular basis for telomerase regulation is highly complex and entails multiple layers of control. While the major target of enzyme regulation is the catalytic subunit TERT, the RNA subunit of telomerase is also implicated in telomerase control. In addition, alterations in gene dosage and alternative isoforms of core telomerase components have been described. Finally, telomerase localization,...

  14. Evaluation of Hepcidin Isoforms in Hemodialysis Patients by a Proteomic Approach Based on SELDI-TOF MS

    Oliviero Olivieri; Antonio Lupo; Nicola Martinelli; Albino Poli; Nicola Tessitore; Valeria Bedogna; Annalisa Castagna; Federica Zaninotto; Domenico Girelli; Natascia Campostrini

    2010-01-01

    The hepatic iron regulator hormone hepcidin consists, in its mature form, of 25 amino acids, but two other isoforms, hepcidin-20 and hepcidin-22, have been reported, whose biological meaning remains poorly understood. We evaluated hepcidin isoforms in sera from 57 control and 54 chronic haemodialysis patients using a quantitative proteomic approach based on SELDI-TOF-MS. Patients had elevated serum levels of both hepcidin-25 and hepcidin-20 as compared to controls (geometric means: 7.52 versu...

  15. Differential expression of intracellular and secreted osteopontin isoforms by murine macrophages in response to Toll-like receptor agonists.

    Zhao, Wei; Wang, Lijuan; Zhang, Lei; Yuan, Chao; Kuo, Paul C.; Gao, Chengjiang

    2015-01-01

    Osteopontin (OPN), expressed by various immune cells, modulates both innate and adaptive immune responses. Different immune cells have shown differential expression of the two isoforms of OPN: secreted form of OPN (sOPN) and intracellular form of OPN (iOPN). However, the molecular mechanisms that control opn gene expression and the OPN isoforms produced by immune cells remain largely unknown. In this study, we demonstrate that OPN mRNA and protein expression are significantly up-regulated upo...

  16. Influence of Temperature, Hypercapnia, and Development on the Relative Expression of Different Hemocyanin Isoforms in the Common Cuttlefish Sepia officinalis

    Strobel, Anneli; Hu, Marian Y.A.; Gutowska, Magdalena A.; Lieb, Bernhard; Lucassen, Magnus; Melzner, Frank; Pörtner, Hans-Otto; Mark, Felix Christopher

    2012-01-01

    The cuttlefish Sepia officinalis expresses several hemocyanin isoforms with potentially different pH optima, indicating their reliance on efficient pH regulation in the blood. Ongoing ocean warming and acidification could influence the oxygen-binding properties of respiratory pigments in ectothermic marine invertebrates. This study examined whether S. officinalis differentially expresses individual hemocyanin isoforms to maintain optimal oxygen transport during development and ...

  17. Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2

    Lee, Yun-Han; Seo, Daekwan; Choi, Kyung-Ju;

    2014-01-01

    Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit H...

  18. A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

    Ioannis Gavvovidis

    Full Text Available Biallelic mutations in MCPH1 cause primary microcephaly (MCPH with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL and a second transcript lacking the six 3' exons (MCPH1Δe9-14. Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1Δe9-14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level.

  19. Unraveling complex interplay between heat shock factor 1 and 2 splicing isoforms.

    Sylvain Lecomte

    Full Text Available Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs. The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and β, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the β isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short β isoform of HSF2 exerts a negative role on HSF1β-dependent transactivation. To further assess the impact of HSF2β inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.

  20. The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

    Lili Li

    Full Text Available Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREB(αΔ mice. Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ mice. CREB(αΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ. CREB(αΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

  1. Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

    De Santanu

    2012-01-01

    Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

  2. Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities.

    Luis J Cocka

    2012-09-01

    Full Text Available Tetherin (BST-2/CD317/HM1.24 is an IFN induced transmembrane protein that restricts release of a broad range of enveloped viruses. Important features required for Tetherin activity and regulation reside within the cytoplasmic domain. Here we demonstrate that two isoforms, derived by alternative translation initiation from highly conserved methionine residues in the cytoplasmic domain, are produced in both cultured human cell lines and primary cells. These two isoforms have distinct biological properties. The short isoform (s-Tetherin, which lacks 12 residues present in the long isoform (l-Tetherin, is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 viral budding in the presence of Vpu. s-Tetherin Vpu resistance can be accounted for by the loss of serine-threonine and tyrosine motifs present in the long isoform. By contrast, the l-Tetherin isoform was found to be an activator of nuclear factor-kappa B (NF-κB signaling whereas s-Tetherin does not activate NF-κB. Activation of NF-κB requires a tyrosine-based motif found within the cytoplasmic tail of the longer species and may entail formation of l-Tetherin homodimers since co-expression of s-Tetherin impairs the ability of the longer isoform to activate NF-κB. These results demonstrate a novel mechanism for control of Tetherin antiviral and signaling function and provide insight into Tetherin function both in the presence and absence of infection.

  3. Changes in claudin isoform expression in the gill during salinity shifts and smoltification of Atlantic salmon

    Madsen, Steffen; Tipsmark, Christian Kølbæk

    2008-01-01

    expression was confirmed by RT-QPCR. We examined the expression profile during the parr-smolt transformation (PST) in freshwater and during acclimation to sea water (SW). During PST, claudin 10e expression peaked in May, coinciding with optimal SW tolerance. The other claudin isoforms were not influenced...... during PST. SW-transfer induced a 5-fold increase in expression of claudin 10e, reduced the expression of 27a and 30a and had no overall effect on 28a and 28b isoforms. The study demonstrates for the first time that SW acclimation involves differential regulation of claudin gene expression in the salmon...

  4. p53 isoforms change p53 paradigm

    Bourdon, JC

    2014-01-01

    Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

  5. New isoforms of rat Aquaporin-4

    Moe, Svein Erik; Sorbo, Jan Gunnar; Søgaard, Rikke;

    2008-01-01

    Aquaporin-4 (AQP4) is a brain aquaporin implicated in the pathophysiology of numerous clinical conditions including brain edema. Here we show that rat AQP4 has six cDNA isoforms, formed by alternative splicing. These are named AQP4a-f, where AQP4a and AQP4c correspond to the two classical M1 and M...

  6. Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure.

    Sophie A O Armitage

    Full Text Available 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.

  7. [Preparation and properties of isocitrate lyase isoforms from the cotyledons of Glycine max L].

    Eprintsev, A T; Diachenko, E V; Lykova, T V; Kuen, C T H; Popov, V N

    2010-01-01

    A four-stage purification procedure including ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose has been elaborated for isolation of isocitrate lyase (EC 4.1.3.1) isoforms from the cotyledons of soybean Glycine max L. Electrophoretically homogeneous preparations of two forms of the enzyme with specific activity of 5.28 and 5.81 U/mg protein have been obtained. Comparison of physicochemical, kinetic, and regulation characteristics of the isoforms obtained revealed fundamental differences between them. Thus, the isoform that migrated quickly in PAAG had a much lower affinity to isocitrate (K(M) - 50 microM) than the slowly migrating form (K(M) - 16 microM). It has been shown that the conservation of activity of the isoforms obtained depends on the presence of divalent cations (Mn2+ and Mg2+) in the medium. It is suggested to use the isoforms of isocitrate lyase isolated from soybeans for the development of biosensors for biochemical and kinetic assays. PMID:20198926

  8. Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

    Carl O Olson

    Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

  9. Identification of novel chicken estrogen receptor-alpha messenger ribonucleic acid isoforms generated by alternative splicing and promoter usage.

    Griffin, C; Flouriot, G; Sonntag-Buck, V; Nestor, P; Gannon, F

    1998-11-01

    Using the rapid amplification of complementary DNA ends (RACE) methodology we have identified three new chicken estrogen receptor-alpha (cER alpha) messenger RNA (mRNA) variants in addition to the previously described form (isoform A). Whereas one of the new variants (isoform B) presents a 5'-extremity contiguous to the 5'-end of isoform A, the two other forms (isoforms C and D) are generated by alternative splicing of upstream exons (C and D) to a common site situated 70 nucleotides upstream of the translation start site in the previously assigned exon 1 (A). The 3'-end of exon 1C has been located at position -1334 upstream of the transcription start site of the A isoform (+1). Whereas the genomic location of exon 1D is unknown, 700 bp 5' to this exon were isolated by genomic walking, and their sequence was determined. The transcription start sites of the cER alpha mRNA isoforms were defined. In transfection experiments, the regions immediately upstream of the A-D cER alpha mRNA isoforms were shown to possess cell-specific promoter activities. Three of these promoters were down-regulated in the presence of estradiol and ER alpha protein. It is concluded, therefore, that the expression of the four different cER alpha mRNA isoforms is under the control of four different promoters. Finally, RT-PCR, S1 nuclease mapping, and primer extension analysis of these different cER alpha mRNA isoforms revealed a differential pattern of expression of the cER alpha gene in chicken tissues. Together, the results suggest that alternative 5'-splicing and promoter usage may be mechanisms used to modulate the levels of expression of the chicken ER alpha gene in a tissue-specific and/or developmental stage-specific manner. PMID:9794473

  10. Entropy-based model for miRNA isoform analysis.

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  11. Differential expression of Na+, K(+)-ATPase α-1 isoforms during seawater acclimation in the amphidromous galaxiid fish Galaxias maculatus.

    Urbina, Mauricio A; Schulte, Patricia M; Bystriansky, Jason S; Glover, Chris N

    2013-04-01

    Inanga (Galaxias maculatus) is an amphidromous fish with a well-known capacity to withstand a wide range of environmental salinities. To investigate the molecular mechanisms facilitating acclimation of inanga to seawater, several isoforms of the Na(+), K(+)-ATPase ion transporter were identified. This included three α-1 (a, b and c), an α-2 and two α-3 (a and b) isoforms. Phylogenetic analysis showed that the inanga α-1a and α-1b formed a clade with the α-1a and α-1b isoforms of rainbow trout, while another clade contained the α-1c isoforms of these species. The expression of all the α-1 isoforms was modulated after seawater exposure (28‰). In gills, the expression of the α-1a isoform was progressively down-regulated after seawater exposure, while the expression of the α-1b isoform was up-regulated. The α-1c isoform behaved similarly to the α-1a, although changes were less dramatic. Physiological indicators of salinity acclimation matched the time frame of the changes observed at the molecular level. A 24-h osmotic shock period was highlighted by small increases in plasma osmolality, plasma Na(+) and a decrease in muscle tissue water content. Thereafter, these values returned close to their pre-exposure (freshwater) values. Na(+), K(+)-ATPase activity showed a decreasing trend over the first 72 h following seawater exposure, but activity increased after 240 h. Our results indicate that inanga is an excellent osmoregulator, an ability that is conferred by the rapid activation of physiological and molecular responses to salinity change. PMID:23142926

  12. Lipoprotein lipase isoelectric point isoforms in humans

    Badia-Villanueva, M.; Carulla, P.; Carrascal, M.;

    2014-01-01

    characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC-MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of......-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and...

  13. Functional studies of sodium pump isoforms

    Clausen, Michael Jakob

    The Na+,K+-ATPase is an essential ion pump found in all animal cells. It uses the energy from ATP hydrolysis to export three Na+ and import two K+, both against their chemical gradients and for Na+ also against the electrical potential. Mammals require four Na+,K+-ATPase isoforms that each have...... synthesized cohorts of pumps from the Golgi apparatus to the plasma membrane....

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

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo, E-mail: kawamots@mail.nih.gov

    2014-08-08

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

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

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

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

    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 first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice. PMID:26643381

  17. Isoformes du domaine N-terminal du suppresseur de tumeur p53 : sur l'activité transcriptionnelle de p53 et expression dans les mélanomes cutanés

    Hafsi, Hind

    2012-01-01

    The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms....

  18. Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain [v1; ref status: indexed, http://f1000r.es/1mg

    Lara Kaddoum

    2013-10-01

    Full Text Available Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene.  MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2 that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.

  19. Immunocytochemical studies of the distribution of alpha and pi isoforms of glutathione S-transferase in cystic renal diseases.

    Hiley, C G; Otter, M; Bell, J; Strange, R C; Keeling, J W

    1994-01-01

    We describe immunohistochemical studies of the expression of alpha and pi class glutathione S-transferases (GSTs) in normal fetal kidneys. These define, in greater detail, changes in expression of alpha isoforms in the proximal tubule. At about 36 weeks of gestation expression of alpha isoforms was down-regulated in the distal tubules and collecting ducts while pi was expressed throughout the nephron. Tubular expression of alpha isoforms was restricted to the part adjacent to the glomerulus; cells farthest from the glomerulus were negative. After 40 weeks of gestation, alpha isoforms were expressed along the entire proximal tubule, while pi was restricted to the distal tubule and collecting ducts. GST expression was also studied in multicystic renal dysplasia, autosomal recessive polycystic kidney disease, and autosomal dominant polycystic kidney disease to determine whether the patterns of expression of alpha and pi isoforms allow identification of the origin of the cysts that characterize these diseases. Cysts were lined by epithelia that were strongly positive for alpha and pi isoforms. The epithelia of noncystic nephrons in renal cystic dysplasia demonstrated delayed maturity, suggesting that GST expression was dependent on the stage of development and not length of gestation. PMID:8066005

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

    Pio Ruben

    2010-12-01

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

  1. Identification of alternatively spliced Dab1 and Fyn isoforms in pig

    Yuan Jihong

    2011-02-01

    Full Text Available Abstract Background Disabled-1 (Dab1 is an adaptor protein that is essential for the intracellular transduction of Reelin signaling, which regulates the migration and differentiation of postmitotic neurons during brain development in vertebrates. Dab1 function depends on its tyrosine phosphorylation by Src family kinases, especially Fyn. Results We have isolated alternatively spliced forms of porcine Dab1 from brain (sDab1 and liver (sDab1-Li and Fyn from brain (sFyn-B and spleen (sFyn-T. Radiation hybrid mapping localized porcine Dab1 (sDab1 and Fyn (sFyn to chromosomes 6q31-35 and 1p13, respectively. Real-time quantitative RT-PCR (qRT-PCR demonstrated that different isoforms of Dab1 and Fyn have tissue-specific expression patterns, and sDab1 and sFyn-B display similar temporal expression characteristics in the developing porcine cerebral cortex and cerebellum. Both sDab1 isoforms function as nucleocytoplasmic shuttling proteins. It was further shown that sFyn phosphorylates sDab1 at tyrosyl residues (Tyr 185, 198/200 and 232, whereas sDab1-Li was phosphorylated at Tyr 185 and Tyr 197 (corresponding to Y232 in sDab1 in vitro. Conclusions Alternative splicing generates natural sDab1-Li that only carries Y185 and Y197 (corresponding to Y232 in sDab1 sites, which can be phosphorylated by Fyn in vitro. sDab1-Li is an isoform that is highly expressed in peripheral organs. Both isoforms are suggested to be nucleocytoplasmic shuttling proteins. Our results imply that the short splice form sDab1-Li might regulate cellular responses to different cell signals by acting as a dominant negative form against the full length sDab1 variant and that both isoforms might serve different signaling functions in different tissues.

  2. The absence of dystrophin brain isoform expression in healthy human heart ventricles explains the pathogenesis of 5' X-linked dilated cardiomyopathy

    Neri Marcella

    2012-03-01

    Full Text Available Abstract Background In X-linked dilated cardiomyopathy due to dystrophin mutations which abolish the expression of the M isoform (5'-XLDC, the skeletal muscle is spared through the up-regulation of the Brain (B isoform, a compensatory mechanism that does not appear to occur in the heart of affected individuals. Methods We quantitatively studied the expression topography of both B and M isoforms in various human heart regions through in-situ RNA hybridization, Reverse-Transcriptase and Real-Time PCR experiments. We also investigated the methylation profile of the B promoter region in the heart and quantified the B isoform up regulation in the skeletal muscle of two 5'-XLDC patients. Results Unlike the M isoform, consistently detectable in all the heart regions, the B isoform was selectively expressed in atrial cardiomyocytes, but absent in ventricles and in conduction system structures. Although the level of B isoform messenger in the skeletal muscle of 5'-XLDC patients was lower that of the M messenger present in control muscle, it seems sufficient to avoid an overt muscle pathology. This result is consistent with the protein level in XLDC patients muscles we previously quantified. Methylation studies revealed that the B promoter shows an overall low level of methylation at the CG dinucleotides in both atria and ventricles, suggesting a methylation-independent regulation of the B promoter activity. Conclusions The ventricular dilatation seen in 5'-XLDC patients appears to be functionally related to loss of the M isoform, the only isoform transcribed in human ventricles; in contrast, the B isoform is well expressed in heart but confined to the atria. Since the B isoform can functionally replace the M isoform in the skeletal muscle, its expression in the heart could potentially exert the same rescue function. Methylation status does not seem to play a role in the differential B promoter activity in atria and ventricles, which may be governed by

  3. Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function.

    Jobling, Andrew Ian; Wan, Ran; Gentle, Alex; Bui, Bang Viet; McBrien, Neville Anthony

    2009-03-01

    the specific nature of TGF-beta isoform expression, which reflects the differences in tissue structure and function. While TGF-beta isoforms are involved in scleral regulation during myopia development in mammals, they do not have a primary role in the retinal and choroidal signals. Thus, the regulation of eye growth via the retinoscleral cascade involves more than one factor, which is likely to be tissue-specific in nature. PMID:19046968

  4. FOXP3 and CTLA-4 : how isoforms regulate immunological tolerance

    Liu, Sang

    2015-01-01

    The maintenance of immunological tolerance is vital for preventing the immune system to damage normal tissues and physiological function of the body. CD4+FOXP3+ regulatory T (Treg) cells can suppress immune responses in a dominant manner and are essential for immunological tolerance. Although many pathways and molecules have been attributed to the suppressive function of Treg cells, the exact nature of the Treg cell-mediated suppression program is still elusive. In this ...

  5. Evolutionary dynamics of gene and isoform regulation in mammalian tissues*

    Merkin, Jason; Russell, Caitlin; CHEN, PING; Burge, Christopher B.

    2012-01-01

    Most mammalian genes produce multiple distinct mRNAs through alternative splicing, but the extent of splicing conservation is not clear. To assess tissue-specific transcriptome variation across mammals, we sequenced cDNA from 9 tissues from 4 mammals and one bird in biological triplicate, at unprecedented depth. We find that while tissue-specific gene expression programs are largely conserved, alternative splicing is well conserved in only a subset of tissues and is frequently lineage-specifi...

  6. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    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. Both Myosin-10 isoforms are required for radial neuronal migration in the developing cerebral cortex.

    Ju, Xing-Da; Guo, Ye; Wang, Nan-Nan; Huang, Ying; Lai, Ming-Ming; Zhai, Yan-Hua; Guo, Yu-Guang; Zhang, Jian-Hua; Cao, Rang-Juan; Yu, Hua-Li; Cui, Lei; Li, Yu-Ting; Wang, Xing-Zhi; Ding, Yu-Qiang; Zhu, Xiao-Juan

    2014-05-01

    During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration. PMID:23300110

  8. A mutation in a skin-specific isoform of SMARCAD1 causes autosomal-dominant adermatoglyphia.

    Nousbeck, Janna; Burger, Bettina; Fuchs-Telem, Dana; Pavlovsky, Mor; Fenig, Shlomit; Sarig, Ofer; Itin, Peter; Sprecher, Eli

    2011-08-12

    Monogenic disorders offer unique opportunities for researchers to shed light upon fundamental physiological processes in humans. We investigated a large family affected with autosomal-dominant adermatoglyphia (absence of fingerprints) also known as the "immigration delay disease." Using linkage and haplotype analyses, we mapped the disease phenotype to 4q22. One of the genes located in this interval is SMARCAD1, a member of the SNF subfamily of the helicase protein superfamily. We demonstrated the existence of a short isoform of SMARCAD1 exclusively expressed in the skin. Sequencing of all SMARCAD1 coding and noncoding exons revealed a heterozygous transversion predicted to disrupt a conserved donor splice site adjacent to the 3' end of a noncoding exon uniquely present in the skin-specific short isoform of the gene. This mutation segregated with the disease phenotype throughout the entire family. Using a minigene system, we found that this mutation causes aberrant splicing, resulting in decreased stability of the short RNA isoform as predicted by computational analysis and shown by RT-PCR. Taken together, the present findings implicate a skin-specific isoform of SMARCAD1 in the regulation of dermatoglyph development. PMID:21820097

  9. Localization and functional characterization of the human NKCC2 isoforms

    Carota, I; Theilig, F; Oppermann, M;

    2010-01-01

    inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

  10. Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry

    Linda V. Hjørnevik

    2015-12-01

    Full Text Available Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s. A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

  11. Androgen receptor isoforms in human and rat prostate

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  12. p53 isoform Δ113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish

    Chen, Jun; Ng, Sok Meng; Chang, Changqing; Zhang, Zhenhai; Bourdon, Jean-Christophe; Lane, David P; Peng, Jinrong

    2009-01-01

    p53 is a well-known tumor suppressor and is also involved in processes of organismal aging and developmental control. A recent exciting development in the p53 field is the discovery of various p53 isoforms. One p53 isoform is human Δ133p53 and its zebrafish counterpart Δ113p53. These N-terminal-truncated p53 isoforms are initiated from an alternative p53 promoter, but their expression regulation and physiological significance at the organismal level are not well understood. We show here that ...

  13. Characterization of Alien isoforms in vertebrates : Caracterización de isoformas de Alien en vertebrados

    Tenbaum, Stephan

    2002-01-01

    Alien protein isoforms have been described to be involved in a number of biological processes. Alienalpha is a corepressor of the thyroid hormone receptor mediating transcriptional repression in a ligand-sensitive manner. Furthermore, Alienalpha is a corepressor for the orphan receptor DAX1 and the vitamin-D3 receptor. Alienbetta/CSN2 is part of the COP9-signalosome complex that acts in protein phosphorylation, protein degradation and cell cycle regulation. The major goal of this...

  14. SASD: the Synthetic Alternative Splicing Database for identifying novel isoform from proteomics

    Zhang, Fan; Drabier, Renee

    2013-01-01

    Background Alternative splicing is an important and widespread mechanism for generating protein diversity and regulating protein expression. High-throughput identification and analysis of alternative splicing in the protein level has more advantages than in the mRNA level. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from pro...

  15. Truffles regulate plant root morphogenesis via the production of auxin and ethylene.

    Splivallo, Richard; Fischer, Urs; Göbel, Cornelia; Feussner, Ivo; Karlovsky, Petr

    2009-08-01

    Truffles are symbiotic fungi that form ectomycorrhizas with plant roots. Here we present evidence that at an early stage of the interaction, i.e. prior to physical contact, mycelia of the white truffle Tuber borchii and the black truffle Tuber melanopsorum induce alterations in root morphology of the host Cistus incanus and the nonhost Arabidopsis (Arabidopsis thaliana; i.e. primary root shortening, lateral root formation, root hair stimulation). This was most likely due to the production of indole-3-acetic acid (IAA) and ethylene by the mycelium. Application of a mixture of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and IAA fully mimicked the root morphology induced by the mycelium for both host and nonhost plants. Application of the single hormones only partially mimicked it. Furthermore, primary root growth was not inhibited in the Arabidopsis auxin transport mutant aux1-7 by truffle metabolites while root branching was less effected in the ethylene-insensitive mutant ein2-LH. The double mutant aux1-7;ein2-LH displayed reduced sensitivity to fungus-induced primary root shortening and branching. In agreement with the signaling nature of truffle metabolites, increased expression of the auxin response reporter DR5GFP in Arabidopsis root meristems subjected to the mycelium could be observed, confirming that truffles modify the endogenous hormonal balance of plants. Last, we demonstrate that truffles synthesize ethylene from l-methionine probably through the alpha-keto-gamma-(methylthio)butyric acid pathway. Taken together, these results establish the central role of IAA and ethylene as signal molecules in truffle/plant interactions. PMID:19535471

  16. Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

    Wong, Marty Kwok-Shing; Pipil, Supriya; Kato, Akira; Takei, Yoshio

    2016-09-01

    Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl(-) channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney. PMID:27322796

  17. Identification of a novel TDRD7 isoforms

    Filonenko V. V.

    2011-12-01

    Full Text Available The aim of our study was to investigate the tudor domain-containing protein 7 (TDRD7 subcellular localization, which could be linked to diverse functions of this protein within the cell. Methods. In this study we employed cell imaging technique for detecting TDRD7 subcellular localization, Western blot analysis of HEK293 cell fractions with anti-TDRD7 monoclonal antibodies and bioinformatical search of possible TDRD7 isoforms in Uniprot, Ensemble, UCSC databases. Results. We have observed specific TDRD7-containing structures in cytoplasm as well as in the nucleus in HEK293 cells. The Western blot analysis of subcellular fractions (cytoplasm, mitochondria, nucleus allowed us to detect three lower immunoreactive bands, with the aproximate molecular weight of 130, 110 and 60 kDa (we termed them as TDRD7, TDRD7 and TDRD7 and specific subcellular localization. The bioinformatical analysis of TDRD7 primary structure allowed us to determine two alternative transcripts from TDRD7 gene coding for proteins with calculated molecular weight of 130 and 60 kDa. Conclusion. The presented data demonstrate the existence at protein level of potential TDRD7 isoforms: TDRD7, TDRD7 and TDRD7. The expression profile of these splice variants and their role in cells remains to be elucidated.

  18. Multiple, but Concerted Cellular Activities of the Human Protein Hap46/BAG-1M and Isoforms

    Ulrich Gehring

    2009-03-01

    Full Text Available The closely related human and murine proteins Hap46/BAG-1M and BAG-1, respectively, were discovered more than a decade ago by molecular cloning techniques. These and the larger isoform Hap50/BAG-1L, as well as shorter isoforms, have the ability to interact with a seemingly unlimited array of proteins of completely unrelated structures. This problem was partially resolved when it was realized that molecular chaperones of the hsp70 heat shock protein family are major primary association partners, binding being mediated by the carboxy terminal BAG-domain and the ATP-binding domain of hsp70 chaperones. The latter, in turn, can associate with an almost unlimited variety of proteins through their substrate-binding domains, so that ternary complexes may result. The protein folding activity of hsp70 chaperones is affected by interactions with Hap46/BAG-1M or isoforms. However, there also exist several proteins which bind to Hap46/BAG-1M and isoforms independent of hsp70 mediation. Moreover, Hap46/BAG-1M and Hap50/BAG-1L, but not the shorter isoforms, can bind to DNA in a sequence-independent manner by making use of positively charged regions close to their amino terminal ends. This is the molecular basis for their effects on transcription which are of major physiological relevance, as discussed here in terms of a model. The related proteins Hap50/BAG-1L and Hap46/BAG-1M may thus serve as molecular links between such diverse bioactivities as regulation of gene expression and protein quality control. These activities are coordinated and synergize in helping cells to cope with conditions of external stress. Moreover, they recently became markers for the aggressiveness of several cancer types.

  19. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

    Wouter Eilers

    2014-01-01

    Full Text Available We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis and slow-type muscle (soleus for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02. In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII.

  20. Immunodetection of nmt55/p54nrb isoforms in human breast cancer

    We previously identified and characterized a novel 55 kDa nuclear protein, termed nmt55/p54nrb, whose expression was decreased in a subset of human breast tumors. The objective of this study was to determine if this reduced expression in human breast tumors was attributed to the regulation of mRNA transcription or the presence of altered forms of this protein. Northern blot analysis and ribonuclease protection assay indicated that nmt55/p54nrb mRNA is expressed at varying levels in estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast tumors suggesting that reduced expression of nmt55/p54nrb protein in ER- tumors was not due to transcriptional regulation. To determine if multiple protein isoforms are expressed in breast cancer, we utilized Western blot and immunohistochemical analyses, which revealed the expression of an nmt55/p54nrb protein isoform in a subset of ER+ tumors. This subset of ER+ human breast tumors expressed an altered form of nmt55/p54nrb that was undetectable with an amino-terminal specific antibody suggesting that this isoform contains alterations or modifications within the amino terminal domain. Our study indicates that nmt55/p54nrb protein is post-transcriptionally regulated in human breast tumors leading to reduced expression in ER- tumors and the expression of an amino terminal altered isoform in a subset of ER+ tumors. The potential involvement of nmt55/p54nrb in RNA binding and pre-mRNA splicing may be important for normal cell growth and function; thus, loss or alteration of protein structure may contribute to tumor growth and progression

  1. The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

    The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5

  2. MXI1-0, an Alternatively Transcribed Mxi1 Isoform, Is Overexpressed in Glioblastomas

    Lars D. Engstrom

    2004-09-01

    Full Text Available The c-Myc transcription factor regulates expression of genes related to cell growth, division, and apoptosis. Will, a member of the Mad family, represses transcription of c-Myc-regulated genes by mediating chromatin condensation via histone deacetylase and the Sin3 corepressor. Mxi1 is a c-Myc antagonist and suppresses cell proliferation in vitro. Here, we describe the identification of MXI1-0, a novel Mxi1 isoform that is alternatively transcribed from an upstream exon. MXI1-0 and Mxi1 have different amino-terminal sequences, but share identical Max- and DNA-binding domains. Both isoforms are able to bind Max, to recognize E-box binding sites, and to interact with Sin3. Despite these similarities and in contrast to Will, MXl10 is predominantly localized to the cytoplasm and fails to repress c-Myc-dependent transcription. Although MXI1-0 and Mxi1 are coexpressed in both human and mouse cells, the relative levels of MXI1-0 are higher in primary glioblastoma tumors than in normal brain tissue. This variation in the levels of MXI1-0 and Mxi1 suggests that MXI1-0 may modulate the Myc-inhibitory activity of Will. The identification of MXI1-0 as an alternatively transcribed Mxi1 isoform has significant implications for the interpretation of previous Mxi1 studies, particularly those related to the phenotype of the mxi1 knockout mouse.

  3. Expression of Monocarboxylate Transporter Isoforms in Rat Skeletal Muscle Under Hypoxic Preconditioning and Endurance Training.

    Saxena, Saurabh; Shukla, Dhananjay; Bansal, Anju

    2016-03-01

    Previously, we have reported the regulation of monocarboxylate transporters (MCT)1 and MCT4 by physiological stimuli such as hypoxia and exercise. In the present study, we have evaluated the effect of hypoxic preconditioning and training on expression of different MCT isoforms in muscles. We found the increased mRNA expression of MCT1, MCT11, and MCT12 after hypoxic preconditioning with cobalt chloride and training. However, the expression of other MCT isoforms increased marginally or even reduced after hypoxic preconditioning. Only the protein expression of MCT1 increased after hypoxia preconditioning. MCT2 protein expression increased after training only and MCT4 protein expression decreased both in preconditioning and hypoxic training. Furthermore, we found decreased plasma lactate level during hypoxia preconditioning (0.74-fold), exercise (0.78-fold), and hypoxia preconditioning along with exercise (0.67-fold), which indicates increased lactate uptake by skeletal muscle. The protein-protein interactions with hypoxia inducible factor-1 and MCT isoforms were also evaluated, but no interaction was found. In conclusion, we say that almost all MCTs are expressed in red gastrocnemius muscle at the mRNA level and their expression is regulated differently under hypoxia preconditioning and exercise condition. PMID:26716978

  4. A novel FADS1 isoform potentiates FADS2-mediated production of eicosanoid precursor fatty acids.

    Park, Woo Jung; Kothapalli, Kumar S D; Reardon, Holly T; Lawrence, Peter; Qian, Shu-Bing; Brenna, J Thomas

    2012-08-01

    The fatty acid desaturase (FADS) genes code for the rate-limiting enzymes required for the biosynthesis of long-chain polyunsaturated fatty acids (LCPUFA). Here we report discovery and function of a novel FADS1 splice variant. FADS1 alternative transcript 1 (FADS1AT1) enhances desaturation of FADS2, leading to increased production of eicosanoid precursors, the first case of an isoform modulating the enzymatic activity encoded by another gene. Multiple protein isoforms were detected in primate liver, thymus, and brain. In human neuronal cells, their expression patterns are modulated by differentiation and result in alteration of cellular fatty acids. FADS1, but not FADS1AT1, localizes to endoplasmic reticulum and mitochondria. Ribosomal footprinting demonstrates that all three FADS genes are translated at similar levels. The noncatalytic regulation of FADS2 desaturation by FADS1AT1 is a novel, plausible mechanism by which several phylogenetically conserved FADS isoforms may regulate LCPUFA biosynthesis in a manner specific to tissue, organelle, and developmental stage. PMID:22619218

  5. B cell receptor-mediated apoptosis of human lymphocytes is associated with a new regulatory pathway of Bim isoform expression.

    Mouhamad, Shahul; Besnault, Laurence; Auffredou, Marie Thérèse; Leprince, Corinne; Bourgeade, Marie Françoise; Leca, Gérald; Vazquez, Aimé

    2004-02-15

    Studies in Bim-deficient mice have shown that the proapoptotic molecule Bim plays a key role in the control of B cell homeostasis and activation. However, the role of Bim in human B lymphocyte apoptosis is unknown. We show in this study that, depending on the degree of cross-linking, B cell receptors can mediate both Bim-dependent and apparent Bim-independent apoptotic pathways. Cross-linked anti-mu Ab-mediated activation induces an original pathway governing the expression of the various Bim isoforms. This new pathway involves the following three sequential steps: 1) extracellular signal-regulated kinase-dependent phosphorylation of the BimEL isoform, which is produced in large amounts in healthy B cells; 2) proteasome-mediated degradation of phosphorylated BimEL; and 3) increased expression of the shorter apoptotic isoforms BimL and BimS. PMID:14764673

  6. Primary structure, tissue distribution, and chromosomal localization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3)

    Valtavaara, M; Szpirer, C; Szpirer, J; Myllylä, R

    1998-05-22

    We report characterization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3, LH3). The cDNA clones encode a polypeptide of 738 amino acids, including a signal peptide. The amino acid sequence has a high overall identity with LH1 and LH2, the isoforms characterized earlier. Conserved regions are present in the carboxyl-terminal portion of the isoforms and also in the central part of the molecules. Histidine and asparagine residues, which are conserved in the other isoforms and are known to be required for enzymatic activity, are also conserved in the novel isoform. The gene for LH3 (PLOD3) has been assigned to human chromosome 7q36 and rat chromosome 12. Gene expression of LH3 is highly regulated in adult human tissues. A strong hybridization signal, corresponding to an mRNA 2.75 kilobases in size, is obtained in heart, placenta and pancreas on multiple tissue RNA blots. Expression of the cDNA in vitro results in the synthesis of a protein that hydroxylates lysyl residues in collagenous sequences in a non-triple helical conformation. PMID:9582318

  7. PKD3 is the predominant protein kinase D isoform in mouse exocrine pancreas and promotes hormone-induced amylase secretion.

    Chen, L Andy; Li, Jing; Silva, Scott R; Jackson, Lindsey N; Zhou, Yuning; Watanabe, Hiroaki; Ives, Kirk L; Hellmich, Mark R; Evers, B Mark

    2009-01-23

    The protein kinase D (PKD) family of serine/threonine kinases, which can be activated by gastrointestinal hormones, consists of three distinct isoforms that modulate a variety of cellular processes including intracellular protein transport as well as constitutive and regulated secretion. Although isoform-specific functions have been identified in a variety of cell lines, the expression and function of PKD isoforms in normal, differentiated secretory tissues is unknown. Here, we demonstrate that PKD isoforms are differentially expressed in the exocrine and endocrine cells of the pancreas. Specifically, PKD3 is the predominant isoform expressed in exocrine cells of the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly expressed in the pancreatic islets. Within isolated mouse pancreatic acinar cells, PKD3 undergoes rapid membrane translocation, trans-activating phosphorylation, and kinase activation after gastrointestinal hormone or cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs viaaCa2+-independent, diacylglycerol- and protein kinase C-dependent mechanism. PKD phosphorylation can also be induced by physiologic concentrations of secretagogues and by in vivo stimulation of the pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK, ribosomal S6 kinase) signaling and significantly enhances cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a novel distinction between the exocrine and endocrine cells of the pancreas and further identify PKD3 as a signaling molecule that promotes hormone-stimulated amylase secretion. PMID:19028687

  8. Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts

    Yonatan Stelzer

    2015-04-01

    Full Text Available Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5′ RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.

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

    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

  10. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach; Christensen, Hans Erik Mølager

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed......, purified and the kinetic properties have been studied and are compared. Substrate inhibition by tryptophan is observed for isoform 1 but not for isoform 2. Large differences are observed in the K m,tetrahydrobiopterin values for the two isoforms, being >10 times larger for isoform 1 compared to isoform 2....